US20200181220A1 - Cytokine conjugates for the treatment of proliferative and infectious diseases - Google Patents

Cytokine conjugates for the treatment of proliferative and infectious diseases Download PDF

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US20200181220A1
US20200181220A1 US16/634,479 US201816634479A US2020181220A1 US 20200181220 A1 US20200181220 A1 US 20200181220A1 US 201816634479 A US201816634479 A US 201816634479A US 2020181220 A1 US2020181220 A1 US 2020181220A1
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polypeptide
modified
cell
isolated
amino acid
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Jerod Ptacin
Carolina E. CAFFARO
Marcos MILLA
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Synthorx Inc
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Synthorx Inc
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Definitions

  • T cells Distinct populations of T cells modulate the immune system to maintain immune homeostasis and tolerance.
  • regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological self-reactivity while cytotoxic T cells target and destroy infected cells and/or cancerous cells.
  • modulation of the different populations of T cells provides an option for treatment of a disease or indication.
  • cytokine conjugates and use in the treatment of one or more indication.
  • also described herein include interleukin 2 (IL-2) conjugates and use in the treatment of one or more indications.
  • the one or more indications comprise cancer or a pathogenic infection.
  • described herein are methods of modulating the interaction between IL-2 and IL-2 receptor to stimulate or expand specific T cell, Natural Killer (NK) cell, and/or Natural killer T (NKT) cell populations.
  • pharmaceutical compositions and kits that comprise one or more interleukin conjugates (e.g., IL-2 conjugates) described herein.
  • an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO: 1.
  • the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64.
  • the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine.
  • the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid.
  • the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa
  • the IL-2 conjugate has a decreased affinity to IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to a wild-type IL-2 polypeptide.
  • the decreased affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2R ⁇ relative to a wild-type IL-2 polypeptide.
  • the decreased affinity 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, 500-fold, 1000-fold, or more relative to a wild-type IL-2 polypeptide.
  • the conjugating moiety impairs or blocks the binding of IL-2 with IL-2R ⁇ .
  • the conjugating moiety comprises a water-soluble polymer.
  • the additional conjugating moiety comprises a water-soluble polymer.
  • each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.
  • each of the water-soluble polymers independently comprises PEG.
  • the PEG is a linear PEG or a branched PEG.
  • each of the water-soluble polymers independently comprises a polysaccharide.
  • the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • each of the water-soluble polymers independently comprises a glycan.
  • each of the water-soluble polymers independently comprises polyamine.
  • the conjugating moiety comprises a protein.
  • the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises an albumin, a transferrin, or a 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 IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide.
  • each of the polypeptides independently comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.
  • the isolated and purified IL-2 polypeptide is modified by glutamylation.
  • the conjugating moiety is directly bound to the isolated and purified IL-2 polypeptide.
  • the conjugating moiety is indirectly bound to the isolated and purified IL-2 polypeptide through a linker.
  • the linker comprises a homobifunctional linker.
  • the homobifunctional linker comprises Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (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′-(2
  • DFDNPS 4,4′-difluoro-3,3′-dinitrophenylsulfone
  • BASED bis-[ ⁇ -(4-azidosalicylamido)ethyl]disulfide
  • formaldehyde glutaraldehyde
  • 1,4-butanediol diglycidyl ether 1,4-butanediol diglycidyl ether
  • adipic acid dihydrazide carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, ⁇ , ⁇ ′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylene-bis(iodoacetamide).
  • the linker comprises a heterobifunctional linker.
  • the heterobifunctional linker comprises 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- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[ ⁇ -methyl- ⁇ -(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohex
  • the linker comprises a cleavable linker, optionally comprising a dipeptide linker.
  • the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.
  • the linker comprises a non-cleavable linker.
  • the linker comprises a maleimide group, optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).
  • the linker further comprises a spacer.
  • the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof.
  • the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.
  • the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.
  • an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating moiety; wherein the IL-2 conjugate has a decreased affinity to an IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to a wild-type IL-2 polypeptide.
  • the conjugating moiety is bound to an amino acid residue that interacts with IL-2R ⁇ .
  • the conjugating moiety is bound to an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO: 1.
  • the conjugating moiety comprises a water-soluble polymer.
  • the additional conjugating moiety comprises a water-soluble polymer.
  • each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.
  • each of the water-soluble polymers independently comprises PEG.
  • the PEG is a linear PEG or a branched PEG.
  • each of the water-soluble polymers independently comprises a polysaccharide.
  • the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • each of the water-soluble polymers independently comprises a glycan.
  • each of the water-soluble polymers independently comprises polyamine.
  • the conjugating moiety comprises a protein.
  • the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises an albumin, a transferrin, or a 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 IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide.
  • each of the polypeptides independently comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.
  • the isolated and purified IL-2 polypeptide is modified by glutamylation.
  • the conjugating moiety is directly bound to the isolated and purified IL-2 polypeptide.
  • the conjugating moiety is indirectly bound to the isolated and purified IL-2 polypeptide through a linker.
  • the linker comprises a homobifunctional linker.
  • the homobifunctional linker comprises Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (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′-(2
  • DFDNPS 4,4′-difluoro-3,3′-dinitrophenylsulfone
  • BASED bis-[3-(4-azidosalicylamido)ethyl]disulfide
  • formaldehyde glutaraldehyde
  • 1,4-butanediol diglycidyl ether 1,4-butanediol diglycidyl ether
  • adipic acid dihydrazide carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, ⁇ , ⁇ ′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylene-bis(iodoacetamide).
  • the linker comprises a heterobifunctional linker.
  • the heterobifunctional linker comprises 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- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[ ⁇ -methyl- ⁇ -(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohex
  • the linker comprises a cleavable linker, optionally comprising a dipeptide linker.
  • the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys.
  • the linker comprises a non-cleavable linker.
  • the linker comprises a maleimide group, optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).
  • the linker further comprises a spacer.
  • the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof.
  • the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.
  • the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.
  • IL-2 interleukin 2
  • IL-2R ⁇ interleukin 2 receptor ⁇
  • IL-2R ⁇ interleukin 2 receptor ⁇
  • the position of the at least one unnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from K35, K64, V69, N71, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from F42, K43, F44, E62, and P65, wherein the residue positions correspond to the positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1.
  • the at least one unnatural amino acid is a lysine analogue; comprises an aromatic side chain; comprises an azido 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.
  • the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-
  • the at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA of the orthogonal synthetase/tRNA pair comprises at least one unnatural nucleobase.
  • the modified IL-2 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid.
  • the conjugating moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide.
  • 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(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.
  • the water-soluble polymer comprises a PEG molecule.
  • the PEG molecule is a linear PEG.
  • the PEG molecule is a branched PEG.
  • the water-soluble polymer comprises a polysaccharide.
  • the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • the lipid comprises a fatty acid.
  • the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the fatty acid is a saturated fatty acid.
  • the protein comprises an albumin, a transferrin, or a transthyretin.
  • the protein comprises a TLR agonist.
  • the protein comprises an antibody or its binding fragments thereof. In some embodiments, the antibody or its binding fragments thereof comprises an Fc portion of an antibody.
  • the peptide comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.
  • the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the modified IL-2 through a linker.
  • the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.
  • the decrease in binding affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% decrease in binding affinity to IL-2R ⁇ relative to a wild-type IL-2 polypeptide.
  • the decrease in 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 relative to IL-2R ⁇ relative to a wild-type IL-2 polypeptide.
  • 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.
  • the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.
  • the N-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the C-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the modified IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the modified IL-2 polypeptide with the decrease in binding affinity to IL-2R ⁇ is capable of expanding CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, Natural killer T (NKT) cell populations, or a combination thereof.
  • the conjugating moiety impairs or blocks the binding of IL-2 with IL-2R ⁇ .
  • activation of CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population via the IL-2R ⁇ complex by the modified IL-2 polypeptide is not significantly different than activation of said cell population by a wild-type IL-2 polypeptide, and wherein the potency of the modified IL-2 polypeptide is at least 1-fold higher than a potency of the wild-type IL-2 polypeptide.
  • the modified IL-2 polypeptide expands CD4+T regulatory (Treg) cells by less than 20%, 15%, 10%, 5%, 1%, or 0.1% when said activator is in contact with said cell population. In some embodiments, the modified IL-2 polypeptide does not expand Treg cells in said cell population.
  • IL-2 interleukin 2
  • IL-2R ⁇ interleukin 2 receptor ⁇
  • IL-2R ⁇ interleukin 2 receptor ⁇
  • IL-2R ⁇ interleukin 2 receptor ⁇
  • the difference in receptor signaling potency is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.
  • the position of the at least one unnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from K35, K64, V69, N71, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from F42, K43, F44, E62, and P65, wherein the residue positions correspond to the positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1.
  • the at least one unnatural amino acid is a lysine analogue; comprises an aromatic side chain; comprises an azido 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.
  • the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-
  • the at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA of the orthogonal synthetase/tRNA pair comprises at least one unnatural nucleobase.
  • the modified IL-2 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid.
  • the conjugating moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide.
  • 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(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.
  • the water-soluble polymer comprises a PEG molecule.
  • the PEG molecule is a linear PEG.
  • the PEG molecule is a branched PEG.
  • the water-soluble polymer comprises a polysaccharide.
  • the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • the lipid comprises a fatty acid.
  • the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the fatty acid is a saturated fatty acid.
  • the protein comprises an albumin, a transferrin, or a transthyretin.
  • the conjugating moiety comprises a TLR agonist.
  • the protein comprises an antibody or its binding fragments thereof.
  • the antibody or its binding fragments thereof comprises an Fc portion of an antibody.
  • the peptide comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.
  • the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the modified IL-2 through a linker.
  • the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.
  • the decrease in binding affinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% decrease in binding affinity to IL-2Ra relative to a wild-type IL-2 polypeptide.
  • the decrease in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more to IL-2R ⁇ relative to a wild-type IL-2 polypeptide.
  • 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.
  • the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.
  • the N-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the C-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the modified IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the modified IL-2 polypeptide with the decrease in binding affinity to IL-2R ⁇ is capable of expanding CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, Natural killer T (NKT) cell populations, or a combination thereof.
  • the conjugating moiety or the unnatural amino acid impairs or blocks the binding of IL-2 with IL-2R ⁇ .
  • activation of CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population via the IL-2R ⁇ complex by the modified IL-2 polypeptide retains significant potency of activation of said cell population relative to a wild-type IL-2 polypeptide.
  • the receptor signaling potency of the modified IL-2 polypeptide to the IL-2R ⁇ complex is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ complex.
  • the receptor signaling potency of the modified IL-2 polypeptide the IL-2R ⁇ complex is lower than a receptor signaling potency of the wild-type IL-2 polypeptide the IL-2R ⁇ complex.
  • the modified IL-2 polypeptide exhibits a first receptor signaling potency to IL-2R ⁇ and a second receptor signaling potency to IL-2R ⁇ y, and wherein the first receptor signaling potency 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, or higher than the second receptor signaling potency.
  • the first receptor signaling potency of the modified IL-2 polypeptide is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ , and the second receptor signaling potency of the modified IL-2 polypeptide is lower than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ y. In some embodiments, the first receptor signaling potency of the modified IL-2 polypeptide is at least 1-fold lower than a receptor signaling potency of the wild-type IL-2 polypeptide.
  • IL-2 interleukin 2
  • the isolated and modified IL-2 polypeptide exhibits a first receptor signaling potency to an IL-2 ⁇ signaling complex and a second receptor signaling potency to an IL-2 ⁇ signaling complex, and wherein a difference between the first receptor signaling potency and the second receptor signaling potency is less than 10-fold.
  • the difference in receptor signaling potency is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.
  • the position of the at least one unnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from K35, K64, V69, N71, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from F42, K43, F44, E62, and P65, wherein the residue positions correspond to the positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1.
  • the at least one unnatural amino acid is a lysine analogue; comprises an aromatic side chain; comprises an azido 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.
  • the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-
  • the at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA of the orthogonal synthetase/tRNA pair comprises at least one unnatural nucleobase.
  • the modified IL-2 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid.
  • the conjugating moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide.
  • 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(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.
  • the water-soluble polymer comprises a PEG molecule.
  • the PEG molecule is a linear PEG.
  • the PEG molecule is a branched PEG.
  • the water-soluble polymer comprises a polysaccharide.
  • the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • the lipid comprises a fatty acid.
  • the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the fatty acid is a saturated fatty acid.
  • the protein comprises an albumin, a transferrin, or a transthyretin.
  • the conjugating moiety comprises a TLR agonist.
  • the protein comprises an antibody or its binding fragments thereof.
  • the antibody or its binding fragments thereof comprises an Fc portion of an antibody.
  • the peptide comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.
  • the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the modified IL-2 through a linker.
  • the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.
  • the decrease in binding affinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% decrease in binding affinity to IL-2Ra relative to a wild-type IL-2 polypeptide.
  • the decrease in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more to IL-2R ⁇ relative to a wild-type IL-2 polypeptide.
  • 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.
  • the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.
  • the N-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the C-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the modified IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the modified IL-2 polypeptide with the decrease in binding affinity to IL-2R ⁇ is capable of expanding CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, Natural killer T (NKT) cell populations, or a combination thereof.
  • the conjugating moiety or the unnatural amino acid impairs or blocks the binding of IL-2 with IL-2R ⁇ .
  • activation of CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population via the IL-2R ⁇ complex by the modified IL-2 polypeptide retains significant potency of activation of said cell population relative to a wild-type IL-2 polypeptide.
  • the receptor signaling potency of the modified IL-2 polypeptide to the IL-2R ⁇ complex is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ complex.
  • the receptor signaling potency of the modified IL-2 polypeptide the IL-2R ⁇ complex is lower than a receptor signaling potency of the wild-type IL-2 polypeptide the IL-2R ⁇ complex.
  • the modified IL-2 polypeptide exhibits a first receptor signaling potency to IL-2R ⁇ and a second receptor signaling potency to IL-2R ⁇ y, and wherein the first receptor signaling potency 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, or higher than the second receptor signaling potency.
  • the first receptor signaling potency of the modified IL-2 polypeptide is higher than a receptor signaling potency of the wild-type L-2 polypeptide to the IL-2R ⁇ , and the second receptor signaling potency of the modified IL-2 polypeptide is lower than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ . In some embodiments, the first receptor signaling potency of the modified IL-2 polypeptide is at least 1-fold lower than a receptor signaling potency of the wild-type IL-2 polypeptide.
  • IL-2 interleukin 2
  • the isolated and modified IL-2 polypeptide provides a first EC50 value for activating IL-2 ⁇ signaling complex and a second EC50 value for activating IL-2 ⁇ signaling complex, and wherein a difference between the first EC50 and the second EC50 value is less than 10-fold.
  • the difference is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.
  • the difference in receptor signaling potency is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.
  • the position of the at least one unnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from K35, K64, V69, N71, M104, C105, and Y107, wherein the residue positions correspond to the positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein the residue positions correspond to the positions 37, 38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1.
  • the position of the at least one unnatural amino acid is selected from F42, K43, F44, E62, and P65, wherein the residue positions correspond to the positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1.
  • the at least one unnatural amino acid is a lysine analogue; comprises an aromatic side chain; comprises an azido 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.
  • the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-
  • the at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA of the orthogonal synthetase/tRNA pair comprises at least one unnatural nucleobase.
  • the modified IL-2 polypeptide is covalently attached to a conjugating moiety through the at least one unnatural amino acid.
  • the conjugating moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide.
  • 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(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.
  • the water-soluble polymer comprises a PEG molecule.
  • the PEG molecule is a linear PEG.
  • the PEG molecule is a branched PEG.
  • the water-soluble polymer comprises a polysaccharide.
  • the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • the lipid comprises a fatty acid.
  • the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the fatty acid is a saturated fatty acid.
  • the protein comprises an albumin, a transferrin, or a transthyretin.
  • the conjugating moiety comprises a TLR agonist.
  • the protein comprises an antibody or its binding fragments thereof.
  • the antibody or its binding fragments thereof comprises an Fc portion of an antibody.
  • the peptide comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.
  • the conjugating moiety is indirectly bound to the at least one unnatural amino acid of the modified IL-2 through a linker.
  • the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.
  • the decrease in binding affinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% decrease in binding affinity to IL-2Ra relative to a wild-type IL-2 polypeptide.
  • the decrease in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more to IL-2R ⁇ relative to a wild-type IL-2 polypeptide.
  • 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.
  • the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof.
  • the N-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the C-terminal deletion comprises a 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the modified IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the modified IL-2 polypeptide with the decrease in binding affinity to IL-2R ⁇ is capable of expanding CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, Natural killer T (NKT) cell populations, or a combination thereof.
  • the conjugating moiety or the unnatural amino acid impairs or blocks the binding of IL-2 with IL-2R ⁇ .
  • activation of CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population via the IL-2R ⁇ complex by the modified IL-2 polypeptide retains significant potency of activation of said cell population relative to a wild-type IL-2 polypeptide.
  • the receptor signaling potency of the modified IL-2 polypeptide to the IL-2R ⁇ complex is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ complex.
  • the receptor signaling potency of the modified IL-2 polypeptide the IL-2R ⁇ complex is lower than a receptor signaling potency of the wild-type IL-2 polypeptide the IL-2R ⁇ complex.
  • the modified IL-2 polypeptide exhibits a first receptor signaling potency to IL-2R ⁇ and a second receptor signaling potency to IL-2R ⁇ , and wherein the first receptor signaling potency 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, or higher than the second receptor signaling potency.
  • the first receptor signaling potency of the modified IL-2 polypeptide is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ , and the second receptor signaling potency of the modified IL-2 polypeptide is lower than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ . In some embodiments, the first receptor signaling potency of the modified IL-2 polypeptide is at least 1-fold lower than a receptor signaling potency of the wild-type IL-2 polypeptide.
  • a pharmaceutical composition comprising: an IL-2 conjugate described above; and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is formulated for parenteral administration.
  • a method of treating a proliferative disease or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an isolated and modified IL-2 polypeptide described above, an IL-2 conjugate described above, an IL-2R ⁇ binding protein described above, an activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell described above, or a pharmaceutical composition described above.
  • the proliferative disease or condition is a cancer.
  • the cancer is a solid tumor cancer.
  • the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.
  • the cancer is a hematologic malignancy.
  • the hematologic malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B
  • the method further comprises administering an additional therapeutic agent.
  • the isolated and modified IL-2 polypeptide, the IL-2 conjugate, the IL-2R ⁇ binding protein, the activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell, or the pharmaceutical composition and the additional therapeutic agent are administered simultaneously.
  • the isolated and modified IL-2 polypeptide, the IL-2 conjugate, the IL-2R ⁇ binding protein, the activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell, or the pharmaceutical composition and the additional therapeutic agent are administered sequentially.
  • the isolated and modified IL-2 polypeptide, the IL-2 conjugate, the IL-2R ⁇ binding protein, the activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell, or the pharmaceutical composition is administered prior to the additional therapeutic agent.
  • the isolated and modified IL-2 polypeptide, the IL-2 conjugate, the IL-2R ⁇ binding protein, the activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell, or the pharmaceutical composition is administered after the administration of the additional therapeutic agent.
  • a method of expanding a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population comprising: contacting a cell population with an isolated and modified IL-2 polypeptide described above, an IL-2 conjugate described above, an IL-2R ⁇ binding protein described above, an activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell described above, or a pharmaceutical composition described above for a time sufficient to induce formation of a complex with an IL-2R ⁇ , thereby stimulating the expansion of the Teff and/or NK cell population.
  • the isolated and modified IL-2 polypeptide described above, the IL-2 conjugate described above, the IL-2R ⁇ binding protein described above, the activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell described above, or the pharmaceutical composition described above expands CD4+T regulatory (Treg) cells by less than 20%, 15%, 10%, 5%, or 1% in the CD3+ cell population compared to an expansion of CD4+ Treg cells in the CD3+ cell population contacted with a wild-type IL-2 polypeptide.
  • the isolated and modified IL-2 polypeptide described above, the IL-2 conjugate described above, the IL-2R ⁇ binding protein described above, the activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell described above, or the pharmaceutical composition described above does not expand CD4+ Treg cells in the cell population.
  • the ratio of the Teff cells to Treg cells in the cell population after incubation with the isolated and modified IL-2 polypeptide described above, the IL-2 conjugate described above, the IL-2R ⁇ binding protein described above, the activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell described above, or the pharmaceutical composition described above is about or at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1.
  • the method is an in vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method.
  • a method of expanding a CD4+ helper cell population, a CD8+ effector na ⁇ ve and/or memtory cell population, a Natural Killer (NK) cell population, a Natural killer T (NKT) cell population, or a combination thereof comprising: (a) contacting a cell with an L-2 conjugate described above; and (b) interacting the L-2 with IL-2R ⁇ and IL-2R ⁇ subunits to form an IL-2/IL-2R ⁇ complex; wherein the IL-2 conjugate has a decreased affinity to IL-2R subunit, and wherein the IL-2/IL-2R ⁇ complex stimulates the expansion of CD4+ helper cells, CD8+ effector na ⁇ ve and/or memtory cells, NK cells, NKT cells, or a combination thereof.
  • kits comprising an isolated and modified IL-2 polypeptide described above, an L-2 conjugate described above, an IL-2R ⁇ binding protein described above, an activator of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell described above, or a pharmaceutical composition comprising an L-2 conjugate described above.
  • kit comprising a polynucleic acid molecule encoding an IL-2 polypeptide described above.
  • FIG. 1 shows exemplary unnatural amino acids. This figure is adapted from FIG. 2 of Young et al., “Beyond the canonical 20 amino acids: expanding the genetic lexicon,” J. of Biological Chemistry 285(15): 11039-11044 (2010).
  • FIG. 2A - FIG. 2B illustrate exemplary unnatural amino acids.
  • FIG. 2A illustrates exemplary lysine derivatives.
  • FIG. 2B illustrates exemplary phenylalanine derivatives.
  • FIG. 3A - FIG. 3D illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) have been genetically encoded in proteins ( FIG. 3A —UAA #1-42; FIG. 3B —UAA #43-89; FIG. 3C —UAA #90-128; FIG. 3D —UAA #129-167).
  • FIGS. 5A-5D are adopted from Table 1 of Dumas et al., Chemical Science 2015, 6, 50-69.
  • FIG. 4A - FIG. 4B show surface plasmon resonance (SPR) analysis of P65_30kD, P65_5kD, E62_30kD, and E62_5kD PEG conjugates.
  • FIG. 4A shows SPR analysis of IL-2 variants binding to immobilized IL-2 R ⁇ .
  • FIG. 4B shows SPR analysis of IL-2 variants binding to immobilized IL-2 R ⁇ .
  • FIG. 5A - FIG. 5C show exemplary IL-2 variant dose response curves for pSTAT5 signaling in human LRS primary cell populations.
  • FIG. 5A native IL-2
  • FIG. 5B E62_30kD
  • FIG. 5C P65_30kD.
  • FIG. 6 shows the mean (+SD) plasma concentration versus time profiles following a single IV bolus dose of aldesleukin (L-2), E62_5, E62_30 and P65_30 to C57BL/6 mice.
  • FIG. 7 shows percentage of pSTAT5+CD8+ T cells vs time cells in peripheral blood following treatment with a single IV bolus dose of P65_30 or aldesleukin to C57BL/6 mice.
  • FIG. 8A - FIG. 8C show percentage of CD8+ T cells ( FIG. 8A ), NK cells ( FIG. 8B ) and CD4+ Treg cells ( FIG. 8C ) in the PBMC population following treatment with a single IV bolus dose of P65_30 or aldesleukin (IL-2). Blood was drawn via cardiac puncture at the time points indicated and immune cell populations were assessed by flow cytometry. Each data point represents an average from 3 replicates at each time point, +SEM.
  • FIG. 9A - FIG. 9B show differences between P65_30 and IL-2 (aldesleukin) in the stimulation of memory CD8+CD44+ T cell proliferation within the CD3+ population following treatment with a single IV bolus dose of P65_30 or aldesleukin (IL-2). Blood was drawn via cardiac puncture at the time points indicated and immune cell populations were assessed by flow cytometry. Data were analyzed using unpaired Student t test. *** designate P values ⁇ 0.001.
  • FIG. 9A shows memory CD8+CD44+ T cell proliferation at 72, 96 and 120 hours.
  • FIG. 9B shows flow cytometry analysis of those cells at the 120 h time point.
  • FIG. 10 shows the increase in tumor-infiltrating lymphocytes (TILs) vs time in C57B16 mice bearing syngeneic B16F10 tumors following treatment with a single IV bolus dose of P65_30.
  • FIG. 10A shows percentage of NK, CD8+T and CD4+T reg cells in P65_30-treated vs untreated (vehicle) animals at Day 5 of treatment.
  • FIG. 10B shows the ratio of CD8+/CD4+ Treg cells in P65_30-treated and control (vehicle) animals. Data were analyzed using unpaired Student t test. *** designate P values ⁇ 0.001.
  • FIG. 11A - FIG. 11B show plasma levels of mouse IL-2, TNF- ⁇ , IFN ⁇ , IL-5 and IL-6 following treatment with a single IV bolus dose of P65_30 or aldesleukin (IL-2) at increasing levels (0.01-5 mg/kg).
  • the concentration of each cytokine in plasma was determined via ELISA (Abcam, Cambridge, UK).
  • N 3 mice and samples were collected at 4, 34 and 72 h post-dose.
  • FIG. 11A shows cytokine levels for aldesleukin-dosed animals and FIG. 11B for P65 30-dosed animals.
  • FIG. 12 shows white blood cell, lymphocyte, and eosinophil counts (mean ⁇ SD) following a single IV dose of P65_30kD to male Cynomolgus monkeys.
  • Cytokines comprise a family of cell signaling proteins such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors playing 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 different stromal cells. In some instances, cytokines modulate the balance between humoral and cell-based immune responses.
  • Interleukins are signaling proteins which modulate the development and differentiation of T and B lymphocytes, cell 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, interleukins 1-13, interleukin 15, and interleukin 17.
  • Interleukin 2 is a pleiotropic type-1 cytokine whose structure comprises a 15.5 kDa four ⁇ -helix bundle.
  • the precursor form of IL-2 is 153 amino acid residues in length, with the first 20 amino acids forming a signal peptide and residues 21-153 forming the mature form.
  • IL-2 is produced primarily by CD4+ T cells post antigen stimulation and to a lesser extent, by CD8+ cells, Natural Killer (NK) cells, and Natural killer T (NKT) cells, activated dendritic cells (DCs), and mast cells.
  • IL-2 signaling occurs through interaction with specific combinations of IL-2 receptor (IL-2R) subunits, IL-2R ⁇ (also known as CD25), IL-2R3 (also known as CD122), and IL-2R ⁇ (also known as CD 132).
  • IL-2R ⁇ also known as CD25
  • IL-2R3 also known as CD122
  • IL-2R ⁇ also known as CD 132
  • Interaction of IL-2 with the IL-2R ⁇ forms the “low-affinity” IL-2 receptor complex with a K d of about 10 ⁇ 8 M.
  • Interaction of IL-2 with IL-2R3 and IL-2R ⁇ forms the “intermediate-affinity” IL-2 receptor complex with a K d of about 10-9 M.
  • Interaction of IL-2 with all three subunits, IL-2R ⁇ , IL-2R ⁇ , and IL-2R ⁇ forms the “high-affinity” IL-2 receptor complex with a K d of about >10 ⁇ 11 M.
  • IL-2 signaling via the “high-affinity” IL-2R ⁇ complex modulates the activation and proliferation of regulatory T cells.
  • Regulatory T cells or CD4 + CD25 + Foxp3 + regulatory T (Treg) cells, mediate maintenance of immune homeostasis by suppression of effector cells such as CD4 + T cells, CD8 + T cells, B cells, NK cells, and NKT cells.
  • Treg cells are generated from the thymus (tTreg cells) or are induced from na ⁇ ve T cells in the periphery (pTreg cells). In some cases, Treg cells are considered as the mediator of peripheral tolerance.
  • IL-2 signaling via the “intermediate-affinity” IL-2R ⁇ complex modulates the activation and proliferation of CD8 + effector T (Teff) cells, NK cells, and NKT cells.
  • CD8 + Teff cells also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, Tcon, or killer T cells
  • NK and NKT cells are types of lymphocytes that, similar to CD8 + Teff cells, target cancerous cells and pathogen-infected cells.
  • IL-2 signaling is utilized to modulate T cell responses and subsequently for treatment of a cancer.
  • IL-2 is administered in a high-dose form to induce expansion of Teff cell populations for treatment of a cancer.
  • high-dose IL2 further leads to concomitant stimulation of Treg cells that dampen anti-tumor immune responses.
  • High-dose IL-2 also induces toxic adverse events mediated by the engagement of IL-2R alpha chain-expressing cells in the vasculature, including type 2 innate immune cells (ILC-2), eosinophils and endothelial cells. This leads to eosinophilia, capillary leak and vascular leak syndrome VLS).
  • lymphocytes e.g., CD4+ helper cells, CD8+ effector na ⁇ ve and memory cells, NK cells, or NKT cells
  • the cytokine comprises an interleukin, an interferon, or a tumor necrosis factor.
  • the cytokine is a cytokine conjugate, e.g., an interleukin conjugate, an interferon conjugate, or a tumor necrosis factor conjugate.
  • described herein comprise pharmaceutical compositions and kits comprising one or more cytokine conjugates described herein.
  • IL-2 is an IL-2 conjugate, which interacts with the “intermediate-affinity” IL-2R ⁇ complex, optionally with a similar potency as the IL-2R ⁇ complex, and with a weakened IL-2R ⁇ interaction relative to wild-type IL-2.
  • further described herein are methods of treating a cancer with use of an IL-2 conjugate described herein.
  • described herein are pharmaceutical compositions and kits which comprise one or more L-2 conjugates described herein.
  • the cytokine comprises an interleukins, a tumor necrosis factor, an interferon, a chemokine, a lymphokine, or a growth factor.
  • the cytokine is an interleukin.
  • the cytokine is an interferon.
  • the cytokine is a tumor necrosis factor.
  • the cytokine is a growth factor.
  • interleukin conjugate in some embodiments, described herein is an interleukin conjugate.
  • exemplary interleukins include, but are not limited to, interleukin 1 ⁇ (IL-1 ⁇ ), 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).
  • interleukin conjugate in which the interleukin is selected from IL-1 ⁇ , IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21.
  • IL-2 conjugates modified at an amino acid position.
  • the modification is to a natural amino acid.
  • the modification is to an unnatural amino acid.
  • described herein is an isolated and modified IL-2 polypeptide that comprises at least one unnatural amino acid.
  • the IL-2 polypeptide is an isolated and purified mammalian IL-2, for example, a rodent IL-2 protein, or a human IL-2 protein.
  • the IL-2 polypeptide is a human IL-2 protein.
  • the L-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • 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 additional cases, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In additional cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 2. In additional cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 2.
  • the IL-2 polypeptide is a truncated variant.
  • the truncation is an N-terminal deletion.
  • the truncation is a C-terminal deletion.
  • the truncation comprises both N-terminal and C-terminal deletions.
  • 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 both termini.
  • the IL-2 polypeptide comprises 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.
  • the IL-2 polypeptide comprises an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 2 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 3 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 4 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 5 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 6 residues.
  • the IL-2 polypeptide comprises an N-terminal deletion of at least or about 7 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 8 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 9 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 10 residues.
  • the IL-2 polypeptide is a functionally active fragment.
  • 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, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the functionally active fragment comprises IL-2 region 10-133, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the functionally active fragment comprises IL-2 region 20-133, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • the functionally active fragment comprises IL-2 region 30-133, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment comprises IL-2 region 10-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment comprises IL-2 region 20-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment comprises IL-2 region 1-130, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment comprises IL-2 region 1-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.
  • an IL-2 conjugate that comprises an isolated, purified, and modified IL-2 polypeptide and a conjugating moiety.
  • the IL-2 conjugate has a decreased affinity to an IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to a wild-type IL-2 polypeptide.
  • the conjugating moiety is bound to an amino acid residue that interacts with IL-2R ⁇ (e.g., at the IL-2/IL-2R ⁇ interface).
  • the conjugating moiety is bound to an amino acid residue that is proximal to the IL-2/IL-2R ⁇ interface (e.g., about 5 ⁇ , about 10 ⁇ , about 15 ⁇ , or about 20 ⁇ away from the IL-2/IL-2R ⁇ interface).
  • the residues involved in the IL-2/IL-2R ⁇ interface comprise L-2 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2R ⁇ subunit.
  • the conjugating moiety is bound to an amino acid residue selected from an amino acid position Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109E110, T111, or A112, in which the numbering of the amino acid residues corresponds to SEQ ID NO: 1.
  • the amino acid position is selected from Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, E61, E62, E68, K64, P65, V69, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, and A112.
  • the amino acid position is selected from N33, P34, K35, T37, R38, M39, T41, F42, K43, F44, Y45, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, M104, C105, E106, Y107, A108, D109, E110, T111, and A112.
  • the amino acid position is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.
  • the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from R38 and K64. In some instances, the amino acid position is selected from E61, E62, and E68.
  • the amino acid position is at K35. In some cases, the amino acid position is at T37. In some cases, the amino acid position is at R38. In some cases, the amino acid position is at T41. In some cases, the amino acid position is at F42. In some cases, the amino acid position is at K43. In some cases, the amino acid position is at F44. In some cases, the amino acid position is at Y45. In some cases, the amino acid position is at E61. In some cases, the amino acid position is at E62. In some cases, the amino acid position is at K64. In some cases, the amino acid position is at E68. In some cases, the amino acid position is at P65. In some cases, the amino acid position is at V69. In some cases, the amino acid position is at L72. In some cases, the amino acid position is at Y107. In some cases, the amino acid position is at L72. In some cases, the amino acid position is at D109.
  • the IL-2 conjugate further comprises an additional mutation.
  • the additional mutation is at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.
  • the amino acid is conjugated to an additional conjugating moiety for increase in serum half-life, stability, or a combination thereof.
  • 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 an unnatural amino acid prior to binding to the additional conjugating moiety.
  • a natural amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine
  • an unnatural amino acid prior to binding to the additional conjugating moiety.
  • the decreased affinity of the modified IL-2 polypeptide to an IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to a wild-type IL-2 polypeptide without the unnatural amino acid modification is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%.
  • the decreased affinity is about 10%.
  • the decreased affinity is about 20%.
  • the decreased affinity is about 40%.
  • the decreased affinity is about 50%.
  • the decreased affinity is about 60%.
  • the decreased affinity is about 80%.
  • the decreased affinity is about 90%.
  • the decreased affinity is about 99%.
  • the decreased affinity is greater than 99%.
  • the decreased affinity is about 80%. In some cases, the decreased affinity is about 100%.
  • the decreased affinity of the modified IL-2 polypeptide to an IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to an equivalent IL-2 polypeptide without the unnatural amino acid modification 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, 1,000-fold, or more.
  • the decreased affinity is about 1-fold.
  • the decreased affinity is about 2-fold.
  • the decreased affinity is about 4-fold.
  • the decreased affinity is about 5-fold.
  • the decreased affinity is about 6-fold. In some cases, the decreased affinity is about 8-fold. In some cases, the decreased affinity is about 10-fold. In some cases, the decreased affinity is about 30-fold. In some cases, the decreased affinity is about 50-fold. In some cases, the decreased affinity is about 100-fold. In some cases, the decreased affinity is about 300-fold. In some cases, the decreased affinity is about 500-fold. In some cases, the decreased affinity is about 1000-fold. In some cases, the decreased affinity is more than 1,000-fold.
  • the modified IL-2 polypeptide does not interact with IL-2R ⁇ . In some instances, the modified IL-2 polypeptide is further conjugated to a conjugating moiety. In some cases, the IL-2 conjugate does not interact with IL-2R ⁇ .
  • the modified IL-2 polypeptide exhibits a first receptor signaling potency to an IL-2 ⁇ signaling complex and a second receptor signaling potency to an IL-2 ⁇ signaling complex, and wherein a difference between the first receptor signaling potency and the second receptor signaling potency is less than 10-fold. In some embodiments, the modified IL-2 polypeptide exhibits a first receptor signaling potency to an IL-2 ⁇ signaling complex and a second receptor signaling potency to an IL-2 ⁇ signaling complex, and wherein a difference between the first receptor signaling potency and the second receptor signaling potency is less than 5-fold.
  • the difference is less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.
  • the modified IL-2 polypeptide is a partial agonist, e.g., an agonist that activates a receptor (e.g., an IL-2 ⁇ signaling complex or an IL-2 ⁇ signaling complex) but has only a partial efficacy at the receptor relative to a full agonist.
  • the modified IL-2 polypeptide is a full agonist, e.g., an agonist that activates a receptor (e.g., an IL-2 ⁇ signaling complex or an TL-2 ⁇ signaling complex) at a maximum response.
  • a full agonist e.g., an agonist that activates a receptor (e.g., an IL-2 ⁇ signaling complex or an TL-2 ⁇ signaling complex) at a maximum response.
  • the receptor signaling potency is measured by an EC50 value.
  • the modified IL-2 polypeptide provides a first EC50 value for activating IL-2 ⁇ signaling complex and a second EC50 value for activating IL-2 ⁇ signaling complex, and wherein a difference between the first EC50 and the second EC50 value is less than 10-fold.
  • the modified IL-2 polypeptide provides a first EC50 value for activating IL-2 ⁇ signaling complex and a second EC50 value for activating IL-2 ⁇ signaling complex, and wherein a difference between the first EC50 and the second EC50 value is less than 5-fold. In some cases, the difference is less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.
  • the receptor signaling potency is measured by an ED50 value.
  • the modified IL-2 polypeptide provides a first ED50 value for activating IL-2 ⁇ signaling complex and a second ED50 value for activating IL-2 ⁇ signaling complex, and wherein a difference between the first ED50 and the second ED50 value is less than 10-fold.
  • the modified IL-2 polypeptide provides a first ED50 value for activating IL-2 ⁇ signaling complex and a second ED50 value for activating IL-2 ⁇ signaling complex, and wherein a difference between the first ED50 and the second ED50 value is less than 5-fold. In some cases, the difference is less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.
  • the conjugating moiety is linked to the N-terminus or the C-terminus of an IL-2 polypeptide, either directly or indirectly through a linker peptide.
  • the conjugating moiety e.g., a polymer, a protein, or a peptide
  • the conjugating moiety is genetically fused to the IL-2, at the N-terminus or the C-terminus of IL-2, and either directly or indirectly through a linker peptide.
  • the conjugating moiety is linked to the N-terminus or the C-terminus amino acid residue.
  • the conjugating moiety is linked to a reactive group that is bound to the N-terminus or C-terminus amino acid residue.
  • the IL-2 conjugate with reduced binding affinity to IL-2R ⁇ is capable of expanding CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell populations.
  • the conjugating moiety impairs or blocks binding of IL-2 with IL-2R ⁇ .
  • activation of CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population via the IL-2R ⁇ complex by the modified IL-2 polypeptide retains significant potency of activation of said cell population relative to a wild-type IL-2 polypeptide.
  • the activation by the modified IL-2 polypeptide is equivalent to that of the wild-type IL-2 polypeptide.
  • the activation by the modified IL-2 polypeptide is higher than that of the wild-type IL-2 polypeptide.
  • the receptor signaling potency of the modified IL-2 polypeptide to the IL-2R ⁇ complex is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ complex. In some cases, the receptor signaling potency of the modified IL-2 polypeptide is at least 1-fold higher than the respective potency of the wild-type IL-2 polypeptide.
  • the receptor signaling potency of the modified IL-2 polypeptide is about or at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-folod, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, or higher than the respective potency of the wild-type IL-2 polypeptide.
  • the dose or concentration of the modified IL-2 polypeptide used for achieveing a similar level of activation of the CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population as a wild-type 11-2 polypeptide is lower than a dose or concentration used for the wild-type IL-2 polypeptide.
  • activation of CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population via the IL-2R ⁇ complex by the modified IL-2 polypeptide retains significant potency of activation of said cell population by a wild-type IL-2 polypeptide.
  • the receptor signaling potency of the modified IL-2 polypeptide the IL-2R ⁇ complex is lower than a receptor signaling potency of the wild-type IL-2 polypeptide the IL-2R ⁇ complex.
  • the receptor signaling potency of the modified IL-2 polypeptide is about or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 50-fold lower than the respective potency of the wild-type IL-2 polypeptide.
  • the modified IL-2 polypeptide exhibits a first receptor signaling potency to IL-2R ⁇ and a second receptor signaling potency to IL-2R ⁇ .
  • the first receptor signaling potency to IL-2R ⁇ is an improved potency relative to a wild-type IL-2 polypeptide.
  • the second receptor signaling potency to IL-2R ⁇ is an impaired potency relative to the wild-type IL-2 polypeptide.
  • the modified IL-2 polypeptide exhibits a first receptor signaling potency to IL-2R ⁇ and a second receptor signaling potency to IL-2R ⁇ , and wherein the first receptor signaling potency 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 higher than the second receptor signaling potency.
  • the first receptor signaling potency is at least 1-fold or higher than the second receptor signaling potency.
  • the first receptor signaling potency is at least 2-fold or higher than the second receptor signaling potency.
  • the first receptor signaling potency is at least 5-fold or higher than the second receptor signaling potency. In some instances, the first receptor signaling potency is at least 10-fold or higher than the second receptor signaling potency. In some instances, the first receptor signaling potency is at least 20-fold or higher than the second receptor signaling potency. In some instances, the first receptor signaling potency is at least 50-fold or higher than the second receptor signaling potency. In some instances, the first receptor signaling potency is at least 100-fold or higher than the second receptor signaling potency. In some instances, the first receptor signaling potency is at least 500-fold or higher than the second receptor signaling potency.
  • the first receptor signaling potency is at least 1000-fold or higher than the second receptor signaling potency. In some instances, the first receptor signaling potency of the modified IL-2 polypeptide is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ , and the second receptor signaling potency of the modified IL-2 polypeptide is lower than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2R ⁇ . In some cases, both receptor signaling potencies are lower than their respective potencies in a wild-type IL-2 polypeptide. In other cases, both receptor signaling potencies are higher than their respective potencies in a wild-type IL-2 polypeptide.
  • the IL-2 conjugate decreases a toxic adverse event in a subject administered with the IL-2 conjugate.
  • exemplary toxic adverse events include eosinophilia, capillary leak, and vascular leak syndrome (VLS).
  • VLS vascular leak syndrome
  • the IL-2 conjugate decreases the occurance of a toxic adverse event in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.
  • the IL-2 conjugate decreases the severity of a toxic adverse event in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.
  • the toxic adverse event is eosinophilia.
  • the IL-2 conjugate decreases the occurance of eosinophilia in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type L-2 or aldesleukin.
  • the L-2 conjugate decreases the severity of eosinophilia in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.
  • the toxic adverse event is capillary leak.
  • the IL-2 conjugate decreases the occurance of capillary leak in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.
  • the IL-2 conjugate decreases the severity of capillary leak in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.
  • the toxic adverse event is VLS.
  • the IL-2 conjugate decreases the occurance of VLS in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.
  • the IL-2 conjugate decreases the severity of VLS in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.
  • the IL-2 conjugate comprises a plasma half-life of greater than 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 greater than 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the IL-2 conjugate comprises a plasma half-life of from about 1 hour to about 7 days, from about 12 hours to about 7 days, from about 18 hours to about 7 days, from about 24 hours to about 7 days, from about 1 hours to about 5 days, from about 12 hours to about 5 days, from about 24 hours to about 5 days, from about 2 days to about 5 days, or from about 2 days to about 3 days.
  • the IL-2 conjugate comprises a plasma half-life of 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.
  • the IL-2 conjugate comprises a plasma half-life that is capable of proliferating and/or expanding a CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, NK cell, NKT cell, or a combination thereof, but does not exert a deleterious effect such as apoptosis.
  • the IL-2 conjugate comprises an extended plasma half-life, e.g., 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 relative to a wild-type IL-2.
  • the IL-2 conjugate comprises an extended plasma half-life, e.g., 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 relative to a wild-type IL-2.
  • 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 relative to a wild-type IL-2.
  • 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
  • the IL-2 conjugate comprises an extended plasma half-life, e.g., 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 relative to aldesleukin.
  • the IL-2 conjugate comprises an extended plasma half-life, e.g., 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 relative to aldesleukin.
  • 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 relative to aldesleukin.
  • 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
  • the IL-2 conjugate comprises an extended plasma half-life with a reduced toxicity. In some instances, 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 a reduced toxicity. In some instances, 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 a reduced toxicity.
  • the IL-2 conjugate comprises an extended plasma half-life of 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 with a reduced toxicity.
  • 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 reduced relative to a wild-type IL2. 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 relative to a wild-type IL-2.
  • the IL-2 conjugate comprises an extended plasma half-life with a reduced toxicity. In some instances, 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 a reduced toxicity. In some instances, 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 a reduced toxicity.
  • the IL-2 conjugate comprises an extended plasma half-life of 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 with a reduced toxicity.
  • 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 reduced relative to aldesleukin. 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 relative to aldesleukin.
  • the IL-2 conjugate comprises a conjugating moiety in which the size (e.g., the volumn or length) of the conjugating moiety enhances plasma stability but does not reduce potency.
  • the size of the conjugating moiety extends 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.
  • the size of the conjugating moiety extends 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.
  • the size of the conjugating moiety extends plasma half-life 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.
  • the size of the conjugating moiety reduces the potency by less than 5%, 4%, 3%, 2%, 1%, or less relative to aldesleukin.
  • the IL-2 conjugate comprises a conjugating moiety in which the size (e.g., the volumn or length) of the conjugating moiety enhances plasma stability and potency.
  • the size of the conjugating moiety extends 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.
  • the size of the conjugating moiety extends 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.
  • the size of the conjugating moiety extends plasma half-life 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.
  • the size of the conjugating moiety further enhances the potency by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more relative to aldesleukin.
  • an IL-2 conjugate comprising an unnatural amino acid covalently attached to a conjugating moiety, wherein the unnatural amino acid is located in region 35-107, and wherein the region 35-107 corresponds to residues K35-Y107 of SEQ ID NO: 1.
  • an interleukin 2 ⁇ receptor (IL-2R ⁇ ) binding protein wherein the binding affinity for an interleukin 2 ⁇ receptor (IL-2R ⁇ ) of said binding protein is less than that of wild-type human IL-2 (hIL-2), wherein the binding affinity for an interleukin 2 ⁇ receptor (IL-2R ⁇ ) of said binding protein is less than that of wild-type human IL-2 (hIL-2).
  • an interleukin 2 ⁇ receptor (IL-2R ⁇ ) binding protein wherein the binding affinity for an interleukin 2 ⁇ receptor (IL-2R ⁇ ) of said binding protein is less than that of wild-type human IL-2 (hIL-2), and wherein said binding protein comprises at least one unnatural amino acid.
  • said 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.
  • the at least one unnatural amino acid is located in region 35-107, and wherein the region 35-107 corresponds to residues K35-Y107 of SEQ ID NO: 1.
  • an IL-2/IL-2R ⁇ complex comprising a modified IL-2 polypeptide comprising a mutation and an IL-2R ⁇ , wherein the modified IL-2 polypeptide has a reduced binding affinity toward IL-2R ⁇ , and wherein the reduced binding affinity is compared to a binding affinity between a wild-type IL-2 polypeptide and IL-2R ⁇ .
  • the modified IL-2 polypeptide further comprises a conjugating moiety covalently attached to site of mutation.
  • the site of mutation comprises an amino acid mutated to a natural amino acid.
  • the site of mutation comprises an amino acid mutated to a cysteine residue.
  • the site of mutation comprises an amino acid mutated to a lysine residue.
  • an IL-2/IL-2R ⁇ complex comprising a modified IL-2 polypeptide comprising an unnatural amino acid and an IL-2R ⁇ , wherein the modified IL-2 polypeptide has a reduced binding affinity toward IL-2R ⁇ , and wherein the reduced binding affinity is compared to a binding affinity between a wild-type IL-2 polypeptide and IL-2R ⁇ .
  • the modified IL-2 polypeptide further comprises a conjugating moiety covalently attached to the unnatural amino acid.
  • an IL-2/IL-2R ⁇ complex comprising a modified IL-2 polypeptide comprising an unnatural amino acid and an IL-2R ⁇ , wherein the modified IL-2 polypeptide has a reduced receptor signaling potency toward IL-2R ⁇ , and wherein the reduced receptor signaling potency is compared to a receptor signaling potency between a wild-type IL-2 polypeptide and IL-2R ⁇ .
  • the modified IL-2 polypeptide further comprises a conjugating moiety covalently attached to the unnatural amino acid.
  • IL-2 modified interleukin 2
  • the mutation is to a natural amino acid. In other instances, the mutation is to an unnatural amino acid.
  • IL-2 modified interleukin 2
  • said activator expands CD4+T regulatory (Treg) cells by less than 20%, 15%, 10%, 5%, 1%, or less than 0.1% when said activator is in contact with said CD3+ cell population compared to an expansion of CD4+ Treg cells in the CD3+ cell population contacted with a wild-type IL-2 polypeptide. In some instances, said activator does not expand Treg cells in said cell population.
  • said cell population is an in vivo cell population. In some instances, said cell population is an in vitro cell population. In some instances, said cell population is an ex vivo cell population.
  • a method of expanding a CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, Natural Killer (NK) cell, or a Natural killer T (NKT) cell population comprising contacting said cell population with a therapeutically effective amount of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, Natural Killer (NK) cell, or a Natural killer T (NKT) cell activator, in which said activator comprises a modified interleukin 2 (IL-2) polypeptide comprising at least one mutation, thereby expanding the CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population.
  • IL-2 modified interleukin 2
  • the mutation is to a natural amino acid. In other instances, the mutation is to an unnatural amino acid.
  • a method of expanding a CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, Natural Killer (NK) cell, or a Natural killer T (NKT) cell population comprising contacting said cell population with a therapeutically effective amount of a CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, Natural Killer (NK) cell, or a Natural killer T (NKT) cell activator, in which said activator comprises a modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid, thereby expanding the CD4+ helper cell, CD8+ effector na ⁇ ve and memory T cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population.
  • IL-2 modified interleukin 2
  • IL-15 conjugates modified at an amino acid position.
  • IL-15 regulates activation and proliferation of T cells and NK cells.
  • the IL-15 conjugate comprises an isolated and purified IL-15 polypeptide and a conjugating moiety.
  • the IL-15 conjugate has a decreased affinity to an IL-15 receptor relative to a wild-type IL-15 polypeptide.
  • the conjugating moiety is bound to an amino acid residue that interacts with the IL-15 receptor (e.g., at an IL-15/IL-15R ⁇ interface) or with IL-2R ⁇ subunits.
  • the conjugating moiety is bound to an amino acid residue that is proximal to the IL-15/IL-15R ⁇ interface (e.g., about 5 ⁇ , about 10 ⁇ , about 15 ⁇ , or about 20 ⁇ away from the IL-15/IL-15R ⁇ interface) or proximal to the IL-2R ⁇ subunits.
  • the residues involved in the IL-15/IL-15R interface comprise IL-15 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-15R.
  • residues involved in the IL-15/IL-2R ⁇ interface comprises IL-15 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2Ry subunits.
  • the conjugating moiety is linked to the N-terminus or the C-terminus of the IL-15 polypeptide (e.g., to the N-terminus or C-terminus amino acid residue or to a reactive group bound to the terminal amino acid residue), either directly or indirectly through a linker peptide.
  • the conjugating moiety modulates the interaction between IL-15 and IL-15R.
  • the IL-15 conjugate upregulates distinct population(s) of tumor infiltrating lymphocytes through IL-15/IL-15R signaling.
  • the IL-15 conjugate promotes a decrease in the proliferation and/or expansion of tumor associated lymphocytes.
  • the IL-15 conjugate modulates immune activity.
  • cytokine conjugates modified at an amino acid position.
  • cytokines include, but are not limited to, IL-1 ⁇ , IL-7, IL-10, IL-12, IL-18, IL-21, an INF (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , or IFN- ⁇ ), and a TNF (e.g., TNF ⁇ or CD40L).
  • the cytokine conjugate comprises an isolated and purified cytokine polypeptide and a conjugating moiety.
  • the cytokine conjugate has a decreased affinity to its respective receptor relative to its wild-type cytokine.
  • the conjugating moiety is bound to an amino acid residue that is proximal to the receptor interface (e.g., about 5 ⁇ , about 10 ⁇ , about 15 ⁇ , or about 20 ⁇ away from the receptor interface).
  • the conjugating moiety is linked to the N-terminus or the C-terminus of the cytokine, either directly or indirectly through a linker peptide.
  • the conjugating moiety modulates the interaction between cytokine and its respective receptor.
  • the cytokine conjugate upregulates distinct population(s) of tumor infiltrating lymphocytes.
  • the cytokine conjugate promotes a decrease in the proliferation and/or expansion of tumor associated lymphocytes.
  • the cytokine conjugate modulates immune activity.
  • cytokine conjugate precursors comprising a mutant cytokine (such as IL-2), wherein one or more amino acids have been mutated from the wild type amino acid.
  • a cytokine precursor is not conjugated.
  • Such mutations variously comprise additions, deletions, or substitutions.
  • the mutation comprises substitution to a different natural amino acid.
  • the mutant cytokine comprises a mutation at amino acid position Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, or A112, in which the numbering of the amino acid residues corresponds to SEQ ID NO: 1.
  • the amino acid position is selected from Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, E61, E62, E68, K64, P65, V69, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, and A112.
  • the amino acid position is selected from N33, P34, K35, T37, R38, M39, T41, F42, K43, F44, Y45, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, M104, C105, E106, Y107, A108, D109, E110, T111, and A112.
  • the amino acid position is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.
  • the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from R38 and K64. In some instances, the amino acid position is selected from E61, E62, and E68.
  • the amino acid position is at K35. In some cases, the amino acid position is at T37. In some cases, the amino acid position is at R38. In some cases, the amino acid position is at T41. In some cases, the amino acid position is at F42. In some cases, the amino acid position is at K43. In some cases, the amino acid position is at F44. In some cases, the amino acid position is at Y45. In some cases, the amino acid position is at E61. In some cases, the amino acid position is at E62. In some cases, the amino acid position is at K64. In some cases, the amino acid position is at E68. In some cases, the amino acid position is at P65. In some cases, the amino acid position is at V69.
  • a cytokine mutant comprises a conjugation moiety, wherein the conjugation moiety is attached to a mutated site in the mutant cytokine.
  • Cytokine mutants described herein often comprise one or more mutations to natural amino acids.
  • a cytokine mutant comprises SEQ ID NO: 1, and at least one mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and an E62K mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and an E62C mutation.
  • the cytokine mutant comprises SEQ ID NO:1 and an E62A mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and an E62I mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and an E62L mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and an E62Y mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62W mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62N mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62R mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62D mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62Q mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62G mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and an E62H mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62M mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62F mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62P mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62S mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62T mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62V mutation.
  • a cytokine mutant comprises SEQ ID NO:1, and at least one mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65K mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65C mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65A mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65I mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65L mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65Y mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65W mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65N mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65R mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65D mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65Q mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65G mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65H mutation.
  • the cytokine mutant comprises SEQ ID NO: 1 and a P65M mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65F mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65E mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65S mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65T mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65V mutation.
  • a cytokine conjugate described herein comprises a cytokine (e.g., IL-2, or other cytokine) that is fused to a peptide or protein (fusion).
  • the peptide or protein is an antibody or antibody fragment.
  • a cytokine conjugate described herein comprises a cytokine (e.g., IL-2, or other cytokine) that is fused to an antibody, or its binding fragments thereof.
  • a cytokine described herein is fused to multiple proteins or peptides.
  • a cytokine conjugate comprises a cytokine fusion to a protein or peptide, and at least one conjugating moiety.
  • an antibody or its binding fragments thereof comprise a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab 2 , F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • a cytokine conjugate comprises a fused peptide or protein is attached by a linker.
  • the linker is a peptide.
  • a cytokine conjugate comprises an N-terminal peptide or protein fusion.
  • a cytokine conjugate comprises a C-terminal peptide or protein fusion.
  • the cytokine fused to the peptide or protein is further conjugated to one or more conjugation moieties described below.
  • the cytokine conjugate comprises a fusion to an scFv, bis-scFv, (scFv) 2 , dsFv, or sdAb fusion.
  • the fusion comprises a scFv.
  • the cytokine conjugate comprises a fusion to bis-scFv.
  • the cytokine conjugate comprises a fusion to (scFv) 2 .
  • the cytokine conjugate comprises a fusion to dsFv.
  • the cytokine conjugate comprises a fusion to sdAb.
  • the cytokine fused to the scFv, bis-scFv, (scFv) 2 , dsFv, or sdAb is further conjugated to one or more conjugation moieties described below.
  • the cytokine conjugate comprises a fusion to an Fc portion of an antibody, e.g., of IgG, IgA, IgM, IgE, or IgD. In some instances, the cytokine conjugate comprises a fusion to an Fc portion of IgG (e.g., IgG 1 , IgG 3 , or IgG 4 ). In some cases, the cytokine fused to the Fc portion is further conjugated to one or more conjugation moieties described below.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • the cytokine polypeptide is fused to a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab 2 , F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • dsFv single-domain antibody
  • sdAb single-domain antibody
  • Ig NAR camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically
  • the cytokine polypeptide is fused to an Fc portion of an antibody. In additional cases, the cytokine polypeptide is fused to an Fc portion of IgG (e.g., IgG 1 , IgG 3 , or IgG 4 ). In some cases, the cytokine fused to the antibody, or its binding fragments thereof is further conjugated to one or more conjugation moieties described below.
  • an IL-2 polypeptide is fused to an antibody, or its binding fragments thereof.
  • the IL-2 polypeptide is fused to a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab 2 , F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • the IL-2 polypeptide is fused to an Fc portion of an antibody. In additional cases, the IL-2 polypeptide is fused to an Fc portion of IgG (e.g., IgG 1 , IgG 3 , or IgG 4 ). In some cases, the IL-2 polypeptide fused to the antibody, or its binding fragments thereof is further conjugated to one or more conjugation moieties described below.
  • an amino acid residue described herein (e.g., within a cytokine such as IL-2) is mutated to lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine prior to binding to (or reacting with) a conjugating moiety.
  • the side chain of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine may bind to a conjugating moiety described herein.
  • the amino acid residue is mutated to cysteine, lysine, or histidine.
  • 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, an unnatural amino acid is not conjugated with a conjugating moiety. In some embodiments, a cytokine described herein comprises an unnatural amino acid, wherein the cytokine is conjugated to the protein, wherein the point of attachment is not the unnatural amino acid.
  • an amino acid residue described herein (e.g., within a cytokine such as IL-2) is mutated to an unnatural amino acid prior to binding to a conjugating moiety.
  • the mutation to an unnatural amino acid prevents or minimizes a self-antigen response of the immune system.
  • the term “unnatural amino acid” refers to an amino acid other than the 20 amino acids that occur naturally in protein.
  • 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-Boronophenylalanine
  • the unnatural amino acid comprises a selective reactive group, or a reactive group for site-selective labeling of a target polypeptide.
  • the chemistry is a biorthogonal reaction (e.g., biocompatible and selective reactions).
  • the chemistry is a Cu(I)-catalyzed or “copper-free” alkyne-azide triazole-forming reaction, the Staudinger ligation, inverse-electron-demand Diels-Alder (IEDDA) reaction, “photo-click” chemistry, or a metal-mediated process such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.
  • the unnatural amino acid comprises a photoreactive group, which crosslinks, upon irradiation with, e.g., UV.
  • the unnatural amino acid comprises a photo-caged amino acid.
  • the unnatural amino acid is apara-substituted, meta-substituted, or an ortho-substituted amino acid derivative.
  • the unnatural amino acid comprises p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), 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
  • the unnatural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxy-phenylalanine, or 3-iodotyrosine.
  • the unnatural amino acid is phenylselenocysteine.
  • the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide containing phenylalanine derivative.
  • the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide containing lysine derivative.
  • the unnatural amino acid comprises an aromatic side chain.
  • the unnatural amino acid does not comprise an aromatic side chain.
  • the unnatural amino acid comprises an azido group.
  • the unnatural amino acid comprises a Michael-acceptor group.
  • Michael-acceptor groups comprise an unsaturated moiety capable of forming a covalent bond through a 1,2-addition reaction.
  • Michael-acceptor groups comprise electron-deficient alkenes or alkynes.
  • Michael-acceptor groups include but are not limited to alpha, beta unsaturated: ketones, aldehydes, sulfoxides, sulfones, nitriles, imines, or aromatics.
  • the unnatural amino acid is dehydroalanine.
  • the unnatural amino acid comprises an aldehyde or ketone group.
  • the unnatural amino acid is a lysine derivative comprising an aldehyde or ketone group.
  • the unnatural amino acid is a lysine derivative comprising one or more O, N, Se, or S atoms at the beta, ganma, or delta position. In some instances, the unnatural amino acid is a lysine derivative comprising O, N, Se, or S atoms at the gamma position.
  • the unnatural amino acid is a lysine derivative wherein the epilson N atom is replaced with an oxygen atom.
  • the unnatural amino acid is a lysine derivative that is not naturally-occuring post-translationally modified lysine.
  • the unnatural amino acid is an amino acid comprising a side chain, wherein the sixth atom from the alpha position comprises a carbonyl group. In some instances, the unnatural amino acid is an amino acid comprising a side chain, wherein the sixth atom from the alpha position comprises a carbonyl group, and the fifth atom from the alpha position is a nitrogen. In some instances, the unnatural amino acid is an amino acid comprising a side chain, wherein the seventh atom from the alpha position is an oxygen atom.
  • the unnatural amino acid is a serine derivative comprising selenium.
  • the unnatural amino acid is selenoserine (2-amino-3-hydroselenopropanoic acid).
  • the unnatural amino acid is 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid.
  • the unnatural amino acid is 2-amino-3-(phenylselanyl)propanoic acid.
  • the unnatural amino acid comprises selenium, wherein oxidation of the selenium results in the formation of an unnatural amino acid comprising an alkene.
  • the unnatural amino acid comprises a cyclooctynyl group.
  • the unnatural amino acid comprises a transcycloctenyl group.
  • the unnatural amino acid comprises a norbornenyl group.
  • the unnatural amino acid comprises a cyclopropenyl group.
  • the unnatural amino acid comprises a diazirine group.
  • the unnatural amino acid comprises a tetrazine group.
  • the unnatural amino acid is a lysine derivative, wherein the side-chain nitrogen is carbamylated. In some instances, the unnatural amino acid is a lysine derivative, wherein the side-chain nitrogen is acylated. In some instances, the unnatural amino acid is 2-amino-6- ⁇ [(tert-butoxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6- ⁇ [(tert-butoxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is N6-Boc-N6-methyllysine. In some instances, the unnatural amino acid is N6-acetyllysine.
  • the unnatural amino acid is pyrrolysine. In some instances, the unnatural amino acid is N6-trifluoroacetyllysine. In some instances, the unnatural amino acid is 2-amino-6- ⁇ [(benzyloxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6- ⁇ [(p-iodobenzyloxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6- ⁇ [(p-nitrobenzyloxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is N6-prolyllysine.
  • the unnatural amino acid is 2-amino-6- ⁇ [(cyclopentyloxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine. In some instances, the unnatural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some instances, the unnatural amino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some instances, the unnatural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine.
  • the unnatural amino acid is 2-amino-6- ⁇ [(2-azidocyclopentyloxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is N6-((2-azidoethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is 2-amino-6- ⁇ [(2-nitrobenzyloxy)carbonyl]amino ⁇ hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6- ⁇ [(2-cyclooctynyloxy)carbonyl]amino ⁇ hexanoic acid.
  • the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. In some instances, the unnatural amino acid is 2-amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid. In some instances, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-(pentenyl-5-oxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-((but-3-yn-1-yloxy)carbonyl)-lysine.
  • the unnatural amino acid is N6-((pent-4-yn-1-yloxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6-(thiazolidine-4-carbonyl)lysine. In some instances, the unnatural amino acid is 2-amino-8-oxononanoic acid. In some instances, the unnatural amino acid is 2-amino-8-oxooctanoic acid. In some instances, the unnatural amino acid is N6-(2-oxoacetyl)lysine.
  • the unnatural amino acid is N6-propionyllysine. In some instances, the unnatural amino acid is N6-butyryllysine, In some instances, the unnatural amino acid is N6-(but-2-enoyl)lysine, In some instances, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl)lysine.
  • the unnatural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is cysteinyllysine. In some instances, the unnatural amino acid is N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine.
  • the unnatural amino acid is N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((meta nitrobenyloxy)N6-methylcarbonyl)lysine. In some instances, the unnatural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6-((cyclohept-3-en-1-yloxy)carbonyl)-L-lysine.
  • the unnatural amino acid is 2-amino-3-(((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a repurposed amber, opal, or ochre stop codon.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a 4-base codon.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a repurposed rare sense codon.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a synthetic codon comprising an unnatural nucleic acid.
  • the unnatural amino acid is incorporated into the cytokine by an orthogonal, modified synthetase/tRNA pair.
  • orthogonal pairs comprise an unnatural synthetase that is capable of charging the unnatural tRNA with the unnatural amino acid, while minimizing charging of a) other endogenous amino acids onto the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs.
  • Such orthogonal pairs comprise tRNAs that are capable of being charged by the unnatural synthetase, while avoiding being charged with a) other endogenous amino acids by endogenous synthetases.
  • such pairs are identified from various organisms, such as bacteria, yeast, Archaea, or human sources.
  • an orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from two different organisms. In some embodiments, an orthogonal synthetase/tRNA pair comprising components that prior to modification, promote translation of two different amino acids. In some embodiments, an orthogonal synthetase is a modified alanine synthetase. In some embodiments, an orthogonal synthetase is a modified arginine synthetase. In some embodiments, an orthogonal synthetase is a modified asparagine synthetase.
  • an orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, an orthogonal synthetase is a modified cysteine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamic acid synthetase. In some embodiments, an orthogonal synthetase is a modified alanine glycine. In some embodiments, an orthogonal synthetase is a modified histidine synthetase.
  • an orthogonal synthetase is a modified leucine synthetase. In some embodiments, an orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, an orthogonal synthetase is a modified lysine synthetase. In some embodiments, an orthogonal synthetase is a modified methionine synthetase. In some embodiments, an orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, an orthogonal synthetase is a modified proline synthetase.
  • an orthogonal synthetase is a modified serine synthetase. In some embodiments, an orthogonal synthetase is a modified threonine synthetase. In some embodiments, an orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, an orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, an orthogonal synthetase is a modified valine synthetase. In some embodiments, an orthogonal synthetase is a modified phosphoserine synthetase.
  • an orthogonal tRNA is a modified alanine tRNA. In some embodiments, an orthogonal tRNA is a modified arginine tRNA. In some embodiments, an orthogonal tRNA is a modified asparagine tRNA. In some embodiments, an orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, an orthogonal tRNA is a modified cysteine tRNA. In some embodiments, an orthogonal tRNA is a modified glutamine tRNA. In some embodiments, an orthogonal tRNA is a modified glutamic acid tRNA. In some embodiments, an orthogonal tRNA is a modified alanine glycine.
  • an orthogonal tRNA is a modified histidine tRNA. In some embodiments, an orthogonal tRNA is a modified leucine tRNA. In some embodiments, an orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, an orthogonal tRNA is a modified lysine tRNA. In some embodiments, an orthogonal tRNA is a modified methionine tRNA. In some embodiments, an orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, an orthogonal tRNA is a modified proline tRNA. In some embodiments, an orthogonal tRNA is a modified serine tRNA.
  • an orthogonal tRNA is a modified threonine tRNA. In some embodiments, an orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, an orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, an orthogonal tRNA is a modified valine tRNA. In some embodiments, an orthogonal tRNA is a modified phosphoserine tRNA.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair.
  • aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/ B. stearothermophilus tRNA CUA pairs, E. coli LeuRS (Ec-Leu)/ B. stearothermophilus tRNA CUA pairs, and pyrrolysyl-tRNA pairs.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Mj-TyrRS/tRNA pair.
  • exemplary UAAs that can be incorporated by a Mj-TyrRS/tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoyphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronopheylalanine; and o-nitrobenzyltyrosine.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Ec-Tyr/tRNA CUA or a Ec-Leu/tRNA CUA pair.
  • exemplary UAAs that can be incorporated by a Ec-Tyr/tRNA CUA or a Ec-Leu/tRNA CUA pair include, but are not limited to, phenylalanine derivatives containing benzophenoe, ketone, iodide, or azide substituents; O-propargyltyrosine; ⁇ -aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3-(naphthalene-2-ylamino)-2-amino-propanoic acid.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a pyrrolysyl-tRNA pair.
  • the PylRS is obtained from an archaebacterial, e.g., from a methanogenic archaebacterial.
  • the 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- ⁇ - D -prolyl- L -lysine, and N- ⁇ -cyclopentyloxycarbonyl- L -lysine; N- ⁇ -Acryloyl- L -lysine; N- ⁇ -[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]- L -lysine; and N- ⁇ -(1-methylcyclopro-2-enecarboxamido)lysine.
  • amide and carbamate substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)he
  • an unnatural amino acid is incorporated into a cytokine described herein (e.g., the IL polypeptide) by a synthetase disclosed in U.S. Pat. Nos. 9,988,619 and 9,938,516.
  • Exemplary UAAs tha can be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, heteroaralkyl unnatural amino acids, and others.
  • such UAAs comprise pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycle.
  • Such amino acids in some embodiments comprise azides, tetrazines, or other chemical group capable of conjugation to a coupling partner, such as a water soluble moiety.
  • a coupling partner such as a water soluble moiety.
  • 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 a cell-free translation system.
  • an unnatural amino acid is incorporated into a cytokine described herein (e.g., the IL polypeptide) by a naturally occurring synthetase.
  • an unnatural amino acid is incorporated into a cytokine by an organism that is auxotrophic for one or more amino acids.
  • synthetases corresponding to the auxotrophic amino acid are capable of charging the corresponding tRNA with an unnatural amino acid.
  • the unnatural amino acid is selenocysteine, or a derivative thereof.
  • the unnatural amino acid is selenomethionine, or a derivative thereof.
  • the unnatural amino acid is an aromatic amino acid, wherein the aromatic amino acid comprises an aryl halide, such as an iodide.
  • the unnatural amino acid is structurally similar to the auxotrophic amino acid.
  • the unnatural amino acid comprises an unnatural amino acid illustrated in FIG. 1 .
  • the unnatural amino acid comprises a lysine or phenylalanine derivative or analogue. In some instances, the unnatural amino acid comprises a lysine derivative or a lysine analogue. In some instances, the unnatural amino acid comprises a pyrrolysine (Pyl). In some instances, the unnatural amino acid comprises a phenylalanine derivative or a phenylalanine analogue. In some instances, 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 instances, the unnatural amino acid comprises an unnatural amino acid illustrated in FIG. 2 (e.g., FIG. 2A and FIG. 2B ).
  • the unnatural amino acid comprises an unnatural amino acid illustrated in FIG. 3A — FIG. 3D (adopted from Table 1 of Dumas et al., Chemical Science 2015, 6, 50-69).
  • an unnatural amino acid incorporated into a cytokine described herein is disclosed in U.S. Pat. Nos. 9,840,493; 9,682,934; US 2017/0260137; U.S. Pat. No. 9,938,516; or US 2018/0086734.
  • Exemplary UAAs that can be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl, and lysine derivative unnatural amino acids.
  • such UAAs comprise pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycle.
  • Such amino acids in some embodiments comprise azides, tetrazines, or other chemical group capable of conjugation to a coupling partner, such as a water soluble moiety.
  • a UAA comprises an azide attached to an aromatic moiety via an alkyl linker.
  • an alkyl linker is a C 1 -C 10 linker.
  • a UAA comprises a tetrazine attached to an aromatic moiety via an alkyl linker.
  • a UAA comprises a tetrazine attached to an aromatic moiety via an amino group. In some embodiments, a UAA comprises a tetrazine attached to an aromatic moiety via an alkylamino group. In some embodiments, a UAA comprises an azide attached to the terminal nitrogen (e.g., N6 of a lysine derivative, or N5, N4, or N3 of a derivative comprising a shorter alkyl side chain) of an amino acid side chain via an alkyl chain. In some embodiments, a UAA comprises a tetrazine attached to the terminal nitrogen of an amino acid side chain via an alkyl chain.
  • a UAA comprises an azide or tetrazine attached to an amide via an alkyl linker.
  • the UAA is an azide or tetrazine-containing carbamate or amide of 3-aminoalanine, serine, lysine, or derivative thereof.
  • such UAAs are incorporated into cytokines in-vivo. In some embodiments, such UAAs are incorporated into cytokines in a cell-free system.
  • conjugating moieties that are bound to one or more cytokines (e.g., interleukins, IFNs, or TNFs) described supra.
  • the conjugating moiety is a molecule that perturbs the interaction of a cytokine with its receptor.
  • the conjugating moiety is any molecule that when bond to the cytokine, enables the cytokine conjugate to modulate an immune response.
  • the conjugating moiety is bound to the cytokine through a covalent bond.
  • a cytokine described herein is attached to a conjugating moiety with a triazole group.
  • a cytokine described herein is attached to a conjugating moiety with a dihydropyridazine or pyridazine group.
  • the conjugating moiety comprises a water-soluble polymer.
  • the conjugating moiety comprises a protein or a binding fragment thereof.
  • the conjugating moiety comprises a peptide.
  • the conjugating moiety comprises a nucleic acid.
  • the conjugating moiety comprises a small molecule.
  • the conjugating moiety comprises a bioconjugate (e.g., a TLR agonist such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2 ⁇ , CFA, or Flagellin).
  • a TLR agonist such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist
  • a synthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2 ⁇ , CFA, or Flagellin.
  • the conjugating moiety blocks cytokine interaction with one or more cytokine domains or subunits with its cognate receptor(s).
  • cytokine conjugates described herein comprise multiple conjugating moieties.
  • a conjugating moiety is attached to an unnatural or natural amino acid in the cytokine peptide.
  • a cytokine conjugate comprises a conjugating moiety attached to a natural amino acid.
  • a cytokine conjugate is attached to an unnatural amino acid in the cytokine peptide.
  • a conjugating moiety is attached to the N or C terminal amino acid of the cytokine peptide.
  • a first conjugating moiety is attached to an unnatural or natural amino acid in the cytokine peptide
  • a second conjugating moiety is attached to the N or C terminal amino acid of the cytokine peptide.
  • a single conjugating moiety is attached to multiple residues of the cytokine peptide (e.g. a staple).
  • a conjugating moiety is attached to both the N and C terminal amino acids of the cytokine peptide.
  • a conjugating moiety descried herein is a water-soluble polymer.
  • the water-soluble polymer is a nonpeptidic, nontoxic, and biocompatible.
  • a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as a cytokine moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.
  • a water-soluble polymer is further non-immunogenic.
  • a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.
  • an undesired immune response e.g., the formation of antibodies
  • an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.
  • 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 and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazolines (“POZ”) (which are described in WO 2008/106186), poly(N-acryloylmorpholine), and combinations of any of the for the
  • the water-soluble polymer is not limited to a particular structure.
  • the water-soluble polymer is linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages.
  • the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
  • 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.
  • Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons.
  • Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 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 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,
  • Branched versions of the water-soluble polymer e.g., a branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers
  • the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight average molecular weight of less than about 6,000 Daltons.
  • PEGs will typically comprise a number of (OCH 2 CH 2 ) monomers [or (CH 2 CH 2 O) monomers, depending on how the PEG is defined].
  • the number of repeating units is identified by the subscript “n” in “(OCH 2 CH 2 )n.”
  • the value of (n) typically falls within one or more of the following ranges: from 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.
  • n the number of repeating units
  • the water-soluble polymer is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower C 1-6 alkoxy group, or a hydroxyl group.
  • a relatively inert group such as a lower C 1-6 alkoxy group, or a hydroxyl group.
  • mPEG methoxy-PEG
  • mPEG is a linear form of PEG wherein one terminus of the polymer is a methoxy (—OCH 3 ) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified.
  • exemplary water-soluble polymers include, but are not limited to, linear or branched discrete PEG (dPEG) from Quanta Biodesign, Ltd; linear, branched, or forked PEGs from Nektar Therapeutics; and Y-shaped PEG derivatives from JenKem Technology.
  • dPEG linear or branched discrete PEG
  • Nektar Therapeutics linear, branched, or forked PEGs from Nektar Therapeutics
  • Y-shaped PEG derivatives from JenKem Technology.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a water-soluble polymer selected from poly(alkylene glycols) such as polyethylene glycol (“PEG”), poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly( ⁇ -hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene, polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and a combination thereof.
  • PEG polyethylene glycol
  • PPG poly(prop
  • the cytokine polypeptide is conjugated to PEG (e.g., PEGylated). In some instances, the cytokine polypeptide is conjugated to PPG. In some instances, the cytokine polypeptide is conjugated to POZ. In some instances, the cytokine polypeptide is conjugated to PVP.
  • an IL-2 polypeptide described herein is conjugated to a water-soluble polymer selected from poly(alkylene glycols) such as polyethylene glycol (“PEG”), poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene, polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and a combination thereof.
  • poly(alkylene glycols) such as polyethylene glycol (“PEG”), poly(propylene
  • the IL-2 polypeptide is conjugated to PEG (e.g., PEGylated). In some instances, the IL-2 polypeptide is conjugated to PPG. In some instances, the IL-2 polypeptide is conjugated to POZ. In some instances, the IL-2 polypeptide is conjugated to PVP.
  • a water-soluble polymer comprises a polyglycerol (PG).
  • the polyglycerol is a hyperbranched 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)).
  • HPG hyperbranched PG
  • the polyglycerol is a linear PG (LPG).
  • LPG linear PG
  • the polyglycerol is a midfunctional PG, a linear-block-hyperbranched PG (e.g., as described by Wurm et.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a cytokine polypeptide described herein is conjugated to a PG, e.g., a HPG, a LPG, a midfunctional PG, a linear-block-hyperbranched PG, or a side-chain functional PG.
  • the cytokine is an IL-2 polypeptide.
  • the IL-2 polypeptide is conjugated to a PG, a midfunctional PG, a linear-block-hyperbranched PG.
  • a water-soluble polymer is a degradable synthetic PEG alternative.
  • degradable synthetic PEG alternatives include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; PEG derivatives comprising comonomers comprising degradable linkage such as poly[(ethylene oxie)-co-(methylene ethylene oxide)][P(EO-co-MEO)], cyclic ketene acetals such as 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene-1,3-dioxepane (MDO), and 2-methylene-4-phenyl-1,3-dioxolane (MPDL) copolymerized with OEGMA; or poly-( ⁇ -caprolactone)-graft-poly(ethylene oxide) (PC
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a degradable synthetic PEG alternative such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-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.
  • the cytokine is an IL-2 polypeptide.
  • the IL-2 polypeptide is conjugated to a degradable synthetic PEG alternative, such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-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.
  • a degradable synthetic PEG alternative such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-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.
  • a water-soluble polymer comprises a poly(zwitterions).
  • exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methyacryloyloxyethyl phosphorylcholine) (PMPC).
  • a cytokine e.g., an interleukin, IFN, or TNF
  • the cytokine is an IL-2 polypeptide.
  • the IL-2 polypeptide is conjugated to a poly(zwitterion) such as PSBMA, PCBMA, or PMPC.
  • a water-soluble polymer comprises a polycarbonate.
  • exemplary polycarbones include, but are not limited to, pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC 6 F 5 ).
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a cytokine is conjugated to a polycarbonate such as MTC-OC 6 F 5 .
  • the cytokine is an IL-2 polypeptide.
  • the IL-2 polypeptide is conjugated to a polycarbonate such as MTC-OC 6 F 5 .
  • a water-soluble polymer comprises a polymer hybrid, such as for example, a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxylcontaining and/or zwitterionic derivatized polymer (e.g., a hydroxylcontaining and/or zwitterionic derivatized PEG polymer).
  • a polymer hybrid such as for example, a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxylcontaining and/or zwitterionic derivatized polymer (e.g., a hydroxylcontaining and/or zwitterionic derivatized PEG polymer).
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a polymer hybrid such as a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxylcontaining and/or zwitterionic derivatized polymer (e.g., a hydroxylcontaining and/or zwitterionic derivatized PEG polymer).
  • the cytokine is an IL-2 polypeptide.
  • the IL-2 polypeptide is conjugated to a polymer hybrid such as a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxylcontaining and/or zwitterionic derivatized polymer (e.g., a hydroxylcontaining and/or zwitterionic derivatized PEG polymer).
  • a polymer hybrid such as a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxylcontaining and/or zwitterionic derivatized polymer (e.g., a hydroxylcontaining and/or zwitterionic derivatized PEG polymer).
  • a 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).
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to dextran.
  • an IL-2 polypeptide is conjugated to PSA.
  • 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.
  • a water-soluble polymer comprises a glycan.
  • glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to N-linked glycans.
  • an IL-2 polypeptide is conjugated to O-linked glycans.
  • an IL-2 polypeptide is conjugated to glycolipids.
  • an IL-2 polypeptide is conjugated to O-GlcNAc.
  • an IL-2 polypeptide is conjugated to glycosaminoglycans.
  • a water-soluble polymer comprises a polyoxazoline polymer.
  • a polyoxazoline polymer is a linear synthetic polymer, and similar to PEG, comprises a low polydispersity.
  • a polyoxazoline polymer is a polydispersed polyoxazoline polymer, characterized with an average molecule weight.
  • the average molecule weight of a 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.
  • a polyoxazoline polymer comprises poly(2-methyl 2-oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), or poly(2-propyl 2-oxazoline) (PPOZ).
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to a polyoxazoline polymer.
  • an IL-2 polypeptide is conjugated to PMOZ.
  • an IL-2 polypeptide is conjugated to PEOZ.
  • an IL-2 polypeptide is conjugated to PPOZ.
  • a water-soluble polymer comprises a polyacrylic acid polymer.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an L-2 polypeptide is conjugated to a polyacrylic acid polymer.
  • a water-soluble polymer comprises polyamine.
  • Polyamine is an organic polymer comprising two or more primary amino groups.
  • a polyamine includes a branched polyamine, a linear polyamine, or cyclic polyamine.
  • a polyamine is a low-molecular-weight linear polyamine.
  • Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to polyamine.
  • an IL-2 polypeptide is conjugated to putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, or piperazine.
  • a water-soluble polymer is described in U.S. Pat. Nos. 7,744,861, 8,273,833, and 7,803,777.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to a linker described in U.S. Pat. Nos. 7,744,861, 8,273,833, or 7,803,777.
  • a conjugating moiety descried herein is a lipid.
  • the lipid is a fatty acid.
  • the fatty acid is a saturated fatty acid.
  • the fatty acid is an unsaturated fatty acid.
  • Exemplary fatty acids include, but are not limited to, fatty acids comprising from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the lipid binds to one or more serum proteins, thereby increasing serum stability and/or serum half-life.
  • the lipid is conjugated to IL-2.
  • the lipid is a fatty acid, e.g., a saturated fatty acid or an unsaturated fatty acid.
  • the fatty acid is from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms.
  • the fatty acid comprises about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length.
  • the fatty acid comprises caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylic acid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic acid),
  • the L-2 lipid conjugate enhances serum stability and/or serum half-life.
  • a conjugating moiety descried herein is a protein or a binding fragment thereof.
  • Exemplary proteins include albumin, transferrin, or transthyretin.
  • the protein or a binding fragment thereof comprises an antibody, or its binding fragments thereof.
  • a cytokine conjugate comprises a protein or a binding fragment thereof.
  • an IL-2 conjugate comprising a protein or a binding fragment thereof has an increased serum half-life, and/or stability.
  • an IL-2 conjugate comprising a protein or a binding fragment thereof has a reduced IL-2 interaction with one or more IL-2R subunits.
  • the protein or a binding fragment thereof blocks IL-2 interaction with one or more IL-2R subunits.
  • the conjugating moiety is albumin.
  • Albumin is a family of water-soluble globular proteins. It is commonly found in blood plasma, comprising about 55-60% of all plasma proteins.
  • Human serum albumin (HSA) is a 585 amino acid polypeptide in which the 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 further comprises a binding site, which can interact either reversibly or irreversibly with endogenous ligands such as long- and medium-chain fatty acids, bilirubin, or hemin, or exogenous compounds such as heterocyclic or aromatic compounds.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • albumin e.g., an interleukin, IFN, or TNF
  • HSA human serum albumin
  • the cytokine polypeptide is conjugated to a functional fragment of albumin.
  • an IL-2 polypeptide is conjugated to albumin.
  • the IL-2 polypeptide is conjugated to human serum albumin (HSA).
  • HSA human serum albumin
  • the IL-2 polypeptide is conjugated to a functional fragment of albumin.
  • the conjugating moiety is transferrin.
  • Transferrin is a 679 amino acid polypeptide that is about 80 kDa in size and comprises two Fe3+ binding sites with one at the N-terminal domain and the other at the C-terminal domain.
  • human transferrin has a half-life of about 7-12 days.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • transferrin e.g., transferrin
  • the cytokine polypeptide is conjugated to human transferrin.
  • the cytokine polypeptide is conjugated to a functional fragment of transferrin.
  • an IL-2 polypeptide is conjugated to transferrin. In some cases, the IL-2 polypeptide is conjugated to human transferrin. In additional cases, the IL-2 polypeptide is conjugated to a functional fragment of transferrin.
  • the conjugating moiety is transthyretin (TTR).
  • TTR transthyretin
  • Transthyretin is a transport protein located in the serum and cerebrospinal fluid which transports the thyroid hormone thyroxine (T 4 ) and retinol-binding protein bound to retinol.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • a cytokine polypeptide is conjugated to transthyretin (via one of its termini or via an internal hinge region).
  • the cytokine polypeptide is conjugated to a functional fragment of transthyretin.
  • an IL-2 polypeptide is conjugated to transthyretin (via one of its termini or via an internal hinge region). In some cases, the IL-2 polypeptide is conjugated to a functional fragment of transthyretin.
  • the conjugating moiety is an antibody, or its binding fragments thereof.
  • an antibody or its binding fragments thereof comprise a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab 2 , F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • the conjugating moiety comprises a scFv, bis-scFv, (scFv) 2 , dsFv, or sdAb. In some cases, the conjugating moiety comprises a scFv. In some cases, the conjugating moiety comprises a bis-scFv. In some cases, the conjugating moiety comprises a (scFv) 2 . In some cases, the conjugating moiety comprises a dsFv. In some cases, the conjugating moiety comprises a sdAb.
  • the conjugating moiety comprises an Fc portion of an antibody, e.g., of IgG, IgA, IgM, IgE, or IgD. In some instances, the moiety comprises an Fc portion of IgG (e.g., IgG 1 , IgG 3 , or IgG 4 ).
  • a cytokine e.g., an interleukin, IFN, or TNF
  • the cytokine polypeptide is conjugated to a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab 2 , F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • dsFv single-domain antibody
  • sdAb single-domain antibody
  • the cytokine polypeptide is conjugated to an Fc portion of an antibody. In additional cases, the cytokine polypeptide is conjugated to an Fc portion of IgG (e.g., IgG 1 , IgG 3 , or IgG 4 ).
  • IgG e.g., IgG 1 , IgG 3 , or IgG 4 .
  • an IL-2 polypeptide is conjugated to an antibody, or its binding fragments thereof.
  • the IL-2 polypeptide is conjugated to a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab 2 , F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • the IL-2 polypeptide is conjugated to an Fc portion of an antibody. In additional cases, the IL-2 polypeptide is conjugated to an Fc portion of IgG (e.g., IgG 1 , IgG 3 , or IgG 4 ).
  • IgG e.g., IgG 1 , IgG 3 , or IgG 4 .
  • an IL-2 polypeptide is conjugated to a water-soluble polymer (e.g., PEG) and an antibody or binding fragment thereof.
  • the antibody or binding fragments thereof comprises a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab′, divalent Fab 2 , F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • a water-soluble polymer e.g., PEG
  • the antibody or binding fragments thereof comprises a humanized antibody or binding fragment thereof, murine antibody or binding
  • the antibody or binding fragments thereof comprises a scFv, bis-scFv, (scFv) 2 , dsFv, or sdAb. In some cases, the antibody or binding fragments thereof comprises a scFv. In some cases, the antibody or binding fragment thereof guides the L-2 conjugate to a target cell of interest and the water-soluble polymer enhances stability and/or serum half-life.
  • one or more IL-2 polypeptide—water-soluble polymer (e.g., PEG) conjugates are further bound to an antibody or binding fragments thereof.
  • the ratio of the IL-2 conjugate to the 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.
  • the ratio of the IL-2 conjugate to the antibody is about 1:1.
  • the ratio of the IL-2 conjugate to the antibody is about 2:1, 3:1, or 4:1.
  • the ratio of the IL-2 conjugate to the antibody is about 6:1 or higher.
  • the one or more L-2 polypeptide—water-soluble polymer (e.g., PEG) conjugates are directly bound to the antibody or binding fragments thereof.
  • the IL-2 conjugate is indirectly bound to the antibody or binding fragments thereof with a linker.
  • linkers include homobifunctional linkers, heterobifunctional linkers, maleimide-based linkers, zero-trace linkers, self-immolative linkers, spacers, and the like.
  • the antibody or binding fragments thereof is bound either directly or indirectly to the IL-2 polypeptide portion of the IL-2 polypeptide—water-soluble polymer (e.g., PEG) conjugate.
  • the conjugation site of the antibody to the IL-2 polypeptide is at a site that will not impede binding of the IL-2 polypeptide with the IL-2R ⁇ .
  • the conjugation site of the antibody to the IL-2 polypeptide is at a site that partially blocks binding of the IL-2 polypeptide with the IL-2R ⁇ .
  • the conjugation site of the antibody to the IL-2 polypeptide is at a site that will impede or further impede binding of the IL-2 polypeptide with the IL-2R ⁇ .
  • the antibody or binding fragments thereof is bound either directly or indirectly to the water-soluble polymer portion of the IL-2 polypeptide—water-soluble polymer (e.g., PEG) conjugate.
  • a conjugating moiety descried herein is a peptide.
  • the peptide is a non-structured peptide.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • the IL-2 conjugate comprising a peptide has an increased serum half-life, and/or stability.
  • the IL-2 conjugate comprising a peptide has a reduced IL-2 interaction with one or more IL-2R subunits.
  • the peptide blocks IL-2 interaction with one or more IL-2R subunits.
  • the conjugating moiety is a XTENTM peptide (Amunix Operating Inc.) and the modification is referred to as XTENylation.
  • XTENylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a XTENTM peptide (Amunix Operating Inc.), a long unstructured hydrophilic peptide comprising different percentage of six amino acids: Ala, Glu, Gly, Ser, and Thr.
  • a XTENTM peptide is selected based on properties such as expression, genetic stability, solubility, aggregation resistance, enhanced half-life, increased potency, and/or increased in vitro activity in combination with a polypeptide of interest.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to a XTEN peptide.
  • the conjugating moiety is a glycine-rich homoamino acid polymer (HAP) and the modification is referred to as HAPylation.
  • HAPylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a glycine-rich homoamino acid polymer (HAP).
  • the HAP polymer comprises a (Gly 4 Ser) n repeat motif (SEQ ID NO: 3) and sometimes are about 50, 100, 150, 200, 250, 300, or more residues in length.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to HAP.
  • the conjugating moiety is a PAS polypeptide and the modification is referred to as PASylation.
  • PASylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a PAS polypeptide.
  • a PAS polypeptide is a hydrophilic uncharged polypeptide consisting of Pro, Ala and Ser residues.
  • the length of a PAS polypeptide is at least about 100, 200, 300, 400, 500, or 600 amino acids.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to a PAS polypeptide.
  • the conjugating moiety is an elastin-like polypeptide (ELP) and the modification is referred to as ELPylation.
  • ELPylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding an elastin-like polypeptide (ELPs).
  • An ELP comprises a VPGxG repeat motif (SEQ ID NO: 4) in which x is any amino acid except proline.
  • a cytokine e.g., an interleukin, IFN, or TNF
  • an IL-2 polypeptide is conjugated to ELP.
  • the conjugating moiety is a CTP peptide.
  • a CTP peptide comprises a 31 amino acid residue peptide FQSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 5) in which the S* denotes O-glycosylation sites (OPKO).
  • a CTP peptide is genetically fused to a cytokine polypeptide (e.g., an IL-2 polypeptide).
  • a cytokine polypeptide e.g., an IL-2 polypeptide
  • a cytokine polypeptide is conjugated to a CTP peptide.
  • a cytokine e.g., an IL-2 polypeptide
  • glutamylation is a reversible posttranslational modification of glutamate, in which the ⁇ -carboxy group of glutamate forms a peptide-like bond with the amino group of a free glutamate in which the ⁇ -carboxy group extends into a polyglutamate chain.
  • a cytokine e.g., an IL-2 polypeptide
  • a gelatin-like protein (GLK) polymer comprises multiple repeats of Gly-Xaa-Yaa wherein Xaa and Yaa primarily comprise proline and 4-hydroxyproline, respectively.
  • the GLK polymer further comprises amino acid residues Pro, Gly, Glu, Qln, Asn, Ser, and Lys.
  • the length of the GLK polymer is about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150 residues or longer.
  • the conjugating moiety comprises an extracellular biomarker.
  • the extracellular biomarker is a tumor antigen.
  • exemplary extracellular biomarker comprises CD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FR ⁇ , GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc 16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8, and B220.
  • the conjugating moiety is bond or conjugated to the cytokine (e.g., IL-2). In some cases, the conjugating moiety is genetically fused, for example, at the N-terminus or the C-terminus, of the cytokine (e.g., IL-2).
  • the conjugating moiety comprises a molecule from a post-translational modification.
  • post-translational modification include myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypiation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., additional of alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation.
  • the cytokine (e.g., IL-2) is modified by a 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., additional of alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation.
  • a post-translational modification such as myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), gly
  • useful functional reactive groups for conjugating or binding a conjugating moiety to a cytokine polypeptide (e.g., an IL-2 polypeptide) described herein include, for example, zero or higher-order linkers.
  • an unnatural amino acid incorporated into an interleukin described herein comprises a functional reactive group.
  • a linker comprises a functional reactive group that reacts with an unnatural amino acid incorporated into an interleukin described herein.
  • a conjugating moiety comprises a functional reactive group that reacts with an unnatural amino acid incorporated into an interleukin described herein.
  • a conjugating moiety comprises a functional reactive group that reacts with a linker (optionally pre-attached to a cytokine peptide) described herein.
  • a linker comprises a reactive group that reacts with a natural amino acid in a cytokine peptide described herein.
  • higher-order linkers comprise bifunctional linkers, such as homobifunctional linkers or heterobifunctional linkers.
  • Exemplary homobifuctional linkers include, but are not limited to, Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (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′
  • DFDNPS 4,4′-difluoro-3,3′-dinitrophenylsulfone
  • BASED bis-[P3-(4-azidosalicylamido)ethyl]disulfide
  • formaldehyde glutaraldehyde
  • 1,4-butanediol diglycidyl ether adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylb enzidine, benzidine, ⁇ , ⁇ ′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylene-bis(iodoacetamide).
  • the bifunctional linker comprises a heterobifunctional linker.
  • exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross-linkers 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- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[ ⁇ -methyl- ⁇ -(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-
  • the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety (e.g., on a conjugating moiety or on IL-2).
  • electrophilic groups include carbonyl groups-such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride.
  • the reactive functional group is aldehyde.
  • Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • an unnatural amino acid incorporated into an interleukin described herein comprises an electrophilic group.
  • 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.
  • the linker is a peptide linker comprising, e.g., 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 instances, the peptide linker comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or less amino acids. In additional cases, the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.
  • the linker comprises a self-immolative linker moiety.
  • the self-immolative linker moiety comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), or derivatives or analogs thereof.
  • the linker comprises a dipeptide linker moiety and a self-immolative linker moiety.
  • the self-immolative linker moiety is such as described in U.S. Pat. No. 9,089,614 and WIPO Application No. WO2015038426.
  • the cleavable linker is glucuronide. In some embodiments, the cleavable linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker is hydrazine. In some embodiments, the cleavable linker is a reducible linker.
  • the linker comprises a maleimide group.
  • the maleimide group is also referred to as a maleimide spacer.
  • the maleimide group further comprises a caproic acid, forming maleimidocaproyl (mc).
  • the linker comprises maleimidocaproyl (mc).
  • linker is maleimidocaproyl (mc).
  • 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) described above.
  • sMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • sulfo-sMCC sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • the maleimide group is a self-stabilizing maleimide.
  • the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of tiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro-Michael reaction.
  • 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).
  • the linker comprises a self-stabilizing maleimide.
  • the linker is a self-stabilizing maleimide.
  • conjugation reactions are used to conjugate linkers, conjugation moieties, and unnatural amino acids incorporated into cytokine peptides described herein. Such conjugation reactions are often compatible with aqueous conditions, such as “bioorthogonal” reactions.
  • conjugation reactions are mediated by chemical reagents such as catalysts, light, or reactive chemical groups found on linkers, conjugation moieties, or unnatural amino acids.
  • conjugation reactions are mediated by enzymes.
  • a conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123.
  • a conjugation reaction used herein is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417.
  • a conjugation reaction comprises reaction of a ketone or aldehyde with a nucleophile. In some embodiments, a conjugation reaction comprises reaction of a ketone with an aminoxy group to form an oxime. In some embodiments, a conjugation reaction comprises reaction of a ketone with an aryl or heteroaryl amine group to form an imine. In some embodiments, a conjugation reaction comprises reaction of an aldehyde with an aryl or heteroaryl amine group to form an imine. In some embodiments, a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via an oxime.
  • a conjugation reaction comprises a Pictet-Spengler reaction of an aldehyde or ketone with a tryptamine nucleophile. In some embodiments, a conjugation reaction comprises a hydrazino-Pictet-Spengler reaction. In some embodiments, a conjugation reaction comprises a Pictet-Spengler ligation.
  • a conjugation reaction described herein comprises reaction of an azide and a phosphine (Staudinger ligation).
  • the phosphine is an aryl phosphine.
  • the aryl phosphine comprises an ortho ester group.
  • the phosphine comprises the structure methyl 2-(diphenylphosphaneyl)benzoate.
  • a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via an arylamide.
  • a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via an amide.
  • a conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction.
  • the 1,3-dipolar cycloaddition reaction comprises reaction of an azide and a phosphine (“Click” reaction).
  • the conjugation reaction is catalyzed by copper.
  • a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via a triazole.
  • a conjugation reaction described herein comprises reaction of an azide with a strained olefin.
  • a conjugation reaction described herein comprises reaction of an azide with a strained alkyne.
  • a conjugation reaction described herein comprises reaction of an azide with a cycloalkyne, for example, 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.
  • a 1,3-dipolar cycloaddition reaction is catalyzed by light (“photoclick”).
  • a conjugation reaction described herein comprises reaction of a terminal allyl group with a tetrazole and light.
  • a conjugation reaction described herein comprises reaction of a terminal alkynyl group with a tetrazole and light. In some embodiments, a conjugation reaction described herein comprises reaction of an O-allyl amino acid with a tetrazine and light. In some embodiments, a conjugation reaction described herein comprises reaction of O-allyl tyrosine with a tetrazine and light.
  • a conjugation reaction described herein comprises an inverse-electron demand cycloaddition reaction comprising a diene and a dienophile.
  • the diene comprises a tetrazine.
  • the dienophile comprises an alkene.
  • the dienophile comprises an alkyne.
  • the alkyne is a strained alkyne.
  • the alkene is a strained diene.
  • the alkyne is a trans-cyclooctyne.
  • the alkyne is a cyclooctene.
  • the alkene is a cyclopropene. In some embodiments, the alkene is a fluorocyclopropene.
  • a conjugation reaction described herein results in the formation of a cytokine peptide attached to a linker or conjugation moiety via a 6-membered ring heterocycle comprising two nitrogen atoms in the ring.
  • a conjugation reaction described herein comprises an olefin metathesis reaction. In some embodiments, a conjugation reaction described herein comprises reaction of an alkene and an alkyne with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reaction of two alkenes with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reaction of two alkynes with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reaction of an alkene or alkyne with a ruthenium catalyst and an amino acid comprising an allyl group.
  • a conjugation reaction described herein comprises reaction of an alkene or alkyne with a ruthenium catalyst and an amino acid comprising an allyl sulfide or selenide.
  • a ruthenium catalyst is Hoveda-Grubbs 2 nd generation catalyst.
  • an olefin metathesis reaction comprises reaction of one or more strained alkenes or alkynes.
  • a conjugation reaction described herein comprises a cross-coupling reaction.
  • cross-coupling reactions comprise transition metal catalysts, such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or other transition metal catalyst and one or more ligands.
  • transition metal catalysts are water-soluble.
  • a conjugation reaction described herein comprises a Suzuki-Miyaura cross-coupling reaction.
  • a conjugation reaction described herein comprises reaction of an aryl halide (or triflate, or tosylate), an aryl or alkenyl boronic acid, and a palladium catalyst.
  • a conjugation reaction described herein comprises a Sonogashira cross-coupling reaction.
  • a conjugation reaction described herein comprises reaction of an aryl halide (or triflate, or tosylate), an alkyne, and a palladium catalyst.
  • cross-coupling reactions result in attachment of a linker or conjugating moiety to a cytokine peptide via a carbon-carbon bond.
  • a conjugation reaction described herein comprises a deprotection or “uncaging” reaction of a reactive group prior to conjugation.
  • a conjugation reaction described herein comprises uncaging of a reactive group with light, followed by a conjugation reaction.
  • a reactive group is protected with an aralkyl moiety comprising one or more nitro groups.
  • uncaging of a reactive group results in a free amine, sulfide, or other reactive group.
  • a conjugation reaction described herein comprises uncaging of a reactive group with a transition metal catalyst, followed by a conjugation reaction.
  • the transition metal catalyst comprises palladium and one or more ligands.
  • a reactive group is protected with an allyl moiety.
  • a reactive group is protected with an allylic carbamate.
  • a reactive group is protected with a propargylic moiety.
  • a reactive group is protected with a propargyl carbamate.
  • a reactive group is protected with a dienophile, wherein exposure to a diene (such as a tetrazine) results in deprotection of the reactive group.
  • a conjugation reaction described herein comprises a ligand-directed reaction, wherein a ligand (optionally) attached to a reactive group) facilitates the site of conjugation between the reactive group and the cytokine peptide.
  • the ligand is cleaved during or after reaction of the cytokine peptide with the reactive group.
  • the conjugation site of the cytokine peptide is a natural amino acid.
  • the conjugation site of the cytokine peptide is a lysine, cysteine, or serine.
  • the conjugation site of the cytokine peptide is an unnatural amino acid described herein.
  • the reactive group comprises a leaving group, such as an electron-poor aryl or heteroaryl group. In some embodiments the reactive group comprises a leaving group, such as an electron-poor alkyl group that is displaced by the cytokine peptide.
  • a conjugation reaction described herein comprises reaction of a radical trapping agent with a radical species. In some embodiments, a conjugation reaction described herein comprises an oxidative radical addition reaction. In some embodiments, a radical trapping agent is an arylamine. In some embodiments, a radical species is a tyrosyl radical. In some embodiments, radical species are generated by a ruthenium catalyst (such as [Ru(bpy) 3 ]) and light.
  • Enzymatic reactions are optionally used for conjugation reactions described herein.
  • Exemplary enzymatic conjugations include SortA-mediated conjugation, a TGs-mediated conjugation, or an FGE-mediated conjugation.
  • a conjugation reaction described herein comprises native protein ligation (NPL) of a terminal 1-amino-2-thio group with a thioester to form an amide bond.
  • conjugation reactions are described herein for reacting a linker or conjugating moiety with a cytokine peptide, wherein the reaction occurs with a natural (“canonical”) amino acid in the cytokine peptide.
  • the natural amino acid is found at a conjugation position is found in a wild type sequence, or alternatively the position has been mutated.
  • a conjugation reaction comprises formation of a disulfide bond at a cysteine residue.
  • a conjugation reaction comprises a 1,4 Michael addition reaction of a cysteine or lysine.
  • a conjugation reaction comprises a cyanobenzothiazole ligation of a cysteine.
  • a conjugation reaction comprises crosslinking with an acetone moiety, such as 1,3-dichloro-2-propionone.
  • a conjugation reaction comprises a 1,4 Michael addition to a dehydroalanine, formed by reaction of cysteine with O-mesitylenesulfonylhydroxylamine.
  • a conjugation reaction comprises reaction of a tyrosine with a triazolinedione (TAD), or TAD derivative.
  • TAD triazolinedione
  • a conjugation reaction comprises reaction of a tryptophan with a rhodium carbenoid.
  • a method of treating a proliferative disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein.
  • a cytokine conjugate e.g., an IL-2 conjugate
  • the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugating moiety, wherein the IL-2 conjugate has a decreased affinity to an IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to a wild-type IL-2 polypeptide.
  • the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO: 1.
  • the IL-2 conjugate preferentially interact with the IL-2R3 and IL-2R ⁇ subunits to form a IL-2/IL-2R ⁇ complex.
  • the IL-2/IL-2R ⁇ complex stimulates and/or enhances expansion of CD4+ helper cells, CD8+ effector na ⁇ ve and memory T cells, NK cells, and/or NKT cells.
  • the expansion of Teff cells skews the Teff:Treg ratio toward the Teff population.
  • the proliferative disease or condition is a cancer.
  • the cancer is a solid tumor.
  • Exemplary solid tumors include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.
  • the solid tumor is a metastatic cancer.
  • the solid tumor is a relapsed or refractory cancer.
  • a cytokine (e.g., interleukin, IFN, or TNF) conjugate described herein is administered to a subject in need thereof, for treating a solid tumor.
  • the subject has bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.
  • the solid tumor is a metastatic cancer.
  • the solid tumor is a relapsed or refractory cancer.
  • an IL-2 conjugate described herein is administered to a subject in need thereof, for treating a solid tumor.
  • the subject has a bladder cancer, a bone cancer, a brain cancer, a breast cancer, a colorectal cancer, an esophageal cancer, an eye cancer, a head and neck cancer, a kidney cancer, a lung cancer, a melanoma, an ovarian cancer, a pancreatic cancer, or a prostate cancer.
  • the IL-2 conjugate is administered to a subject for the treatment of a bladder cancer.
  • the IL-2 conjugate is administered to a subject for the treatment of a breast cancer.
  • the IL-2 conjugate is administered to a subject for the treatment of a colorectal cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of an esophageal cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a head and neck cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a kidney cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a lung cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a melanoma. In some cases, the IL-2 conjugate is administered to a subject for the treatment of an ovarian cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a pancreatic cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a prostate cancer.
  • the IL-2 conjugate is administered to a subject for the treatment of a metastatic cancer.
  • the metastatic cancer comprises a metastatic bladder cancer, metastatic bone cancer, metastatic brain cancer, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic eye cancer, metastatic head and neck cancer, metastatic kidney cancer, metastatic lung cancer, metastatic melanoma, metastatic ovarian cancer, metastatic pancreatic cancer, or metastatic prostate cancer.
  • the IL-2 conjugate is administered to a subject for the treatment of metastatic bladder cancer, metastatic bone cancer, metastatic brain cancer, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic eye cancer, metastatic head and neck cancer, metastatic kidney cancer, metastatic lung cancer, metastatic melanoma, metastatic ovarian cancer, metastatic pancreatic cancer, or metastatic prostate cancer.
  • the IL-2 conjugate is administered to a subject for the treatment of a relapsed or refractory cancer.
  • the relapsed or refractory cancer comprises a relapsed or refractory bladder cancer, relapsed or refractory bone cancer, relapsed or refractory brain cancer, relapsed or refractory breast cancer, relapsed or refractory colorectal cancer, relapsed or refractory esophageal cancer, relapsed or refractory eye cancer, relapsed or refractory head and neck cancer, relapsed or refractory kidney cancer, relapsed or refractory lung cancer, relapsed or refractory melanoma, relapsed or refractory ovarian cancer, relapsed or refractory pancreatic cancer, or relapsed or refractory prostate cancer.
  • the IL-2 conjugate is administered to a subject for the treatment of a relapsed or refractory bladder cancer, relapsed or refractory bone cancer, relapsed or refractory brain cancer, relapsed or refractory breast cancer, relapsed or refractory colorectal cancer, relapsed or refractory esophageal cancer, relapsed or refractory eye cancer, relapsed or refractory head and neck cancer, relapsed or refractory kidney cancer, relapsed or refractory lung cancer, relapsed or refractory melanoma, relapsed or refractory ovarian cancer, relapsed or refractory pancreatic cancer, or relapsed or refractory prostate cancer.
  • a relapsed or refractory bladder cancer relapsed or refractory bone cancer, relapsed or refractory brain cancer, relapsed or refractory breast cancer
  • the cancer is a treatment-na ⁇ ve cancer.
  • the treatment-na ⁇ ve cancer is a cancer that has not been treated by a therapy.
  • the treatment-na ⁇ ve cancer is a solid tumor, such as bladder cancer, a bone cancer, a brain cancer, a breast cancer, a colorectal cancer, an esophageal cancer, an eye cancer, a head and neck cancer, a kidney cancer, a lung cancer, a melanoma, an ovarian cancer, a pancreatic cancer, or a prostate cancer.
  • described herein is a method of treating a treatment-na ⁇ ve solid tumor in a subject in need thereof which comprises administering to the subject a cytokine conjugate (e.g., an IL-2 conjugate) described herein.
  • a cytokine conjugate e.g., an IL-2 conjugate
  • the cancer is a hematologic malignancy.
  • the hematologic malignancy comprises a leukemia, a lymphoma, or a myeloma.
  • the hematologic malignancy is a T-cell malignancy.
  • the hematological malignancy is a B-cell malignancy.
  • Exemplary hematologic malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,
  • the hematologic malignancy is a metastatic cancer.
  • the metastatic cancer is a metastatic T-cell malignancy or a metastatic B-cell malignancy.
  • the hematologic malignancy is a relapsed or refractory cancer.
  • the relapsed or refractory cancer is a relapsed or refractory T-cell malignancy or a relapsed or refractory B-cell malignancy.
  • a cytokine e.g., interleukin, IFN, or TNF
  • a cytokine described herein is administered to a subject in need thereof, for treating a hematologic malignancy.
  • the subject has a T-cell malignancy.
  • the subject has a B-cell malignancy.
  • the subject has chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary
  • an IL-2 conjugate described herein is administered to a subject in need thereof, for treating a hematologic malignancy.
  • the subject has a T-cell malignancy.
  • the subject has a B-cell malignancy.
  • the subject has chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary
  • the IL-2 conjugate is administered to a subject for the treatment of CLL. In some cases, the IL-2 conjugate is administered to a subject for the treatment of SLL. In some cases, the IL-2 conjugate is administered to a subject for the treatment of FL. In some cases, the IL-2 conjugate is administered to a subject for the treatment of DLBCL. In some cases, the IL-2 conjugate is administered to a subject for the treatment of MCL. In some cases, the IL-2 conjugate is administered to a subject for the treatment of Waldenstrom's macroglobulinemia. In some cases, the IL-2 conjugate is administered to a subject for the treatment of multiple myeloma. In some cases, the IL-2 conjugate is administered to a subject for the treatment of Burkitt's lymphoma.
  • the IL-2 conjugate is administered to a subject for the treatment of a metastatic hematologic malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a metastatic T-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a metastatic B-cell malignancy.
  • the IL-2 conjugate is administered to a subject for the treatment of a metastatic chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B
  • the IL-2 conjugate is administered to a subject for the treatment of a relapsed or refractory hematologic malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a relapsed or refractory T-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of a relapsed or refractory B-cell malignancy.
  • the IL-2 conjugate is administered to a subject for the treatment of a relapsed or refractory chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal
  • an additional therapeutic agent is further administered to the subject.
  • the additional therapeutic agent is administered simultaneously with a cytokine conjugate (e.g., an IL-2 conjugate).
  • the additional therapeutic agent and the IL-2 conjugate are administered sequentially, e.g., the cytokine conjugate (e.g., IL-2 conjugate) is administered prior to the additional therapeutic agent or that the cytokine conjugate (e.g., IL-2 conjugate) is administered after administration of the additional therapeutic agent.
  • the additional therapeutic agent comprises a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, radiation therapy, or a combination thereof.
  • additional therapeutic agents include, but are not limited to, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as daunorubicin, doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors such as topotecan
  • the additional therapeutic agent comprises a first-line therapy.
  • first-line therapy comprises a primary treatment for a subject with a cancer.
  • the cancer is a primary cancer.
  • the cancer is a metastatic or recurrent cancer.
  • the first-line therapy comprises chemotherapy.
  • the first-line treatment comprises radiation therapy.
  • a cytokine conjugate (e.g., IL-2 conjugate) is administered with an additional therapeutic agent selected from an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; an antimetabolite such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; an anthracycline such as daunorubicin, doxorubicin, epirubicin, or idarubicin; a topoisomerase I inhibitor such as topotecan or irinotecan (CPT-11); a topoisome
  • a cytokine conjugate (e.g., IL-2 conjugate) described herein is administered with an inhibitor of the enzyme poly ADP ribose polymerase (PARP).
  • PARP inhibitors include, but are not limited to, olaparib (AZD-2281, Lynparza®, from Astra Zeneca), rucaparib (PF-01367338, Rubraca®, from Clovis Oncology), niraparib (MK-4827, Zejula®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from AbbVie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene).
  • the cytokine conjugate (e.g., IL-2 conjugate) is administered in combination with a PARP inhibitor such as olaparib, rucaparib, niraparib, talazoparib, veliparib, CK-102, E7016, iniparib, or pamiparib.
  • a PARP inhibitor such as olaparib, rucaparib, niraparib, talazoparib, veliparib, CK-102, E7016, iniparib, or pamiparib.
  • a cytokine conjugate e.g., IL-2 conjugate
  • an immune checkpoint inhibitor e.g., IL-2 conjugate
  • exemplary checkpoint inhibitors include:
  • PD-L1 inhibitors such as Genentech's MPDL3280A (RG7446), Anti-mouse PD-L1 antibody Clone 10F.9G2 (Cat # BE0101) from BioXcell, anti-PD-L1 monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol-Meyer's Squibb, MSB0010718C, mouse anti-PD-L1 Clone 29E.2A3, and AstraZeneca's MEDI4736;
  • PD-L2 inhibitors such as GlaxoSmithKline's AMP-224 (Amplimmune), and rHIgM 12B7;
  • PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat # BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMPI-14 (Cat # BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck's MK-3475 anti-mouse PD-1 antibody (Keytruda, pembrolizumab, lambrolizumab), AnaptysBio's anti-PD-1 antibody known as ANB011, antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224, and Pidilizumab (CT-011) from CureTech Ltd;
  • CTLA-4 inhibitors such as Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101), anti-CTLA4 antibody clone 9H10 from Millipore, Pfizer's tremelimumab (CP-675,206, ticilimumab), and anti-CTLA4 antibody clone BNI3 from Abcam;
  • CTLA-4 inhibitors such as Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101), anti-CTLA4 antibody clone 9H10 from Millipore, Pfizer's tremelimumab (CP-675,206, ticilimumab), and anti-CTLA4 antibody clone BNI3 from Abcam;
  • LAG3 inhibitors such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences, IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS-986016, and the LAG-3 chimeric antibody A9H12;
  • B7-H3 inhibitors such as MGA271;
  • KIR inhibitors such as Lirilumab (IPH2101);
  • CD137 inhibitors such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);
  • PS inhibitors such as Bavituximab
  • inhibitors such as an antibody or fragments (e.g., a monoclonal antibody, a human, humanized, or chimeric antibody) thereof, RNAi molecules, or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.
  • an antibody or fragments e.g., a monoclonal antibody, a human, humanized, or chimeric antibody
  • RNAi molecules e.g., RNAi molecules, or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.
  • the cytokine conjugate (e.g., IL-2 conjugate) is administered in combination with pembrolizumab, nivolumab, tremelimumab, or ipilimumab.
  • a cytokine conjugate (e.g., IL-2 conjugate) described herein is administered with an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab.
  • an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab.
  • a cytokine conjugate (e.g., IL-2 conjugate) is administered with an additional therapeutic agent selected from an additional cytokine.
  • the additional cytokine enhances and/or synergizes T effector cell expansion and/or proliferation.
  • the additional cytokine comprises IL-13, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNF ⁇ .
  • the additional cytokine is IL-7.
  • the additional cytokine is IL-15.
  • the additional cytokine is IL-21.
  • the additional cytokine is TNF ⁇ .
  • a cytokine conjugate (e.g., IL-2 conjugate) is administered with an additional therapeutic agent selected from a receptor agonist.
  • the receptor agonist comprises a Toll-like receptor (TLR) ligand.
  • TLR Toll-like receptor
  • the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • the TLR ligand comprises a synthetic ligand such as, for example, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF20, CFA, or Flagellin.
  • the cytokine conjugate (e.g., IL-2 conjugate) is administered with one or more TLR agonists selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.
  • the cytokine conjugate (e.g., IL-2 conjugate) is administered with one or more TLR agonists selected from Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF20, CFA, and Flagellin.
  • a cytokine conjugate (e.g., IL-2 conjugate) is used in conjunction with an adoptive T cell transfer (ACT) therapy.
  • ACT involves identification of autologous T lymphocytes in a subject with, e.g., anti-tumor activity, expansion of the autologous T lymphocytes in vitro, and subsequent reinfusion of the expanded T lymphocytes into the subject.
  • ACT comprises use of allogeneic T lymphocytes with, e.g., anti-tumor activity, expansion of the T lymphocytes in vitro, and subseqent infusion of the expanded allogeneic T lymphocytes into a subject in need thereof.
  • a cytokine conjugate (e.g., IL-2 conjugate) described herein is used in conjunction with an autologous T lymphocytes as part of an ACT therapy.
  • a cytokine conjugate (e.g., IL-2 conjugate) described herein is used in conjunction with an allogeneic T lymphocytes as part of an ACT therapy.
  • the cytokine conjugate (e.g., IL-2 conjugate) is administered simultaneously with the ACT therapy to a subject in need thereof.
  • the cytokine conjugate (e.g., IL-2 conjugate) is administered sequentially with the ACT therapy to a subject in need thereof.
  • a cytokine conjugate (e.g., IL-2 conjugate) is used for an ex vivo activation and/or expansion of an autologous and/or allogenic T cell transfer.
  • the cytokine conjugate e.g., IL-2 conjugate
  • the cytokine conjugate is used to activate and/or expand a sample comprising autologous and/or allogenic T cells and the cytokine conjugate (e.g., IL-2 conjugate) is optionally removed from the sample prior to administering the sample to a subject in need thereof.
  • a cytokine conjugate (e.g., IL-2 conjugate) is administered with a vaccine.
  • a cytokine conjugate e.g., IL-2 conjugate
  • an oncolytic virus e.g., IL-2 conjugate
  • the cytokine conjugate acts as a stimulatory agent to modulate the immune response.
  • the cytokine conjugate e.g., IL-2 conjugate
  • Exemplary oncolytic viruses include T-Vec (Amgen), G47 ⁇ (Todo et al.), JX-594 (Sillajen), CG0070 (Cold Genesys), and Reolysin (Oncolytics Biotech).
  • the cytokine conjugate e.g., IL-2 conjugate
  • an oncolytic virus such as T-Vec, G47A, JX-594, CG0070, or Reolysin.
  • a cytokine conjugate e.g., IL-2 conjugate
  • a radiation therapy is administered in combination with a radiation therapy.
  • a cytokine conjugate (e.g., IL-2 conjugate) is administered in combination with surgery.
  • a method of treating a pathogenic infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein.
  • a cytokine conjugate e.g., an IL-2 conjugate
  • the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugating moiety, wherein the IL-2 conjugate has a decreased affinity to an IL-2 receptor ⁇ (IL-2R ⁇ ) subunit relative to a wild-type IL-2 polypeptide.
  • the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO: 1.
  • the IL-2 conjugate preferentially interact with the IL-2R3 and IL-2R ⁇ subunits to form a IL-2/IL-2R ⁇ complex, which stimulates and/or enhances expansion of CD4+ helper cells, CD8+ effector na ⁇ ve and memory cells, NK cells, and/or NKT cells.
  • the IL-2 conjugate facilitates recognition of pathogenic reservoir by CD8+ T-cells.
  • the pathogenic infection is a viral infection, in which upon treatment with an antiviral therapy, a viral reservoir (e.g., resting CD4+ T cells) persists in a treated host.
  • a viral reservoir e.g., resting CD4+ T cells
  • a cytokine conjugate e.g., an IL-2 conjugate
  • the cytokine conjugate is utilized as a monotherapy to redirect CD8+ T cells to infected resting cells for elimination.
  • the cytokine conjugate (e.g., IL-2 conjugate) is utilized in combination with an additional therapy to redirect CD8+ T cells to infected resting cells for elimination.
  • additional therapy comprises antiviral treatments such as acyclovir, brivudine, docosanol, famciclovir, foscarnet, idoxuridine, penciclovir, trifluridine, valacyclovir, and pritelivir.
  • the virus is a DNA virus or an RNA virus.
  • the DNA viruses include single-stranded (ss) DNA viruses, double-stranded (ds) DNA viruses, or DNA viruses that contain both ss and ds DNA regions.
  • the RNA viruses include single-stranded (ss) RNA viruses or double-stranded (ds) RNA viruses. In some cases, the ssRNA viruses are further classified into positive-sense RNA viruses or negative-sense RNA viruses.
  • Exemplary dsDNA viruses include viruses from the family: Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae, Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfaviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae, Mimiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae, Poxviridae, Sphaerolipovi
  • Exemplary ssDNA viruses include viruses from the family: Anelloviridae, Bacillariodnaviridae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, and Spiraviridae.
  • Exemplary DNA viruses that contain both ss and ds DNA regions include viruses from the group of pleolipoviruses.
  • the pleolipoviruses include Haloarcula hispanica pleomorphic virus 1, Halogeometricum pleomorphic virus 1, Halorubrum pleomorphic virus 1, Halorubrum pleomorphic virus 2, Halorubrum pleomorphic virus 3, and Halorubrum pleomorphic virus 6.
  • Exemplary dsRNA viruses include viruses from the family: Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megavirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Rotavirus, and Totiviridae.
  • Exemplary positive-sense ssRNA viruses include viruses from the family: Alphaflexiviridae, Alphatetraviridae, Alvernaviridae, Arteriviridae, Astroviridae, Barnaviridae, Betaflexiviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Coronaviridae, Dicistroviridae, Flaviviridae, Gammaflexiviridae, Iflaviridae, Leviviridae, Luteoviridae, Marnaviridae, Mesoniviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Picornaviridae, Potyviridae, Roniviridae, Retroviridae, Secoviridae, Togaviridae, Tombusviridae, Tymoviridae, and Virgaviridae.
  • Exemplary negative-sense ssRNA viruses include viruses from the family: Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Nyamiviridae, Ophioviridae, Orthomyxoviridae, Paramyxoviridae, and Rhabdoviridae.
  • the pathogenic infection is caused by Abelson leukemia virus, Abelson murine leukemia virus, Abelson's virus, Acute laryngotracheobronchitis virus, Sydney River virus, Adeno associated virus group, Adenovirus, African horse sickness virus, African swine fever virus, AIDS virus, Aleutian mink disease parvovirus, Alpharetrovirus, Alphavirus, ALV related virus, Amapari virus, Aphthovirus, Aquareovirus, Arbovirus.
  • Arbovirus C arbovirus group A, arbovirus group B, Arenavirus group, Argentine hemorrhagic fever virus, Argentine hemorrhagic fever virus, Arterivirus, Astrovirus, Ateline herpesvirus group, Aujezky's disease virus, Aura virus, Ausduk disease virus, Australian bat lyssavirus, Aviadenovirus, avian erythroblastosis virus, avian infectious bronchitis virus, avian leukemia virus, avian leukosis virus, avian lymphomatosis virus, avian myeloblastosis virus, avian paramyxovirus, avian pneumoencephalitis virus, avian reticuloendotheliosis virus, avian sarcoma virus, avian type C retrovirus group, Avihepadnavirus, Avipoxvirus, B virus, B19 virus, Babanki virus, baboon herpesvirus, baculovirus,
  • the pathogenic infection is caused by a retrovirus.
  • retroviruses include, but are not limited to, human immunodefiency virus (HIV), human T-cell leukemia viruses (HTLV), moloney murine leukemia virus (MuLV), murine mammary tumor virus (MMTV), avian leucosis and sarcoma viruses, or Mason-Pfizer monkey virus.
  • a cytokine conjugate (e.g., an IL-2 conjugate) described herein is administered to a subject with a retroviral infection or during a latency period to reduce and/or eliminate infected cells that are in a resting period.
  • the retrovirus comprises human immunodefiency virus (HIV), human T-cell leukemia viruses (HTLV), moloney murine leukemia virus (MuLV), murine mammary tumor virus (MMTV), avian leucosis and sarcoma viruses, or Mason-Pfizer monkey virus.
  • the cytokine conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a resting period.
  • the cytokine conjugate is an 1L-2 conjugate.
  • the 1L-2 conjugate is administered to a subject with a retroviral infection or during a latency period to reduce and/or eliminate infected cells that are in a resting period.
  • the retrovirus comprises human immunodefiency virus (HIV), human T-cell leukemia viruses (HTLV), moloney murine leukemia virus (MuLV), murine mammary tumor virus (MMTV), avian leucosis and sarcoma viruses, or Mason-Pfizer monkey virus.
  • the IL-2 conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a resting period.
  • the IL-2 conjugate is administered to the subject in combination with an antiretroviral therapy.
  • the retrovirus is HIV.
  • a cytokine conjugate e.g., an IL-2 conjugate described herein is administered to a subject having acquired immune deficiency syndrome (AIDS) or during a latency period to reduce and/or eliminate HIV-infected cells (e.g., CD4+ T cells) that are in a resting period.
  • the cytokine conjugate is an IL-2 conjugate.
  • the IL-2 conjugate is administered to the subject in combination with an antiretroviral therapy.
  • Exemplary HIV antiretroviral therapy includes:
  • NRTIs nucleoside reverse transcriptase inhibitors
  • abacavir emtricitabine
  • lamivudine tenofovir disoproxil fumarate
  • zidovudine zidovudine
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • efavirenz etravirine
  • nevirapine nevirapine
  • rilpivirine non-nucleoside reverse transcriptase inhibitors
  • protease inhibitors such as atazanavir, darunavir, fosamprenavir, ritonavir, saquinavir, and tipranavir;
  • fusion inhibitors such as enfuvirtide
  • CCR5 antagonists such as maraviroc
  • integrase inhibitors such as dolutegravir and raltegravir
  • post-attachment inhibitors such as ibalizumab
  • abacavir and lamivudine such as abacavir and lamivudine; abacavir, dolutegravir, and lamivudine; abacavir, lamivudine, and zidovudine; atazanavir and cobicistat; bictegravir, emtricitabine, and tenofovir alafenamide; darunavir and cobicistat; dolutegravir and rilpivirine; efavirenz, emtricitabine, and tenofovir disoproxil fumarate; efavirenz, lamivudine, and tenofovir disoproxil fumarate; efavirenz, lamivudine, and tenofovir disoproxil fumarate; elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide fumarate; elvit
  • the IL-2 conjugate is administered to the subject in combination with an antiretroviral therapy such as nucleoside reverse transcriptase inhibitors (NRTIs) such as abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, and zidovudine; non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as efavirenz, etravirine, nevirapine, or rilpivirine; protease inhibitors (PIs) such as atazanavir, darunavir, fosamprenavir, ritonavir, saquinavir, and tipranavir; fusion inhibitors such as enfuvirtide; CCR5 antagonists such as maraviroc; integrase inhibitors such as dolutegravir and raltegravir; post-attachment inhibitors such as ibalizumab; pharmacokinetic enhancers
  • the virus is a hepatitis virus, e.g., hepatitis A, B, C, D, or E.
  • a cytokine conjugate e.g., an IL-2 conjugate
  • the cytokine conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a resting period.
  • the cytokine conjugate is an IL-2 conjugate.
  • the IL-2 conjugate is administered to a subject with a hepatitis infection or during a latency period to reduce and/or eliminate infected cells that are in a resting period.
  • the IL-2 conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a resting period.
  • the IL-2 conjugate is administered to the subject in combination with an antiviral therapy.
  • Exemplary antiviral therapy for hepatitis include ribavirin; NS3/4A protease inhibitors such as paritaprevir, simeprevir, and grazoprevir; NS5A protease inhibitors such as ledipasvir, ombitasvir, elbasvir, and daclatasvir; NS5B nucleotide/nucleoside and nonnucleoside polymerase inhibitors such as sofosbuvir and dasabuvir; and combinations such as ledipasvir-sofosbuvir, dasabuvir-ombitasvir-paritaprevir-ritonavir; elbasvir-grazoprevir, ombitasvir-paritaprevir-ritonavir, sofosbuvir-velpatasvir, sofosbuvir-velpatasvir-voxilaprevir, and glecaprevir-pibrentasvir; and interferons such as peg
  • e IL-2 conjugate is administered to the subject in combination with an antiviral therapy such as ribavirin; NS3/4A protease inhibitors such as paritaprevir, simeprevir, and grazoprevir; NS5A protease inhibitors such as ledipasvir, ombitasvir, elbasvir, and daclatasvir; NS5B nucleotide/nucleoside and nonnucleoside polymerase inhibitors such as sofosbuvir and dasabuvir; and combinations such as ledipasvir-sofosbuvir, dasabuvir-ombitasvir-paritaprevir-ritonavir; elbasvir-grazoprevir, ombitasvir-paritaprevir-ritonavir, sofosbuvir-velpatasvir, sofosbuvir-velpatasvir-voxilaprevir, and glecaprevir-pibrenta
  • lymphocyte populations e.g., CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, NK cell, and/or NKT cell populations, or methods of expanding a Treg cell population.
  • the method comprises contacting a cell with a cytokine conjugate described herein and interacting the cytokine with a cytokine receptor to form a complex, wherein the complex stimulates expansion of a distinct lymphocyte population.
  • the method of expanding a CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, Natural Killer (NK) cell, or Natural killer T (NKT) cell population comprises contacting a cell population with an isolated and modified IL-2 polypeptide described above for a time sufficient to induce formation of a complex with an IL-2R ⁇ , thereby stimulating the expansion of the Teff and/or NK cell population.
  • the method of expanding CD4+ helper cell, CD8+ effector na ⁇ ve and memory cell, NK cell, and/or NKT cell populations comprises (a) contacting a cell population with an IL-2 conjugate described herein; and (b) interacting the IL-2 with IL-2R3 and IL-2R ⁇ subunits to form an IL-2/IL-2R ⁇ complex; wherein the IL-2 conjugate has a decreased affinity to IL-2R ⁇ subunit, and wherein the IL-2/IL-2R ⁇ complex stimulates the expansion of CD4+ helper cells, CD8+ effector na ⁇ ve and memory cells, NK cells, and/or NKT cells.
  • the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO: 1.
  • the amino acid position is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107.
  • the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some instances, the amino acid position is selected from R38 and K64. In some instances, the amino acid position is selected from E61, E62, and E68. In some cases, the amino acid position is at E62.
  • the IL-2 conjugate expands CD4+T regulatory (Treg) cells by less than 20%, 15%, 10%, 5%, or 1% in the cell population. In some instances, the IL-2 conjugate does not expand CD4+ Treg cells in the cell population. In some instances, the ratio of the Teff cells to Treg cells in the cell population after incubation with the isolated and modified IL-2 polypeptide is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1.
  • the ratio of the Teff cells to Treg cells in the cell population after incubation with the isolated and modified IL-2 polypeptide is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1.
  • the time sufficient to induce formation of a complex with an IL-2R3 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 instances, the time sufficient to induce formation of a complex with an IL-2R3 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.
  • the method is an in vivo method.
  • the method is an in vitro method.
  • the method is an ex vivo method.
  • the cytokine (e.g., interleukin, IFN, or TNF) polypeptides described herein, either containing a natural amino acid mutation or an unnatural amino acid mutation, are generated recombinantly or are synthesized chemically.
  • the cytokine (e.g., IL-2) polypeptides described herein are generated recombinantly, for example, either by a host cell system, or in a cell-free system.
  • the cytokine (e.g., L-2) polypeptides are generated recombinantly through a host cell system.
  • the host cell is a eukaryotic cell (e.g., mammalian cell, insect cells, yeast cells or plant cell) or a prokaryotic cell (e.g., gram-positive bacterium or a gram-negative bacterium).
  • a eukaryotic host cell is a mammalian host cell.
  • a mammalian host cell is a stable cell line, or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division.
  • a mammalian host cell is a transient cell line, or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division.
  • Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293 H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293FTM cells, Flp-InTM T-RExTM 293 cell line, Flp-InTM-293 cell line, Flp-InTM-3T3 cell line, Flp-InTM-BHK cell line, Flp-InTM-CHO cell line, Flp-InTM-CV-1 cell line, Flp-InTM-Jurkat cell line, FreeStyleTM 293-F cells, FreeStyleTM CHO-S cells, GripTiteTM 293 MSR cell line, GS-CHO cell line, HepaRGTM cells, T-RExTM Jurkat cell line, Per.C6 cells, T-RExTM-293 cell line, T-RExTM-CHO cell line, and T-RExTM-HeLa cell line.
  • an eukaryotic host cell is an insect host cell.
  • exemplary insect host cell include Drosophila S2 cells, Sf9 cells, Sf21 cells, High FiveTM cells, and expresSF+® cells.
  • a eukaryotic host cell is a yeast host cell.
  • yeast host cells include Pichia pastoris yeast strains such as GS 115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strain such as INVScl.
  • an eukaryotic host cell is a plant host cell.
  • the plant cells comprise a cell from algae.
  • Exemplary plant cell lines include strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.
  • a host cell is a prokaryotic host cell.
  • prokaryotic host cells include BL21, Mach1TM, DH10BTM, TOP10, DH5a, DH10BacTM, OmniMaxTM, MegaXTM, DH12STM, INV110, TOP10OF′, INV ⁇ F, TOP10/P3, ccdB Survival, PIR1, PIR2, Stb12TM, Stb13TM, or Stb14TM.
  • suitable polynucleic acid molecules or vectors for the production of an IL-2 polypeptide described herein include any suitable vectors derived from either a eukaryotic or prokaryotic source.
  • Exemplary polynucleic acid molecules or vectors include vectors from bacteria (e.g., E. coli ), insects, yeast (e.g., Pichiapastoris), algae, or mammalian source.
  • Bacterial vectors include, for example, pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.
  • Insect vectors include, for example, pFastBacl, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.
  • FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2
  • MAT vectors such as pPolh-MAT1, or pPolh-MAT2.
  • Yeast vectors include, for example, Gateway® pDESTTM 14 vector, Gateway® pDESTTM 15 vector, Gateway® pDESTTM 17 vector, Gateway® pDESTTM 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA, B, & C Pichiapastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF 1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.
  • Algae vectors include, for example, pChlamy-4 vector or MCS vector.
  • Mammalian vectors include, for example, transient expression vectors or stable expression vectors.
  • Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4.
  • Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.
  • a cell-free system is used for the production of a cytokine (e.g., IL-2) polypeptide described herein.
  • a cell-free system comprises a mixture of cytoplasmic and/or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis.
  • a cell-free system utilizes prokaryotic cell components.
  • a cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis is obtained in a cell-free system based on, for example, Drosophila cell, Xenopus egg, Archaea, or HeLa cells.
  • Exemplary cell-free systems include E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.
  • Cell-free translation systems variously comprise components such as plasmids, mRNA, DNA, tRNAs, 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 yields, increase synthesis rate, increase protein product fidelity, or incorporate unnatural amino acids.
  • cytokines described herein are synthesized using cell-free translation systems described in U.S. Pat. No. 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or U.S. Pat. No. 8,778,631.
  • cell-free translation systems comprise modified release factors, or even removal of one or more release factors from the system.
  • cell-free translation systems comprise a reduced protease concentration.
  • cell-free translation systems comprise modified tRNAs with re-assigned codons used to code for unnatural amino acids.
  • the synthetases described herein for the incorporation of unnatural amino acids are used in cell-free translation systems.
  • tRNAs are pre-loaded with unnatural amino acids using enzymatic or chemical methods before being added to a cell-free translation system.
  • components for a cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organism.
  • a cytokine e.g., IL-2
  • IL-2 cytokine-2
  • a cytokine e.g., IL-2
  • IL-2 IL-2
  • a circularly permuted form either via an expression host system or through a cell-free system.
  • An orthogonal or expanded genetic code can be used in the present disclosure, in which one or more specific codons present in the nucleic acid sequence of a cytokine (e.g., IL-2) polypeptide are allocated to encode the unnatural amino acid so that it can be genetically incorporated into the cytokine (e.g., IL-2) by using an orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA synthetase/tRNA pair is capable of charging a tRNA with an unnatural amino acid and is capable of incorporating that unnatural amino acid into the polypeptide chain in response to the codon.
  • the codon is the codon amber, ochre, opal or a quadruplet codon. In some cases, the codon corresponds to the orthogonal tRNA which will be used to carry the unnatural amino acid. In some cases, the codon is amber. In other cases, the codon is an orthogonal codon.
  • the codon is a quadruplet codon, which can be decoded by an orthogonal ribosome ribo-Q1.
  • the quadruplet codon is as illustrated in Neumann, et al., “Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome,” Nature, 464(7287): 441-444 (2010).
  • a codon used in the present disclosure is a recoded codon, e.g., a synonymous codon or a rare codon that is replaced with alternative codon.
  • the recoded codon is 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).
  • the recoded codon is as described in Ostrov et al., “Design, synthesis, and testing toward a 57-codon genome,” Science 353(6301): 819-822 (2016).
  • unnatural nucleic acids are utilized leading to incorporation of one or more unnatural amino acids into the cytokine (e.g., IL-2).
  • exemplary unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-
  • 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, those that 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 O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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—CH 3 ) uracil, 5-propynyl cytosine, 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 and guanines, 5-
  • nucleic acids comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acids in some cases include one or several heterocyclic bases other than the principal five base components of naturally-occurring nucleic acids.
  • the heterocyclic base includes, in some cases, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo [2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrolo [2, 3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo [2.3-d]pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the nucleic acid via the 9-position, the pyrimidines via the 1-position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1-position.
  • nucleotide analogs are also modified at the phosphate moiety.
  • Modified phosphate moieties include, but are not limited to, those with modification at the linkage between two nucleotides and contains, for example, a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3′-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkage between two nucleotides are through a 3′-5′ linkage or a 2′-5′ linkage, and the linkage contains inverted 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.
  • nucleotides containing modified phosphates include but are not limited to, 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; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.
  • unnatural 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.
  • unnatural nucleic acids include modifications at the 5′-position and the 2′-position of the sugar ring (PCT/US94/02993), such as 5′-CH 2 -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).
  • unnatural nucleic acids include amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3′ linked nucleoside in the dimer (5′ to 3′) comprises a 2′-OCH 3 and a 5′-(S)—CH 3 (Mesmaeker et al., Synlett, 1997, 1287-1290).
  • Unnatural nucleic acids can include 2′-substituted 5′-CH 2 (or O) modified nucleosides (PCT/US92/01020).
  • Unnatural nucleic acids can include 5′-methylenephosphonate DNA and RNA monomers, and dimers (Bohringer et al., Tet.
  • Unnatural nucleic acids can include 5′-phosphonate monomers having a 2′-substitution (US2006/0074035) and other modified 5′-phosphonate monomers (WO1997/35869).
  • Unnatural nucleic acids can include 5′-modified methylenephosphonate monomers (EP614907 and EP629633).
  • Unnatural nucleic acids can include analogs of 5′ or 6′-phosphonate ribonucleosides comprising a hydroxyl group at the 5′ and/or 6′-position (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).
  • Unnatural nucleic acids can include 5′-phosphonate deoxyribonucleoside monomers and dimers having a 5′-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82).
  • Unnatural nucleic acids can include nucleosides having a 6′-phosphonate group wherein the 5′ or/and 6′-position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH 3 ) 3 ) (and analogs thereof); a methyleneamino group (CH 2 NH 2 ) (and analogs thereof) or a cyano group (CN) (and analogs thereof) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med.
  • unnatural nucleic acids also include modifications of the sugar moiety.
  • nucleic acids contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property.
  • nucleic acids comprise a chemically modified ribofuranose ring moiety.
  • Examples of chemically modified ribofuranose rings include, without limitation, addition of substitutent groups (including 5′ and/or 2′ substituent groups; bridging of two ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R 1 )(R 2 ) (R ⁇ H, C 1 -C 12 alkyl or a protecting group); and combinations thereof.
  • Examples of chemically modified sugars can be found in WO2008/101157, US2005/0130923, and WO2007/134181.
  • a modified nucleic acid comprises modified sugars or sugar analogs.
  • the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group.
  • the sugar can be in a pyranosyl or furanosyl form.
  • the sugar moiety may be the furanoside of ribose, deoxyribose, arabinose or 2′-O-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] 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.
  • a sugar modification may include 2′-O-methyl-uridine or 2′-O-methyl-cytidine.
  • Sugar modifications include 2′-O-alkyl-substituted deoxyribonucleosides and 2′-O-ethyleneglycol like ribonucleosides.
  • the preparation of these sugars or sugar analogs and the respective “nucleosides” wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known.
  • Sugar modifications may also be made and combined with other modifications.
  • Modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications.
  • Sugar modifications include, but are not limited to, the following modifications at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 , alkyl or C 2 to C 10 alkenyl and alkynyl.
  • 2′ sugar modifications also include but are not limited to —O[(CH 2 ) n O] m CH 3 , —O(CH 2 ) n OCH 3 , —O(CH 2 ) n NH 2 , —O(CH 2 ) n CH 3 , —O(CH 2 ) n ONH 2 , and —O(CH 2 ) n ON[(CH 2 )n CH 3 )] 2 , where n and m are from 1 to about 10.
  • modifications at the 2′ position include but are not limited to: C 10 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • Modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH 2 and S.
  • Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH 3 , and 2′-O(CH 2 ) 2 OCH 3 substituent groups.
  • the substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—(C 1 -C 10 alkyl), OCF 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 —O—N(R m )(R n ), and O—CH 2 —C( ⁇ O)—N(R m )(R n ), where each R m and R n is, independently, H or substituted or unsubstituted C 1 -C 10 alkyl.
  • nucleic acids described herein include one or more bicyclic nucleic acids.
  • the bicyclic nucleic acid comprises a bridge between the 4′ and the 2′ ribosyl ring atoms.
  • nucleic acids provided herein include one or more bicyclic nucleic acids wherein the bridge comprises a 4′ to 2′ bicyclic nucleic acid.
  • 4′ to 2′ bicyclic nucleic acids include, but are not limited to, one of the formulae: 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 )—S-2′; 4′-(CH 2 ) 2 —O-2′ (ENA); 4′-CH(CH 3 )—O-2′ and 4′-CH(CH2OCH 3 )—O-2′, and analogs thereof (see, U.S. Pat. No. 7,399,845); 4′-C(CH 3 )(CH 3 )—O-2′ and analogs thereof, (see WO2009/006478, WO2008/150729, US2004/0171570, U.S. Pat. No.
  • nucleic acids comprise linked nucleic acids.
  • Nucleic acids can be linked together using any inter nucleic acid linkage.
  • the two main classes of inter nucleic acid linking groups are defined by the presence or absence of a phosphorus atom.
  • Representative phosphorus containing inter nucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P ⁇ S).
  • Non-phosphorus containing inter nucleic acid linking groups include, but are not limited to, methylenemethylimino (—CH 2 —N(CH 3 )—O—CH 2 —), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H) 2 —O—); and N,N*-dimethylhydrazine (—CH 2 —N(CH 3 )—N(CH 3 )).
  • inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates.
  • Unnatural nucleic acids can contain a single modification.
  • Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.
  • Backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in any combination. Other non-phosphate linkages may also be used.
  • backbone modifications e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages
  • backbone modifications can confer immunomodulatory activity on the modified nucleic acid and/or enhance their stability in vivo.
  • a phosphorous derivative is attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like.
  • Exemplary polynucleotides containing modified phosphate linkages 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.
  • backbone modification comprises replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group.
  • modifications include: anionic internucleoside linkage; N3′ to P5′ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179).
  • a modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g. a combination of phosphate linkages such as a combination of phosphodiester and phosphoroth
  • Substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA).
  • PNA aminoethylglycine
  • U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake.
  • Conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (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), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et
  • a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol 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), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-S—H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (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.
  • unnatural nucleic acids further form unnatural base pairs.
  • exemplary unnatural nucleotides capable of forming an unnatural DNA or RNA base pair (UBP) under conditions in vivo includes, but is not limited to, 5SICS, d5SICS, NAM, dNaM, and combinations thereof.
  • unnatural nucleotides include:
  • an unnatural base pair generate an unnatural amino acid described in Dumas et al., “Designing logical codon reassignment—Expanding the chemistry in biology,” Chemical Science, 6: 50-69 (2015).
  • the host cell into which the constructs or vectors disclosed herein are introduced is cultured or maintained in a suitable medium such that the tRNA, the tRNA synthetase and the protein of interest are produced.
  • the medium also comprises the unnatural amino acid(s) such that the protein of interest incorporates the unnatural amino acid(s).
  • the orthogonal tRNA synthetase/tRNA pair charges a tRNA with an unnatural amino acid and incorporates the unnatural amino acid into the polypeptide chain in response to the codon.
  • exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/ B. stearothermophilus tRNA CUA pairs, E. coli LeuRS (Ec-Leu)/ B. stearothermophilus tRNA CUA pairs, and pyrrolysyl-tRNA pairs.
  • a cytokine (e.g., IL-2) polypeptide comprising an unnatural amino acid(s) are prepared by introducing the nucleic acid constructs described herein comprising the tRNA and tRNA synthetase and comprising a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons into a host cell.
  • the host cell is exposed to a physiological solution comprising the unnatural amino acid(s), and the host cells are then maintained under conditions which permit expression of the protein of interest's encoding sequence.
  • the unnatural amino acid(s) is incorporated into the polypeptide chain in response to the codon.
  • one or more unnatural amino acids are incorporated into the cytokine (e.g., IL-2) polypeptide.
  • two or more unnatural amino acids may be incorporated into the cytokine (e.g., IL-2) polypeptide at two or more sites in the protein.
  • cytokine e.g., IL-2
  • multiple codons will need to be incorporated into the encoding nucleic acid sequence at the desired positions such that the tRNA synthetase/tRNA pairs can direct the incorporation of the unnatural amino acids in response to the codon(s).
  • At least 1, 2, 3, 4, or more codon encoding nucleic acids may be incorporated into the nucleic acid sequence of interest.
  • a second or further orthogonal tRNA-tRNA synthetase pair may be used to incorporate the second or further unnatural amino acid; suitably said second or further orthogonal tRNA-tRNA synthetase pair recognizes a different codon in the nucleic acid encoding the protein of interest so that the two or more unnatural amino acids can be specifically incorporated into different defined sites in the protein in a single manufacturing step.
  • two or more orthogonal tRNA-tRNA synthetase pairs may therefore be used.
  • cytokine e.g., IL-2
  • IL-2 cytokine
  • 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.
  • Suitable host cells may include bacterial cells (e.g., E. coli ), but most suitably host cells are eukaryotic cells, for example insect cells (e.g. Drosophila such as Drosophila melanogaster ), yeast cells, nematodes (e.g. Celegans), mice (e.g. Mus musculus ), or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells) or human cells or other eukaryotic cells.
  • suitable host cells are known to those skilled in the art.
  • the host cell is a mammalian cell—such as a human cell or an insect cell.
  • Vector DNA can be introduced into host cells via conventional transformation or transfection techniques.
  • stable cell lines are prepared.
  • a gene that encodes a selectable marker for example, for resistance to antibiotics
  • Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, or methotrexate.
  • Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (for example, cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • the constructs described herein are integrated into the genome of the host cell.
  • An advantage of stable integration is that the uniformity between individual cells or clones is achieved. Another advantage is that selection of the best producers may be carried out. Accordingly, it is desirable to create stable cell lines.
  • the constructs described herein are transfected into a host cell. An advantage of transfecting the constructs into the host cell is that protein yields may be maximized.
  • a cell comprising the nucleic acid construct or the vector described herein.
  • the pharmaceutical composition and formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral, oral, buccal, rectal, sublingual, or transdermal administration routes.
  • parenteral administration comprises intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intratechal administration.
  • the pharmaceutical composition is formulated for local administration. In other instances, the pharmaceutical composition is formulated for systemic administration.
  • the 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 melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • aqueous liquid dispersions self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • the pharmaceutical formulations include a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form.
  • exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like.
  • Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like.
  • PVP polyvinylpyrrollidone
  • the pharmaceutical composition is formulated as an immunoliposome, which comprises a plurality of IL-2 conjugates bound either directly or indirectly to lipid bilayer of liposomes.
  • exemplary lipids include, but are not limited to, fatty acids; phospholipids; sterols such as cholesterols; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, globocides, and cerebrosides; surfactant amines such as stearyl, oleyl, and linoleyl amines.
  • the lipid comprises a cationic lipid.
  • the lipid comprises a phospholipid.
  • Exemplary phospholipids include, but are not limited to, phosphatidic acid (“PA”), phosphatidylcholine (“PC”), phosphatidylglycerol (“PG”), phophatidylethanol amine (“PE”), phophatidylinositol (“PI”), and phosphatidylserine (“PS”), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine (“DAPC”), didecanoyl-L-alpha-phosphatidylcholine (“DDPC”), dielaidoylphosphatidylcholine (“DEPC”), dilauroylphosphatidylcholine (“DLPC”), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine (“DM
  • the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids
  • 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.
  • acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
  • the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range.
  • salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions
  • suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
  • the pharmaceutical formulations include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, glucose, or salts like potassium phosphate, sodium citrate, ammonium sulfate and/or other agents such as heparin to increase the solubility and in vivo stability of polypeptides.
  • the pharmaceutical formulations further include diluent which are used to stabilize compounds because they can provide a more stable environment.
  • Salts dissolved in buffered solutions are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.
  • diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling.
  • Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®, dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar, such as Di-Pac® (Amstar), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's 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.
  • Avicel®
  • the pharmaceutical formulations include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance.
  • disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carb
  • the pharmaceutical formulations include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
  • lactose calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
  • Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing or inhibiting adhesion or friction of materials.
  • Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as CarbowaxTM, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or
  • Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.
  • Solubilizers include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.
  • Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like.
  • Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, polysorbate-20 or Tween® 20, or trometamol.
  • Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g
  • 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), and the like.
  • 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), and the like.
  • Pluronic® Pluronic®
  • Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants is included to enhance physical stability or for other purposes.
  • Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.
  • 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 doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.
  • the pharmaceutical compositions described herein are administered for therapeutic applications.
  • the pharmaceutical composition is administered once per day, twice per day, three times per day or more.
  • the pharmaceutical composition is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per 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.
  • the administration of the composition is given continuously, alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”).
  • the length of the drug holiday varies between 2 days and 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.
  • the dose reduction during a drug holiday is from 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%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.
  • the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated.
  • the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.
  • toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the 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 the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50.
  • Compounds exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
  • kits and articles of manufacture for use with one or more methods and compositions described herein.
  • Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers are formed from a variety of materials such as glass or plastic.
  • the articles of manufacture provided herein contain packaging materials.
  • packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • the container(s) include 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 the delivery of cytokine (e.g., IL-2) polypeptides or cytokine (e.g., IL-2) conjugates.
  • cytokine e.g., IL-2
  • kits further optionally include an identifying description or label or instructions relating to its use in the methods described herein.
  • a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
  • a label is on or associated with the container.
  • a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
  • the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein.
  • the pack for example, contains metal or plastic foil, such as a blister pack.
  • the pack or dispenser device is accompanied by instructions for administration.
  • the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for drugs, or the approved product insert.
  • compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 ⁇ L” means “about 5 ⁇ L” and also “5 ⁇ L.” Generally, the term “about” includes an amount that would be expected to be within experimental error, such as for example, within 15%, 10%, or 5%.
  • the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal.
  • the mammal is a human.
  • the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker.
  • the term “significant” or “significantly” in reference to binding affinity means a change in the binding affinity of the cytokine (e.g., IL-2 polypeptide) sufficient to impact binding of the cytokine (e.g., IL-2 polypeptide) to a target receptor.
  • the term refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
  • the term means 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.
  • the term “significant” or “significantly” in reference to activation of one or more cell populations via a cytokine signaling complex means a change sufficient to activate the cell population.
  • the change to activate the cell population is measured as a receptor signaling potency.
  • an EC50 value may be provided.
  • an ED50 value may be provided.
  • a concentration or dosage of the cytokine may be provided.
  • the term “potency” refers to the amount of a cytokine (e.g., IL-2 polypeptide) required to produce a target effect. In some instances, the term “potency” refers to the amount of cytokine (e.g., IL-2 polypeptide) required to activate a target cytokine receptor (e.g., IL-2 receptor). In other instances, the term “potency” refers to the amount of cytokine (e.g., IL-2 polypeptide) required to activate a target cell population. In some cases, potency is measured as ED50 (Effective Dose 50), or the dose required to produce 50% of a maximal effect. In other cases, potency is measured as EC50 (Effective Concentration 50), or the dose required to produce the target effect in 50% of the population.
  • ED50 Effective Dose 50
  • EC50 Effective Concentration 50
  • PathHunter cell lines were expanded from freezer stocks according to standard procedures. Cells were seeded in a total volume of 20. ⁇ L into white walled, 384-well microplates and incubated for the appropriate time prior to testing.
  • sample For agonist determination, cells were incubated with sample to induce response. Intermediate dilution of sample stocks was performed to generate 5 ⁇ sample in assay buffer. About 5 ⁇ L of 5 ⁇ sample was added to cells and incubated at 37° C. for 6 to 16 hours depending on the assay. Vehicle concentration was 1%.
  • Assay signal was generated through a single addition of 12.5 or 15 ⁇ L (50% v/v) of PathHunter Detection reagent cocktail for agonist and antagonist assays respectively, followed by a one hour incubation at room temperature.
  • activity was detected using a high sensitivity detection reagent (PathHunter Flash Kit) to improve assay performance.
  • an equal volume of detection reagent 25 or 30 uL was added to the wells, followed by a one hour incubation at room temperature.
  • Microplates were read following signal generation with a PerkinElmer EnvisionTM instrument for chemilumine-scent signal detection.
  • % Activity 100% ⁇ (mean RLU of test sample ⁇ mean RLU of vehicle control)/(mean MAX RLU control ligand ⁇ mean RLU of vehicle control).
  • % Inhibition 100% ⁇ (1 ⁇ (mean RLU of test sample ⁇ mean RLU of vehicle control)/(mean RLU of EC80 control ⁇ mean RLU of vehicle control)).
  • Structural data of the IL-2/heterotrimeric receptor signaling complex were used to guide design of nAA-pegylation sites to specifically abrogate the interaction of IL-2 and IL-2 receptor ⁇ subunit (IL-2R ⁇ ).
  • Exemplary IL-2 conjugates were subjected to functional analysis: K35, F42, K43, E62, and P65.
  • the IL-2 conjugates were expressed as inclusion bodies in E. coli , purified and re-folded using standard procedures before site-specifically pegylating the IL-2 product using DBCO-mediated copper-free click chemistry to attach stable, covalent mPEG moieties to the AzK.
  • the IL-2 conjugates were screened for functional activity at Discoverx (Fremont Calif.) using the PathHunter IL-2 Cytokine Receptor assay.
  • This assay uses recombinant human U20S cell line that expresses the IL-2 receptor 3 (IL-2R13) and ⁇ (IL-2R ⁇ ) subunits, each fused to half of the split reporter enzyme 3-galactosidase.
  • a second cell line has been further engineered to express the IL-2R ⁇ subunit. Parallel testing with these two cell lines allows assessment of variant activation of the IL-2 receptor apy as well as the basal py complex.
  • IL-2 agonist activity on the IL-2 ⁇ receptor complex stimulates receptor dimerization and reporter 3-galactosidase reconstitution that results in a chemiluminescent signal.
  • the assay was run in agonist mode to determine the EC 50 of each test article, and comparison of dose-response curve profiles between IL2R ⁇ positive and negative cell types allows determination of the contribution of IL2R ⁇ to the observed activity.
  • Table 1 shows the EC50 data for IL-2 receptor agonism in cell-based screen for 30kD PEGylated IL-2 conjugates.
  • PBMC peripheral blood mononuclear cell
  • pSTATS transcription factor STAT5
  • NK natural killer
  • IL-2 P65_30kD and IL-2 E62_30kD retained potency relative to the native IL-2, with EC 50 values for pSTAT5 production within 4-5 fold of the native IL-2 ( FIG. 5A ).
  • the EC50 values ( ⁇ g/mL) was calculated from dose response curves generated from MFI plots.
  • Study E3826-U1704 included 13 time points (0.13, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h) sacrificing 3 mice per each time point and study E3826-U1803 included 9 time points (2, 8, 12, 24, 48, 72, 120, 168, and 240 h) sacrificing 4-7 mice per each time point.
  • Plasma and blood cells (in both studies) and tumors in study E3826-U1803 were collected for PK and PD analyses.
  • Bioanalysis of plasma samples was performed using a qualified human IL-2 ELISA assay (Abcam, Cambridge, UK). Concentrations of Aldesleukin, E62_30kD and P65_30kD and the internal standard in samples derived from plasma were determined using an ELISA assay.
  • PK data analysis was performed at NW Solutions (Seattle, Wash.). The PK data were imported into Phoenix WinNonlin v6.4 (Certara/Pharsight, Princeton, N.J.) for analysis. The group mean plasma concentration versus time data were analyzed with noncompartmental methods using an IV bolus administration model.
  • the plasma concentration profiles of P65_30kD, E62_30kD, E62_5kD and aldesleukin at 0.3 mg/kg are plotted in FIG. 6 .
  • both P65_30kD and E62_30kD exhibits a superior PK profile relative to aldesleukin as summarized on Table 3.
  • the T max was observed at 0.03 h post-dose (the first measured timepoint after dosing) and mean plasma concentrations were measurable out to 4 h post-dose.
  • the T max was observed at 0.03 h post-dose and mean plasma concentrations were measurable out to 120 h post-dose (the last measured timepoint).
  • the T max was observed at 0.133 hr post-dose and mean plasma concentrations were measurable out to 12 hr post-dose.
  • Exposure based on C max and AUC 0-t was as follows: P65_30kD>>E62_30kD>>E62_5kD>aldesleukin.
  • E62_5kD with a smaller PEG had a PK profile closer to rIL-2.
  • P65_30kD exposure was 5.5 and 200 times higher than aldesleukin based on Cmax and AUC0-t, respectively.
  • P65_30kD demonstrated 23-fold extended t1/2 (13.3 h vs. 0.57 h) and about 198-fold reduced CL (6.58 vs 1300 mL/h/Kg) compared to the aldesleukin.
  • STAT5 phosphorylation and induction of cell proliferation was used as pharmacodynamic readouts to assess the pharmacological profile of P65_30kD relative to its pharmacokinetics.
  • the pSTAT5 PD marker showed good correlation with PK for both P65_30kD and aldesleukin in CD8+ effector T cells (Table 7). Persistent elevation of pSTAT5 was observed in both NK and CD8+ T cells up to 72 h, and up to 24 h in Tregs. pSTAT5 induction returned to baseline after only 2 h in mice dosed with aldesleukin ( FIG. 7 ).
  • FIGS. 8A-8C Phenotypic analysis of CD8+ effector T cells revealed substantial expansion of CD44+ memory cells within this population.
  • Table 8 shows the plasma and tumor drug concentration following a single dose of P65_30kD at 3 mg/kg in B16-F10 tumor-bearing mice.
  • the tumor half-life was twice the plasma half-life (24.4 vs 12.6), indicating that the P65_30kD penetrates the tumor and is retained in the tumor.
  • the tail end of the curves cross showing the plasma eliminates faster than the tumor.
  • the tumor:plasma AUC ratio was 9.7% and 8.4% for the 1 and 3 mg/kg doses respectively.
  • FIG. 10A - FIG. 10B show the expansion of NK and CD+ T cells by P65_30kD in B16F10 tumors.
  • FIG. 10A shows of the percentage of NK cells, CD8+ cells and Tregs in the tumor CD3+ T cell population following treatment with a single IV bolus dose of P65_30kD at 3 g/kg.
  • Tumor samples were analyzed for immune cell populations 5 days after treatment by flow cytometry. Each data point represents an average from 3 replicates at each time point, ⁇ SEM. The cell population data represented is from day 5 Tumor samples and the CD8/Treg ratio was calculated from the day 7 samples.
  • FIG. 10A shows of the percentage of NK cells, CD8+ cells and Tregs in the tumor CD3+ T cell population following treatment with a single IV bolus dose of P65_30kD at 3 g/kg.
  • Tumor samples were analyzed for immune cell populations 5 days after treatment by flow cytometry. Each data point represents an average from 3 replicates at
  • 10B shows the ratio of CD8+ effector over CD4+ regulatory T cells 7 days following treatment with a single IV bolus dose of P65_30kD at 3 mg/kg. Each data point represents an average from 3 replicates at each time point, ⁇ SEM.
  • All plasma samples were analyzed for human IL-2 as well as mouse IL-2, TNF- ⁇ , IFN ⁇ , IL-5, and IL-6 cytokines, employing commercially-available ELISA kits.
  • a major of toxicity associated with High-dose IL-2 is Vascular leak syndrome and associated Cytokine Release Syndrome (CRS).
  • CRS Cytokine Release Syndrome
  • the pharmacokinetic and pharmacodynamic profile of P65_30kD was evaluated in non-na ⁇ ve cynomolgus monkeys following administration of a single intravenous dose at 0.3 mg/kg.
  • the study was conducted at Charles River Laboratories, Inc. (Reno, Nev.) and PK data analysis was performed at NW Solutions (Seattle, Wash.). Blood samples were collected pre-dose and at 21 time points (0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 120, 144, 168, 192 and 240 h post-dose. Both plasma and blood cells were collected for PK and PD analyses. Selected time points were used for PK, PD, cell population and hematology analysis.
  • Table 10 shows P65_30kD PK Parameters in Cynomolgus monkey.
  • Tmax was observed at 0.5 h post-dose (the first measured timepoint after dosing) and mean plasma concentrations were measurable out to 168 h post-dose (the last measure)
  • the t 1/2 and AUC for P65_30kD were 13.6 h and 121000 hr*ng/mL respectively.
  • the evaluation time points correspond to pre-dose at day ⁇ 1 and 1, 3, 6, 8, 10, 12, 14, 17, 19, 21 post-dose.
  • WBC white blood cell
  • IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKN 1 ( homo PKLTRMLTFKFYMPKKATELKHLQCLEEELKPL sapiens ) EEVLNLAQSKNFHLRPRDLISNINVIVLELKGS (mature ETTFMCEYADETATIVEFLNRWITFCQSIISTL form) T IL-2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQ 2 ( homo LEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM sapiens ) PKKATELKHLQCLEEELKPLEEVLNLAQSKNFH (precursor) LRPRDLISNINVIVLELKGSETTFMCEYADETA NCBI Acces- TIVEFLNRWITFCQSIISTLT sion No.: AAB46883.1
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Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
US11077195B2 (en) 2019-02-06 2021-08-03 Synthorx, Inc. IL-2 conjugates and methods of use thereof
WO2021216920A1 (en) 2020-04-22 2021-10-28 Iovance Biotherapeutics, Inc. Systems and methods for coordinating manufacturing of cells for patient-specific immunotherapy
WO2021226085A1 (en) 2020-05-04 2021-11-11 Iovance Biotherapeutics, Inc. Selection of improved tumor reactive t-cells
WO2021226061A1 (en) 2020-05-04 2021-11-11 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of the same in immunotherapy
WO2021262437A3 (en) * 2020-06-21 2022-02-10 Ketan Desai Use of il13 for prevention and treatment of covid-19
WO2022076952A1 (en) 2020-10-06 2022-04-14 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
WO2022076606A1 (en) 2020-10-06 2022-04-14 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
WO2022125941A1 (en) 2020-12-11 2022-06-16 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with braf inhibitors and/or mek inhibitors
WO2022133140A1 (en) 2020-12-17 2022-06-23 Iovance Biotherapeutics, Inc. Treatment with tumor infiltrating lymphocyte therapies in combination with ctla-4 and pd-1 inhibitors
WO2022133149A1 (en) 2020-12-17 2022-06-23 Iovance Biotherapeutics, Inc. Treatment of cancers with tumor infiltrating lymphocytes
WO2022147196A2 (en) 2020-12-31 2022-07-07 Iovance Biotherapeutics, Inc. Devices and processes for automated production of tumor infiltrating lymphocytes
WO2022165260A1 (en) 2021-01-29 2022-08-04 Iovance Biotherapeutics, Inc. Methods of making modified tumor infiltrating lymphocytes and their use in adoptive cell therapy
WO2022170219A1 (en) 2021-02-05 2022-08-11 Iovance Biotherapeutics, Inc. Adjuvant therapy for cancer
WO2022187741A2 (en) 2021-03-05 2022-09-09 Iovance Biotherapeutics, Inc. Tumor storage and cell culture compositions
WO2022198141A1 (en) 2021-03-19 2022-09-22 Iovance Biotherapeutics, Inc. Methods for tumor infiltrating lymphocyte (til) expansion related to cd39/cd69 selection and gene knockout in tils
WO2022204564A2 (en) 2021-03-25 2022-09-29 Iovance Biotherapeutics, Inc. Methods and compositions for t-cell coculture potency assays and use with cell therapy products
WO2022225981A2 (en) 2021-04-19 2022-10-27 Iovance Biotherapeutics, Inc. Chimeric costimulatory receptors, chemokine receptors, and the use of same in cellular immunotherapies
WO2022245754A1 (en) 2021-05-17 2022-11-24 Iovance Biotherapeutics, Inc. Pd-1 gene-edited tumor infiltrating lymphocytes and uses of same in immunotherapy
WO2023004074A2 (en) 2021-07-22 2023-01-26 Iovance Biotherapeutics, Inc. Method for cryopreservation of solid tumor fragments
WO2023009716A1 (en) 2021-07-28 2023-02-02 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with kras inhibitors
WO2023039488A1 (en) 2021-09-09 2023-03-16 Iovance Biotherapeutics, Inc. Processes for generating til products using pd-1 talen knockdown
WO2023049862A1 (en) 2021-09-24 2023-03-30 Iovance Biotherapeutics, Inc. Expansion processes and agents for tumor infiltrating lymphocytes
US11622993B2 (en) 2017-08-03 2023-04-11 Synthorx, Inc. Cytokine conjugates for the treatment of autoimmune diseases
WO2023077015A2 (en) 2021-10-27 2023-05-04 Iovance Biotherapeutics, Inc. Systems and methods for coordinating manufacturing of cells for patient-specific immunotherapy
WO2023086803A1 (en) 2021-11-10 2023-05-19 Iovance Biotherapeutics, Inc. Methods of expansion treatment utilizing cd8 tumor infiltrating lymphocytes
WO2023147488A1 (en) 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Cytokine associated tumor infiltrating lymphocytes compositions and methods
WO2023147486A1 (en) 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Tumor infiltrating lymphocytes engineered to express payloads
WO2023196877A1 (en) 2022-04-06 2023-10-12 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
WO2023201369A1 (en) 2022-04-15 2023-10-19 Iovance Biotherapeutics, Inc. Til expansion processes using specific cytokine combinations and/or akti treatment
WO2023220608A1 (en) 2022-05-10 2023-11-16 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with an il-15r agonist
WO2024011114A1 (en) 2022-07-06 2024-01-11 Iovance Biotherapeutics, Inc. Devices and processes for automated production of tumor infiltrating lymphocytes
WO2024030758A1 (en) 2022-08-01 2024-02-08 Iovance Biotherapeutics, Inc. Chimeric costimulatory receptors, chemokine receptors, and the use of same in cellular immunotherapies
US11919934B2 (en) 2018-02-26 2024-03-05 Synthorx, Inc. IL-15 conjugates and uses thereof
WO2024055017A1 (en) 2022-09-09 2024-03-14 Iovance Biotherapeutics, Inc. Processes for generating til products using pd-1/tigit talen double knockdown
WO2024055018A1 (en) 2022-09-09 2024-03-14 Iovance Biotherapeutics, Inc. Processes for generating til products using pd-1/tigit talen double knockdown

Families Citing this family (72)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018170288A1 (en) 2017-03-15 2018-09-20 Pandion Therapeutics, Inc. Targeted immunotolerance
BR112019024127A2 (pt) 2017-05-24 2020-06-23 Pandion Therapeutics, Inc. Imunotolerância alvejada
JP7325341B2 (ja) 2017-07-11 2023-08-14 シンソークス，インク． 非天然ヌクレオチドの組み込み及びその方法
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
US10174091B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
AU2019297451A1 (en) 2018-07-03 2021-01-28 Marengo Therapeutics, Inc. Anti-TCR antibody molecules and uses thereof
FI3849614T3 (fi) 2018-09-11 2024-02-08 Ambrx Inc Interleukiini-2-polypeptidikonjugaatteja ja niiden käyttöjä
US20210221863A1 (en) * 2018-09-21 2021-07-22 Innovent Biologics (Suzhou) Co., Ltd. Novel interleukin-2 and use thereof
US20220193253A1 (en) * 2019-04-26 2022-06-23 Prolynx Llc Slow-release cytokine conjugates
KR20220035333A (ko) 2019-05-20 2022-03-22 팬디온 오퍼레이션스, 인코포레이티드 Madcam 표적 면역관용
KR20220012296A (ko) 2019-05-20 2022-02-03 싸이튠 파마 암 또는 감염성 질환 치료를 위한 IL-2/IL-15Rβγ 작용제 투여 요법
WO2020257525A1 (en) * 2019-06-20 2020-12-24 Cspc Dophen Corporation Modified il-2 proteins, peg conjugates, and uses thereof
US20220289806A1 (en) * 2019-08-15 2022-09-15 Cytimm Therapeutics, Inc. Modified Interleukin 2 (IL-2) Polypeptides, Conjugates and Uses Thereof
MX2022001776A (es) * 2019-08-15 2022-03-17 Synthorx Inc Terapias de combinacion de inmuno oncologia con conjugados de il-2.
MX2022002053A (es) 2019-08-23 2022-03-17 Synthorx Inc Conjugados de il-15 y sus usos.
CN114853874A (zh) * 2019-09-10 2022-08-05 中国医学科学院北京协和医院 靶向调节t细胞的长效白介素-2及其在治疗自身免疫病中的应用
US20210070827A1 (en) 2019-09-10 2021-03-11 Synthorx, Inc. Il-2 conjugates and methods of use to treat autoimmune diseases
TW202131952A (zh) 2019-11-04 2021-09-01 美商欣爍克斯公司 介白素10接合物及其用途
EP3819307A1 (de) 2019-11-07 2021-05-12 CytoKi Pharma ApS Therapeutische derivate von interleukin-22
CN114524873A (zh) * 2019-12-17 2022-05-24 北京志道生物科技有限公司 白介素-2衍生物
CN115315436A (zh) * 2020-01-10 2022-11-08 明峰治疗股份公司 修饰的il-2多肽及其用途
JP2023511274A (ja) 2020-01-14 2023-03-17 シンセカイン インコーポレイテッド Ｉｌ２オルソログおよび使用法
CA3166509A1 (en) 2020-01-14 2021-07-22 Synthekine, Inc. Biased il2 muteins methods and compositions
AU2021233909A1 (en) * 2020-03-11 2022-09-29 Ambrx, Inc. Interleukin-2 polypeptide conjugates and methods of use thereof
KR20230004682A (ko) * 2020-04-22 2023-01-06 머크 샤프 앤드 돔 엘엘씨 인터류킨-2 수용체 베타 감마c 이량체에 대해 편향되고 비펩티드성 수용성 중합체에 접합된 인간 인터류킨-2 접합체
US20210340586A1 (en) 2020-04-30 2021-11-04 Sutro Biopharma, Inc. Methods of Producing Full-Length Antibodies Using E. coli
JP2023526282A (ja) 2020-05-13 2023-06-21 ボナム セラピューティクス，インク． タンパク質複合体の組成物及びその使用方法
JP2023527919A (ja) 2020-06-03 2023-06-30 アセンディス ファーマ オンコロジー ディヴィジョン エー／エス Ｉｌ－２配列及びその使用
KR20230027235A (ko) 2020-06-25 2023-02-27 신톡스, 인크. Il-2 콘쥬게이트 및 항-egfr 항체를 사용한 면역 종양학 병용 요법
WO2022031884A2 (en) 2020-08-05 2022-02-10 Synthekine, Inc. Il2rg binding molecules and methods of use
KR20230065259A (ko) 2020-08-05 2023-05-11 신테카인, 인크. Il10 수용체 결합 분자 및 사용 방법
EP4192490A1 (de) 2020-08-05 2023-06-14 Synthekine, Inc. Il27ra-bindende moleküle und verfahren zur verwendung
EP4192489A2 (de) 2020-08-05 2023-06-14 Synthekine, Inc. Il2rb-bindende moleküle und verfahren zur verwendung
TW202227480A (zh) 2020-09-14 2022-07-16 美商蘇特羅生物製藥公司 使用無細胞蛋白質合成系統來大規模生產抗體之方法
MX2023004029A (es) 2020-10-09 2023-04-27 Synthorx Inc Terapia de combinacion de inmunoncologia con conjugados de il-2 y pembrolizumab.
AU2021356610A1 (en) 2020-10-09 2023-06-15 Synthorx, Inc. Immuno oncology therapies with il-2 conjugates
CN116963755A (zh) 2020-10-14 2023-10-27 赛缇姆治疗公司 经修饰的白细胞介素2（il-2）多肽以及制备和使用其的方法
JP2023550880A (ja) 2020-10-26 2023-12-06 サイチューン ファーマ 非黒色腫皮膚癌を治療するためのＩＬ－２／ＩＬ－１５Ｒβγアゴニスト
AU2021367887A1 (en) 2020-10-26 2023-06-01 Cytune Pharma IL-2/IL-15Rβү AGONIST FOR TREATING SQUAMOUS CELL CARCINOMA
CN112279906B (zh) * 2020-10-30 2022-09-20 浙江新码生物医药有限公司 人白细胞介素2-聚乙二醇偶联物及其制备方法与应用
WO2022098954A1 (en) 2020-11-06 2022-05-12 Nektar Therapeutics Tlr agonist compounds and related cancer immunotherapy methods
EP4274616A2 (de) 2021-01-11 2023-11-15 Synthekine, Inc. Zusammensetzungen und verfahren im zusammenhang mit rezeptorpaarung
WO2022174102A1 (en) 2021-02-12 2022-08-18 Synthorx, Inc. Lung cancer combination therapy with il-2 conjugates and an anti-pd-1 antibody or antigen-binding fragment thereof
WO2022174101A1 (en) 2021-02-12 2022-08-18 Synthorx, Inc. Skin cancer combination therapy with il-2 conjugates and cemiplimab
WO2022200525A1 (en) 2021-03-26 2022-09-29 Innate Pharma Multispecific proteins comprising an nkp46-binding site, a cancer antgienge binding site fused to a cytokine for nk cell engaging
CN113321722A (zh) * 2021-04-13 2021-08-31 苏州复融生物技术有限公司 白介素2突变体及其应用
WO2022256538A1 (en) 2021-06-03 2022-12-08 Synthorx, Inc. Head and neck cancer combination therapy comprising an il-2 conjugate and cetuximab
WO2022260916A1 (en) * 2021-06-08 2022-12-15 Merck Sharp & Dohme Llc Functional cell-based potency assay for measuring biological activity of interleukin 2 (il-2) analogs
WO2022258691A1 (en) 2021-06-09 2022-12-15 Innate Pharma Multispecific proteins binding to nkg2d, a cytokine receptor, a tumour antigen and cd16a
WO2022258678A1 (en) 2021-06-09 2022-12-15 Innate Pharma Multispecific proteins binding to nkp30, a cytokine receptor, a tumour antigen and cd16a
BR112023025476A2 (pt) 2021-06-09 2024-02-27 Innate Pharma Proteína de ligação multimérica e seu método de produção, composição farmacêutica, molécula de ácido nucleico isolada, vetor de expressão, célula isolada e proteína de ligação para uso
CN117616050A (zh) 2021-06-09 2024-02-27 先天制药公司 与nkp46、细胞因子受体、肿瘤抗原和cd16a结合的多特异性蛋白质
WO2023281485A1 (en) 2021-07-09 2023-01-12 Bright Peak Therapeutics Ag Modified il-2 polypeptides for treatment of inflammatory and autoimmune diseases
WO2023281482A1 (en) 2021-07-09 2023-01-12 Bright Peak Therapeutics Ag Cd20-targeted il-2 and its uses
US20230250181A1 (en) 2021-07-09 2023-08-10 Bright Peak Therapeutics Ag Modified checkpoint inhibitors and uses thereof
WO2023281480A1 (en) 2021-07-09 2023-01-12 Bright Peak Therapeutics Ag Conjugates of checkpoint inhibitors with il-2, and uses thereof
KR20240040134A (ko) 2021-07-09 2024-03-27 브라이트 피크 테라퓨틱스 아게 항체 접합체 및 이의 제조
KR20240043797A (ko) 2021-08-13 2024-04-03 싸이튠 파마 암 치료용 IL-2/IL-15Rβγ 작용제와 항체-약물 접합체 조합
CN113788890B (zh) * 2021-09-01 2022-11-08 浙江新码生物医药有限公司 一种人白细胞介素2-聚乙二醇偶联物及其应用
CN113698468B (zh) * 2021-09-01 2022-10-11 浙江新码生物医药有限公司 人白细胞介素2-聚乙二醇偶联物及其应用
WO2023064802A1 (en) * 2021-10-13 2023-04-20 The Trustees Of Columbia University In The City Of New York Methods for controlling osteocalcin release
WO2023079524A2 (en) * 2021-11-08 2023-05-11 Life Technologies As Polymer-interaction molecule conjugates and methods of use
WO2023122573A1 (en) 2021-12-20 2023-06-29 Synthorx, Inc. Head and neck cancer combination therapy comprising an il-2 conjugate and pembrolizumab
WO2023122750A1 (en) 2021-12-23 2023-06-29 Synthorx, Inc. Cancer combination therapy with il-2 conjugates and cetuximab
WO2023133595A2 (en) 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Methods of ex vivo dosing and administration of lipid particles or viral vectors and related systems and uses
CN114349843A (zh) * 2022-01-18 2022-04-15 浙江博锐生物制药有限公司 白细胞介素-2衍生物及其制备方法和应用
CN114533730A (zh) * 2022-02-17 2022-05-27 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Triapine用于治疗非洲猪瘟病毒感染的应用
WO2023161857A1 (en) 2022-02-23 2023-08-31 Bright Peak Therapeutics Ag Bifunctional cytokine compositions
WO2023193015A1 (en) 2022-04-01 2023-10-05 Sana Biotechnology, Inc. Cytokine receptor agonist and viral vector combination therapies
WO2023202035A1 (zh) * 2022-04-20 2023-10-26 北京大学宁波海洋药物研究院 受体偏向的peg化il-2变体组合及其应用
WO2023235790A1 (en) * 2022-06-01 2023-12-07 University Of Miami Bifunctional il-2 and il-10 fusion proteins and uses thereof
WO2023241653A1 (zh) * 2022-06-17 2023-12-21 舒泰神（北京）生物制药股份有限公司 白介素-2(il-2)突变体及其用途

Family Cites Families (323)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
JPS5927900A (ja) 1982-08-09 1984-02-14 Wakunaga Seiyaku Kk 固定化オリゴヌクレオチド
FR2540122B1 (fr) 1983-01-27 1985-11-29 Centre Nat Rech Scient Nouveaux composes comportant une sequence d'oligonucleotide liee a un agent d'intercalation, leur procede de synthese et leur application
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US4849513A (en) 1983-12-20 1989-07-18 California Institute Of Technology Deoxyribonucleoside phosphoramidites in which an aliphatic amino group is attached to the sugar ring and their use for the preparation of oligonucleotides containing aliphatic amino groups
US5015733A (en) 1983-12-20 1991-05-14 California Institute Of Technology Nucleosides possessing blocked aliphatic amino groups
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
EP0154316B1 (de) 1984-03-06 1989-09-13 Takeda Chemical Industries, Ltd. Chemisch modifizierte Lymphokine und Verfahren zu ihrer Herstellung
US4569790A (en) 1984-03-28 1986-02-11 Cetus Corporation Process for recovering microbially produced interleukin-2 and purified recombinant interleukin-2 compositions
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
FR2567892B1 (fr) 1984-07-19 1989-02-17 Centre Nat Rech Scient Nouveaux oligonucleotides, leur procede de preparation et leurs applications comme mediateurs dans le developpement des effets des interferons
US5430136A (en) 1984-10-16 1995-07-04 Chiron Corporation Oligonucleotides having selectably cleavable and/or abasic sites
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
FR2575751B1 (fr) 1985-01-08 1987-04-03 Pasteur Institut Nouveaux nucleosides de derives de l'adenosine, leur preparation et leurs applications biologiques
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US5656493A (en) 1985-03-28 1997-08-12 The Perkin-Elmer Corporation System for automated performance of the polymerase chain reaction
JPS61225199A (ja) * 1985-03-29 1986-10-06 Kyowa Hakko Kogyo Co Ltd 新規ヒトインタ−ロイキン２ポリペチド誘導体
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US4766106A (en) 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US5206344A (en) 1985-06-26 1993-04-27 Cetus Oncology Corporation Interleukin-2 muteins and polymer conjugation thereof
US5576195A (en) 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
US5093232A (en) 1985-12-11 1992-03-03 Chiron Corporation Nucleic acid probes
US4910300A (en) 1985-12-11 1990-03-20 Chiron Corporation Method for making nucleic acid probes
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
JPS638396A (ja) 1986-06-30 1988-01-14 Wakunaga Pharmaceut Co Ltd ポリ標識化オリゴヌクレオチド誘導体
US4931544A (en) 1986-09-04 1990-06-05 Cetus Corporation Succinylated interleukin-2 for pharmaceutical compositions
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
JP2828642B2 (ja) 1987-06-24 1998-11-25 ハワード フローレイ インスティテュト オブ イクスペリメンタル フィジオロジー アンド メディシン ヌクレオシド誘導体
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US4924624A (en) 1987-10-22 1990-05-15 Temple University-Of The Commonwealth System Of Higher Education 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
DE3738460A1 (de) 1987-11-12 1989-05-24 Max Planck Gesellschaft Modifizierte oligonukleotide
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
JPH03503894A (ja) 1988-03-25 1991-08-29 ユニバーシィティ　オブ　バージニア　アランミ　パテンツ　ファウンデイション オリゴヌクレオチド　ｎ‐アルキルホスホラミデート
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
CA2008096A1 (en) 1989-01-19 1990-07-19 Samuel Rose Nucleic acid amplification using single primer
US4902502A (en) 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5089261A (en) 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US5457183A (en) 1989-03-06 1995-10-10 Board Of Regents, The University Of Texas System Hydroxylated texaphyrins
US5599923A (en) 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
EP0942000B1 (de) 1989-10-24 2004-06-23 Isis Pharmaceuticals, Inc. 2'-Modifizierte Oligonukleotide
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5177198A (en) 1989-11-30 1993-01-05 University Of N.C. At Chapel Hill Process for preparing oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5130302A (en) 1989-12-20 1992-07-14 Boron Bilogicals, Inc. Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5587470A (en) 1990-01-11 1996-12-24 Isis Pharmaceuticals, Inc. 3-deazapurines
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
AU7579991A (en) 1990-02-20 1991-09-18 Gilead Sciences, Inc. Pseudonucleosides and pseudonucleotides and their polymers
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
WO1991014781A1 (en) 1990-03-19 1991-10-03 Henkel Research Corporation METHOD FOR INCREASING THE OMEGA-HYDROXYLASE ACTIVITY IN $i(CANDIDA TROPICALIS)
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
GB9009980D0 (en) 1990-05-03 1990-06-27 Amersham Int Plc Phosphoramidite derivatives,their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
ES2116977T3 (es) 1990-05-11 1998-08-01 Microprobe Corp Soportes solidos para ensayos de hibridacion de acidos nucleicos y metodos para inmovilizar oligonucleotidos de modo covalente.
JPH04126084A (ja) 1990-05-11 1992-04-27 Hoechst Japan Ltd 蛋白質の製造法
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
BR9106702A (pt) 1990-07-27 1993-06-08 Isis Pharmaceuticals Inc Analogo de oligonucleotideos e processos para modular a producao de uma proteina por um organismo e para tratar um organismo
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
IL113519A (en) 1990-08-03 1997-11-20 Sterling Winthrop Inc Oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage, their preparation and pharmaceutical compositions for inhibiting gene expression containing said oligonucleosides
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
CA2092002A1 (en) 1990-09-20 1992-03-21 Mark Matteucci Modified internucleoside linkages
NZ239893A (en) 1990-09-25 1993-11-25 Hoechst Japan A method for introducing a foreign dna into a cell
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
ATE198598T1 (de) 1990-11-08 2001-01-15 Hybridon Inc Verbindung von mehrfachreportergruppen auf synthetischen oligonukleotiden
US5268273A (en) 1990-12-14 1993-12-07 Phillips Petroleum Company Pichia pastoris acid phosphatase gene, gene regions, signal sequence and expression vectors comprising same
US5672697A (en) 1991-02-08 1997-09-30 Gilead Sciences, Inc. Nucleoside 5'-methylene phosphonates
US5595732A (en) 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5389529A (en) 1991-06-12 1995-02-14 Regeneron Pharmaceuticals, Inc. Modified lamβ signal sequence and processes for producing recombinant neurotrophins
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5612199A (en) 1991-10-11 1997-03-18 Behringwerke Ag Method for producing a polynucleotide for use in single primer amplification
DE59208572D1 (de) 1991-10-17 1997-07-10 Ciba Geigy Ag Bicyclische Nukleoside, Oligonukleotide, Verfahren zu deren Herstellung und Zwischenprodukte
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
JP3739785B2 (ja) 1991-11-26 2006-01-25 アイシス ファーマシューティカルズ，インコーポレイティド 修飾されたピリミジンを含有するオリゴマーを使用する増強された三重らせんおよび二重らせんの成形
TW393513B (en) 1991-11-26 2000-06-11 Isis Pharmaceuticals Inc Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
DE9115660U1 (de) 1991-12-18 1992-07-30 Hoechst Ag, 6230 Frankfurt, De
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
FR2687679B1 (fr) 1992-02-05 1994-10-28 Centre Nat Rech Scient Oligothionucleotides.
US5229109A (en) 1992-04-14 1993-07-20 Board Of Regents, The University Of Texas System Low toxicity interleukin-2 analogues for use in immunotherapy
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
EP0577558A2 (de) 1992-07-01 1994-01-05 Ciba-Geigy Ag Carbocyclische Nukleoside mit bicyclischen Ringen, Oligonukleotide daraus, Verfahren zu deren Herstellung, deren Verwendung und Zwischenproduckte
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
TW263437B (de) 1992-09-24 1995-11-21 Takeda Pharm Industry Co Ltd
US6288302B1 (en) 1992-11-04 2001-09-11 National Science Council Of R.O.C. Application of α-amylase gene promoter and signal sequence in the production of recombinant proteins in transgenic plants and transgenic plant seeds
EP0673559A1 (de) 1992-12-14 1995-09-27 Honeywell Inc. Motorsystem mit individuell gesteuerten redundanten wicklungen
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
GB9304618D0 (en) 1993-03-06 1993-04-21 Ciba Geigy Ag Chemical compounds
CA2159631A1 (en) 1993-03-30 1994-10-13 Sanofi Acyclic nucleoside analogs and oligonucleotide sequences containing them
EP0691979A1 (de) 1993-03-31 1996-01-17 Sanofi Neue 5'-substituierte nucleoside und daraus hergestellte oligomere
WO1994022891A1 (en) 1993-03-31 1994-10-13 Sterling Winthrop Inc. Oligonucleotides with amide linkages replacing phosphodiester linkages
DE4311944A1 (de) 1993-04-10 1994-10-13 Degussa Umhüllte Natriumpercarbonatpartikel, Verfahren zu deren Herstellung und sie enthaltende Wasch-, Reinigungs- und Bleichmittelzusammensetzungen
US6294323B1 (en) 1993-04-14 2001-09-25 Behringwerke Ag Self initiating single primer amplification of nucleic acids
GB9311682D0 (en) 1993-06-05 1993-07-21 Ciba Geigy Ag Chemical compounds
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5446137B1 (en) 1993-12-09 1998-10-06 Behringwerke Ag Oligonucleotides containing 4'-substituted nucleotides
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5470719A (en) 1994-03-18 1995-11-28 Meng; Shi-Yuan Modified OmpA signal sequence for enhanced secretion of polypeptides
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US6010871A (en) 1994-09-29 2000-01-04 Ajinomoto Co., Inc. Modification of peptide and protein
JPH0889278A (ja) 1994-09-29 1996-04-09 Ajinomoto Co Inc 遺伝子導入用修飾蛋白質及びその製法
US5712114A (en) 1995-06-06 1998-01-27 Basf Aktiengesellschaft Compositions for expression of proteins in host cells using a preprocollagen signal sequence
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
JP4020429B2 (ja) 1995-06-07 2007-12-12 インヴィトロジェン コーポレーション 操作された組換え部位を使用する組換えクローニング
US6143557A (en) 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
US6261797B1 (en) 1996-01-29 2001-07-17 Stratagene Primer-mediated polynucleotide synthesis and manipulation techniques
GB9606158D0 (en) 1996-03-23 1996-05-29 Ciba Geigy Ag Chemical compounds
JPH09324000A (ja) 1996-06-05 1997-12-16 Suntory Ltd インターロイキン−２受容体を特異的に認識する因子とリボヌクレアーゼとの接合体
US7875733B2 (en) 2003-09-18 2011-01-25 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising 4′-thionucleosides for use in gene modulation
JP3756313B2 (ja) 1997-03-07 2006-03-15 武 今西 新規ビシクロヌクレオシド及びオリゴヌクレオチド類縁体
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
IL135000A0 (en) 1997-09-12 2001-05-20 Exiqon As Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues
US6794499B2 (en) 1997-09-12 2004-09-21 Exiqon A/S Oligonucleotide analogues
US5932474A (en) 1997-10-21 1999-08-03 The Regents Of The University Of California Target sequences for synthetic molecules
US6054271A (en) 1997-10-21 2000-04-25 The Regents Of The University Of California Methods of using synthetic molecules and target sequences
US6008378A (en) 1997-10-21 1999-12-28 The Regents Of The University Of California Synthetic molecules that specifically react with target sequences
DE69834420T2 (de) 1997-10-21 2007-04-19 The Regents Of The University Of California, Oakland Zielsequenzen für synthetische moleküle und verfahren für deren verwendung
WO1999021977A1 (en) 1997-10-24 1999-05-06 Life Technologies, Inc. Recombinational cloning using nucleic acids having recombination sites
DE19813317A1 (de) 1998-03-26 1999-09-30 Roche Diagnostics Gmbh Verbessertes Verfahren zur Primer Extension Präamplifikations-PCR
US6391544B1 (en) 1998-05-15 2002-05-21 Abbott Laboratories Method for using unequal primer concentrations for generating nucleic acid amplification products
US6955807B1 (en) * 1998-05-15 2005-10-18 Bayer Pharmaceuticals Corporation IL-2 selective agonists and antagonists
DZ2788A1 (fr) * 1998-05-15 2003-12-01 Bayer Ag Agonistes et antagonistes selectifs à IL-2.
US6562798B1 (en) 1998-06-05 2003-05-13 Dynavax Technologies Corp. Immunostimulatory oligonucleotides with modified bases and methods of use thereof
WO2000011028A2 (en) 1998-08-21 2000-03-02 Yeda Research And Development Co. Ltd. Anti-inflammatory peptides derived from il-2 and analogues thereof
US6175001B1 (en) 1998-10-16 2001-01-16 The Scripps Research Institute Functionalized pyrimidine nucleosides and nucleotides and DNA's incorporating same
EP2119788A1 (de) 1999-03-02 2009-11-18 Life Technologies Corporation Zusammensetzungen und Verfahren zur Verwendung beim Rekombinationsklonen von Nukleinsäuren
KR100782896B1 (ko) 1999-05-04 2007-12-06 엑시콘 에이/에스 Ｌ-리보-ｌｎａ 유사체
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
MXPA01012500A (es) 1999-06-03 2003-10-14 Bioinnovation Ltd Productos para terapia de gen.
DE60045238D1 (de) 1999-07-15 2010-12-30 Japan Science & Tech Agency Nukleinsäurebasenpaar
WO2001032887A1 (en) 1999-10-29 2001-05-10 Stratagene Compositions and methods utilizing dna polymerases
NZ530816A (en) 1999-12-10 2005-10-28 Invitrogen Corp Use of multiple recombination sites with unique specificity in recombinational cloning
WO2003070918A2 (en) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Rna interference by modified short interfering nucleic acid
US20020001804A1 (en) 2000-02-25 2002-01-03 Wayne Mitchell Genomic analysis of tRNA gene sets
WO2002036626A1 (en) 2000-11-02 2002-05-10 Maxygen Aps Single-chain multimeric polypeptides
WO2002062816A1 (en) 2001-02-07 2002-08-15 Celltech R & D Limited Non-natural nucleotides and dinucleotides
US20020170681A1 (en) 2001-02-23 2002-11-21 Alain Saurer Letter moistener
EP1363927A2 (de) 2001-03-01 2003-11-26 Pharmasset Limited Verfahren zur herstellung von 2',3'-didehydronucleoside
JP2005502322A (ja) 2001-04-19 2005-01-27 ザ スクリップス リサーチ インスティテュート 非天然アミノ酸のインビボ組込み
NZ532027A (en) 2001-10-10 2008-09-26 Neose Technologies Inc Remodeling and glycoconjugation of peptides
US7026440B2 (en) 2001-11-07 2006-04-11 Nektar Therapeutics Al, Corporation Branched polymers and their conjugates
ES2381025T3 (es) * 2001-12-04 2012-05-22 Merck Patent Gmbh Inmunocitocinas con selectividad modulada
AUPR975201A0 (en) 2001-12-24 2002-01-24 Unisearch Limited Enzymatic redox labelling of nucleic acids
US20060074035A1 (en) 2002-04-17 2006-04-06 Zhi Hong Dinucleotide inhibitors of de novo RNA polymerases for treatment or prevention of viral infections
US7745417B2 (en) 2002-07-17 2010-06-29 Riken Nucleosides or nucleotides having novel unnatural bases and use thereof
US7977049B2 (en) 2002-08-09 2011-07-12 President And Fellows Of Harvard College Methods and compositions for extending the life span and increasing the stress resistance of cells and organisms
WO2004044139A2 (en) 2002-11-05 2004-05-27 Isis Parmaceuticals, Inc. Modified oligonucleotides for use in rna interference
AU2003291753B2 (en) 2002-11-05 2010-07-08 Isis Pharmaceuticals, Inc. Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
CA2507189C (en) 2002-11-27 2018-06-12 Sequenom, Inc. Fragmentation-based methods and systems for sequence variation detection and discovery
TWI364295B (en) 2002-12-26 2012-05-21 Mountain View Pharmaceuticals Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity
KR20060003862A (ko) 2003-03-14 2006-01-11 네오스 테크놀로지스, 인크. 수용성분기폴리머 및 그 접합체
US20070026485A1 (en) 2003-04-09 2007-02-01 Neose Technologies, Inc. Glycopegylation methods and proteins/peptides produced by the methods
CN101223272B (zh) 2003-04-17 2013-04-03 斯克利普斯研究院 扩展真核生物遗传密码
WO2004106356A1 (en) 2003-05-27 2004-12-09 Syddansk Universitet Functionalized nucleotide derivatives
WO2005007121A2 (en) 2003-07-18 2005-01-27 Massachusetts Institute Of Technology Mutant interleukin-2(il-2) polypeptides
ES2382807T3 (es) 2003-08-28 2012-06-13 Takeshi Imanishi Nuevos ácidos nucleicos artificiales del tipo de enlace N-O con reticulación
US20050069521A1 (en) * 2003-08-28 2005-03-31 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of interleukin-2 proteins
WO2005026187A1 (ja) 2003-09-10 2005-03-24 Riken 非天然型塩基を有するヌクレオシド又はヌクレオチド及びその利用
PL1675622T3 (pl) 2003-09-17 2017-11-30 Nektar Therapeutics Proleki na bazie wieloramiennego polimeru
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
EP1685246A2 (de) 2003-11-03 2006-08-02 Medical Research Council Polymerase
AU2005211362B2 (en) * 2004-02-02 2008-03-13 Ambrx, Inc. Modified human interferon polypeptides and their uses
JP2007527242A (ja) 2004-03-05 2007-09-27 カイロン コーポレーション 治療剤の患者耐容性を予測するためのインビトロ試験システム
WO2005092928A1 (en) 2004-03-11 2005-10-06 Fresenius Kabi Deutschland Gmbh Conjugates of hydroxyalkyl starch and a protein, prepared by reductive amination
US20060084136A1 (en) 2004-07-14 2006-04-20 Invitrogen Corporation Production of fusion proteins by cell-free protein synthesis
WO2006093526A2 (en) 2004-07-21 2006-09-08 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a modified or non-natural nucleobase
WO2006049297A1 (ja) 2004-11-08 2006-05-11 Riken 新規なヌクレオシド若しくはヌクレオチド誘導体及びその利用
EP1846020B1 (de) 2005-01-27 2013-08-21 Novartis Vaccines and Diagnostics, Inc. Verfahren zur behandlung von nierenzellenkarzinom
TWI376234B (en) 2005-02-01 2012-11-11 Msd Oss Bv Conjugates of a polypeptide and an oligosaccharide
JP5649018B2 (ja) 2005-08-04 2015-01-07 タグシクス・バイオ株式会社 新規人工塩基対及びその利用
CA2642657A1 (en) 2005-12-09 2007-06-14 Riken Method for replicating nucleic acids and novel unnatural base pairs
KR20130042043A (ko) 2006-01-27 2013-04-25 아이시스 파마수티컬즈 인코포레이티드 6-변형된 바이시클릭 핵산 유사체
DK1984382T3 (da) 2006-01-27 2012-09-03 Santaris Pharma As LNA modificerede fosforthiolerede oligonukleotider
CA2641133A1 (en) 2006-02-14 2007-08-23 Basf Se Pyridin-4 -ylmethylamides for combating pests
JP5196378B2 (ja) 2006-02-22 2013-05-15 独立行政法人理化学研究所 サプレッサーｔＲＮＡの合成方法、ＤＮＡ構築物及びそれを用いた非天然型アミノ酸組み込みタンパク質の製造
US20140314864A1 (en) 2006-03-31 2014-10-23 Massachusetts Institute Of Technology System for Targeted Delivery of Therapeutic Agents
EP2395099A3 (de) 2006-05-02 2012-05-16 Allozyne, Inc. Aminosäuresubstituierte Moleküle
DK2066684T3 (da) 2006-05-11 2012-10-22 Isis Pharmaceuticals Inc 5´-Modificerede bicycliske nukleinsyreanaloge
JP2009542592A (ja) 2006-07-06 2009-12-03 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング Ｉｌ−２媒介性免疫応答の有効性を高める組成物および方法
AU2007294124A1 (en) 2006-09-08 2008-03-13 Bayer Schering Pharma Aktiengesellschaft Compounds and methods for 18F labeled agents
MX2009002526A (es) 2006-09-08 2009-04-16 Ambrx Inc Transcripcion de tarn supresor en celulas de vertebrados.
JP2010510794A (ja) 2006-11-28 2010-04-08 ハナル ファーマシューティカル カンパニー リミテッド 修飾型エリスロポエチンポリペプチド及びこの治療用用途
EP2126083A1 (de) 2006-12-06 2009-12-02 Erik Bjerregaard Pedersen Hoogsteen-typ-triplex-bildung mit pyren markierter sonden für den nukleinsäurenachweis im fluoreszenz-assay
WO2008101157A1 (en) 2007-02-15 2008-08-21 Isis Pharmaceuticals, Inc. 5'-substituted-2'-f modified nucleosides and oligomeric compounds prepared therefrom
WO2008106186A2 (en) 2007-02-28 2008-09-04 Serina Therapeutics, Inc. Activated polyoxazolines and compositions comprising the same
EP2130835B1 (de) 2007-03-09 2012-05-23 Riken Verbindung mit aus einem mononukleosid oder mononukleotid abgeleiteter struktur, nukleinsäure-etikettierungssubstanz sowie verfahren und kit zur nukleinsäure-erkennung
WO2008150729A2 (en) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2008154401A2 (en) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
WO2009006478A2 (en) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
CA2707840A1 (en) 2007-08-20 2009-02-26 Allozyne, Inc. Amino acid substituted molecules
US8546556B2 (en) 2007-11-21 2013-10-01 Isis Pharmaceuticals, Inc Carbocyclic alpha-L-bicyclic nucleic acid analogs
JP5679147B2 (ja) 2008-02-07 2015-03-04 独立行政法人理化学研究所 非天然塩基を含む改変ｔＲＮＡ及びその用途
US8426569B2 (en) 2008-03-31 2013-04-23 Riken DNA capable of being amplified by PCR with high selectivity and high efficiency
BRPI0911501A2 (pt) 2008-04-29 2015-07-28 Monsanto Technology Llc Genes e usos para melhoramento de plantas.
WO2009155102A2 (en) 2008-05-30 2009-12-23 Barofold, Inc. Method for derivatization of proteins using hydrostatic pressure
WO2010023670A2 (en) 2008-08-28 2010-03-04 Insight Biopharmaceuticals Ltd. Methods for covalently attaching a polymer to a methionine residue in proteins and peptides
WO2010036698A1 (en) 2008-09-24 2010-04-01 Isis Pharmaceuticals, Inc. Substituted alpha-l-bicyclic nucleosides
WO2010068278A2 (en) 2008-12-10 2010-06-17 The Scripps Research Institute Production of carrier-peptide conjugates using chemically reactive unnatural amino acids
JP5766124B2 (ja) 2009-01-21 2015-08-19 アムジェン インコーポレイテッド 炎症性疾患および自己免疫疾患の処置の組成物および方法
WO2011043385A1 (ja) 2009-10-06 2011-04-14 独立行政法人理化学研究所 特異な塩基対を形成する人工塩基対
WO2011053065A2 (ko) 2009-10-29 2011-05-05 한국과학기술원 카테콜 폴리에틸렌글리콜 유도체와 단백질 또는 펩타이드의 접합체 및 이의 제조방법
EP3091027B1 (de) 2010-04-28 2018-01-17 Ionis Pharmaceuticals, Inc. 5'-modifizierte nukleoside und daraus hergestellte oligomere verbindungen
LT2637694T (lt) 2010-11-12 2021-05-25 Nektar Therapeutics Il-2 fragmento konjugatai ir polimeras
JP6093712B2 (ja) * 2010-12-22 2017-03-08 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー インターロイキン２のスーパーアゴニストおよびアンタゴニスト
PE20140303A1 (es) * 2011-02-10 2014-03-22 Roche Glycart Ag Polipeptidos interleuquina-2 mutantes
EA201892619A1 (ru) 2011-04-29 2019-04-30 Роше Гликарт Аг Иммуноконъюгаты, содержащие мутантные полипептиды интерлейкина-2
US8343752B2 (en) 2011-05-03 2013-01-01 Verdezyne, Inc. Biological methods for preparing adipic acid
ES2907763T3 (es) 2012-08-31 2022-04-26 Sutro Biopharma Inc Aminoácidos modificados que comprenden un grupo azido
US9650621B2 (en) 2012-10-12 2017-05-16 Sutro Biopharma, Inc. Proteolytic inactivation of select proteins in bacterial extracts for improved expression
JP6482471B2 (ja) 2012-12-21 2019-03-13 バイオアライアンス コマンディテール フェンノートシャップ 親水性の自壊性リンカー及びそのコンジュゲート
SG11201508642WA (en) 2013-04-19 2015-11-27 Sutro Biopharma Inc Expression of biologically active proteins in a bacterial cell-free synthesis system using cell extracts with elevated levels of exogenous chaperones
AU2014306271A1 (en) 2013-08-08 2016-03-24 The Scripps Research Institute A method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
WO2015038426A1 (en) 2013-09-13 2015-03-19 Asana Biosciences, Llc Self-immolative linkers containing mandelic acid derivatives, drug-ligand conjugates for targeted therapies and uses thereof
WO2015054590A2 (en) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. NON-NATURAL AMINO ACID tRNA SYNTHETASES FOR PYRIDYL TETRAZINE
WO2015054658A1 (en) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use
DK3055321T3 (en) 2013-10-11 2018-12-17 Sutro Biopharma Inc Non-natural amino acid tRNA synthetases for para-methylazido-1-phenylalanine
DK3129493T3 (da) 2014-04-09 2021-09-27 Scripps Research Inst Import af unaturlige eller modificerede nukleosidtriphosphater ind i celler via nukleinsyre-triphosphat-transportører
US10150802B2 (en) 2014-04-24 2018-12-11 The Board Of Trustees Of The Leland Stanford Junior University Superagonists, partial agonists and antagonists of interleukin-2
HUE043038T2 (hu) 2014-08-11 2019-07-29 Delinia Inc Regulator T-sejteket szelektíven aktiváló, módosított IL-2-változatok autoimmun betegségek kezelésére
US10487133B2 (en) 2014-12-19 2019-11-26 Sutro Biopharma, Inc. Codon optimization for titer and fidelity improvement
WO2016115168A1 (en) 2015-01-12 2016-07-21 Synthorx, Inc. Incorporation of unnatural nucleotides and methods thereof
MX2017012966A (es) 2015-04-10 2018-06-06 Amgen Inc Muteinas de interleuquina 2 para la expansion de celulas t regulatorias.
US11761007B2 (en) 2015-12-18 2023-09-19 The Scripps Research Institute Production of unnatural nucleotides using a CRISPR/Cas9 system
US20190015520A1 (en) 2015-12-21 2019-01-17 Duke University Polymer conjugates having reduced antigenicity and methods of using the same
KR20180133198A (ko) 2016-05-04 2018-12-13 암젠 인크 T-조절 세포의 증식을 위한 인터류킨-2 뮤테인
ES2929047T3 (es) 2016-06-24 2022-11-24 Scripps Research Inst Transportador de nucleósido trifosfato novedoso y usos del mismo
JP7325341B2 (ja) 2017-07-11 2023-08-14 シンソークス，インク． 非天然ヌクレオチドの組み込み及びその方法
NZ761430A (en) 2017-08-03 2024-03-22 Synthorx Inc Cytokine conjugates for the treatment of proliferative and infectious diseases
CN111615396A (zh) 2017-11-21 2020-09-01 小利兰·斯坦福大学董事会 白介素-2的部分激动剂
AU2018394189B2 (en) 2017-12-27 2023-12-21 Kyowa Kirin Co., Ltd. IL-2 variant
JP2021514974A (ja) 2018-02-26 2021-06-17 シンソークス， インコーポレイテッド Ｉｌ−１５コンジュゲートおよびその使用
JP2021519336A (ja) 2018-03-28 2021-08-10 アセンディス ファーマ オンコロジー ディヴィジョン エー／エス Ｉｌ−２コンジュゲート
CA3106858A1 (en) 2018-07-24 2020-01-30 Biontech Rna Pharmaceuticals Gmbh Il2 agonists
FI3849614T3 (fi) 2018-09-11 2024-02-08 Ambrx Inc Interleukiini-2-polypeptidikonjugaatteja ja niiden käyttöjä
US20210221863A1 (en) 2018-09-21 2021-07-22 Innovent Biologics (Suzhou) Co., Ltd. Novel interleukin-2 and use thereof
CN113260374A (zh) 2018-11-08 2021-08-13 新索思股份有限公司 白介素10缀合物及其用途
CA3125529A1 (en) 2019-01-07 2020-07-16 Inhibrx, Inc. Polypeptides comprising modified il-2 polypeptides and uses thereof
BR112021014415A2 (pt) 2019-02-06 2021-09-21 Synthorx, Inc. Conjugados de il-2 e métodos de uso dos mesmos
WO2020201095A1 (en) 2019-03-29 2020-10-08 Institut Curie Interleukin-2 variants with modified biological activity
US20220193253A1 (en) 2019-04-26 2022-06-23 Prolynx Llc Slow-release cytokine conjugates
KR20220034115A (ko) 2019-06-14 2022-03-17 큐진 인크. 암 치료용 신규한 인터루킨-2 변이체
US20220306714A1 (en) 2019-08-12 2022-09-29 AskGene Pharma, Inc. Il-2 fusion proteins that preferentially bind il-2ralpha
MX2022001866A (es) 2019-08-13 2022-03-11 Amgen Inc Muteinas de interleucina-2 para la expansion de celulas t reguladoras.
MX2022001776A (es) 2019-08-15 2022-03-17 Synthorx Inc Terapias de combinacion de inmuno oncologia con conjugados de il-2.
US20220289806A1 (en) 2019-08-15 2022-09-15 Cytimm Therapeutics, Inc. Modified Interleukin 2 (IL-2) Polypeptides, Conjugates and Uses Thereof
MX2022002053A (es) 2019-08-23 2022-03-17 Synthorx Inc Conjugados de il-15 y sus usos.
US20210070827A1 (en) 2019-09-10 2021-03-11 Synthorx, Inc. Il-2 conjugates and methods of use to treat autoimmune diseases
CN114853874A (zh) 2019-09-10 2022-08-05 中国医学科学院北京协和医院 靶向调节t细胞的长效白介素-2及其在治疗自身免疫病中的应用
TW202131952A (zh) 2019-11-04 2021-09-01 美商欣爍克斯公司 介白素10接合物及其用途
JP2023506223A (ja) 2019-12-13 2023-02-15 シンセカイン インコーポレイテッド Ｉｌ－２オルソログおよび使用法
MX2022007605A (es) 2019-12-23 2022-07-19 Synthorx Inc Metodos de preparacion de n6-((2-azidoetoxi)carbonil)lisina.
CN115315436A (zh) 2020-01-10 2022-11-08 明峰治疗股份公司 修饰的il-2多肽及其用途
US20220016252A1 (en) 2020-06-25 2022-01-20 Synthorx, Inc. Immuno oncology combination therapy with il-2 conjugates and anti-egfr antibodies
KR20230027235A (ko) 2020-06-25 2023-02-27 신톡스, 인크. Il-2 콘쥬게이트 및 항-egfr 항체를 사용한 면역 종양학 병용 요법
AU2021356610A1 (en) 2020-10-09 2023-06-15 Synthorx, Inc. Immuno oncology therapies with il-2 conjugates
MX2023004029A (es) 2020-10-09 2023-04-27 Synthorx Inc Terapia de combinacion de inmunoncologia con conjugados de il-2 y pembrolizumab.
WO2022174101A1 (en) 2021-02-12 2022-08-18 Synthorx, Inc. Skin cancer combination therapy with il-2 conjugates and cemiplimab
WO2022256538A1 (en) 2021-06-03 2022-12-08 Synthorx, Inc. Head and neck cancer combination therapy comprising an il-2 conjugate and cetuximab

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11701407B2 (en) 2017-08-03 2023-07-18 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
US11622993B2 (en) 2017-08-03 2023-04-11 Synthorx, Inc. Cytokine conjugates for the treatment of autoimmune diseases
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
US11919934B2 (en) 2018-02-26 2024-03-05 Synthorx, Inc. IL-15 conjugates and uses thereof
US11077195B2 (en) 2019-02-06 2021-08-03 Synthorx, Inc. IL-2 conjugates and methods of use thereof
WO2021216920A1 (en) 2020-04-22 2021-10-28 Iovance Biotherapeutics, Inc. Systems and methods for coordinating manufacturing of cells for patient-specific immunotherapy
WO2021226085A1 (en) 2020-05-04 2021-11-11 Iovance Biotherapeutics, Inc. Selection of improved tumor reactive t-cells
WO2021226061A1 (en) 2020-05-04 2021-11-11 Iovance Biotherapeutics, Inc. Processes for production of tumor infiltrating lymphocytes and uses of the same in immunotherapy
WO2021262437A3 (en) * 2020-06-21 2022-02-10 Ketan Desai Use of il13 for prevention and treatment of covid-19
WO2022076952A1 (en) 2020-10-06 2022-04-14 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
WO2022076606A1 (en) 2020-10-06 2022-04-14 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
WO2022125941A1 (en) 2020-12-11 2022-06-16 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with braf inhibitors and/or mek inhibitors
WO2022133149A1 (en) 2020-12-17 2022-06-23 Iovance Biotherapeutics, Inc. Treatment of cancers with tumor infiltrating lymphocytes
WO2022133140A1 (en) 2020-12-17 2022-06-23 Iovance Biotherapeutics, Inc. Treatment with tumor infiltrating lymphocyte therapies in combination with ctla-4 and pd-1 inhibitors
WO2022147196A2 (en) 2020-12-31 2022-07-07 Iovance Biotherapeutics, Inc. Devices and processes for automated production of tumor infiltrating lymphocytes
WO2022165260A1 (en) 2021-01-29 2022-08-04 Iovance Biotherapeutics, Inc. Methods of making modified tumor infiltrating lymphocytes and their use in adoptive cell therapy
WO2022170219A1 (en) 2021-02-05 2022-08-11 Iovance Biotherapeutics, Inc. Adjuvant therapy for cancer
WO2022187741A2 (en) 2021-03-05 2022-09-09 Iovance Biotherapeutics, Inc. Tumor storage and cell culture compositions
WO2022198141A1 (en) 2021-03-19 2022-09-22 Iovance Biotherapeutics, Inc. Methods for tumor infiltrating lymphocyte (til) expansion related to cd39/cd69 selection and gene knockout in tils
WO2022204564A2 (en) 2021-03-25 2022-09-29 Iovance Biotherapeutics, Inc. Methods and compositions for t-cell coculture potency assays and use with cell therapy products
WO2022225981A2 (en) 2021-04-19 2022-10-27 Iovance Biotherapeutics, Inc. Chimeric costimulatory receptors, chemokine receptors, and the use of same in cellular immunotherapies
WO2022245754A1 (en) 2021-05-17 2022-11-24 Iovance Biotherapeutics, Inc. Pd-1 gene-edited tumor infiltrating lymphocytes and uses of same in immunotherapy
WO2023004074A2 (en) 2021-07-22 2023-01-26 Iovance Biotherapeutics, Inc. Method for cryopreservation of solid tumor fragments
WO2023009716A1 (en) 2021-07-28 2023-02-02 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with kras inhibitors
WO2023039488A1 (en) 2021-09-09 2023-03-16 Iovance Biotherapeutics, Inc. Processes for generating til products using pd-1 talen knockdown
WO2023049862A1 (en) 2021-09-24 2023-03-30 Iovance Biotherapeutics, Inc. Expansion processes and agents for tumor infiltrating lymphocytes
WO2023077015A2 (en) 2021-10-27 2023-05-04 Iovance Biotherapeutics, Inc. Systems and methods for coordinating manufacturing of cells for patient-specific immunotherapy
WO2023086803A1 (en) 2021-11-10 2023-05-19 Iovance Biotherapeutics, Inc. Methods of expansion treatment utilizing cd8 tumor infiltrating lymphocytes
WO2023147486A1 (en) 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Tumor infiltrating lymphocytes engineered to express payloads
WO2023147488A1 (en) 2022-01-28 2023-08-03 Iovance Biotherapeutics, Inc. Cytokine associated tumor infiltrating lymphocytes compositions and methods
WO2023196877A1 (en) 2022-04-06 2023-10-12 Iovance Biotherapeutics, Inc. Treatment of nsclc patients with tumor infiltrating lymphocyte therapies
WO2023201369A1 (en) 2022-04-15 2023-10-19 Iovance Biotherapeutics, Inc. Til expansion processes using specific cytokine combinations and/or akti treatment
WO2023220608A1 (en) 2022-05-10 2023-11-16 Iovance Biotherapeutics, Inc. Treatment of cancer patients with tumor infiltrating lymphocyte therapies in combination with an il-15r agonist
WO2024011114A1 (en) 2022-07-06 2024-01-11 Iovance Biotherapeutics, Inc. Devices and processes for automated production of tumor infiltrating lymphocytes
WO2024030758A1 (en) 2022-08-01 2024-02-08 Iovance Biotherapeutics, Inc. Chimeric costimulatory receptors, chemokine receptors, and the use of same in cellular immunotherapies
WO2024055017A1 (en) 2022-09-09 2024-03-14 Iovance Biotherapeutics, Inc. Processes for generating til products using pd-1/tigit talen double knockdown
WO2024055018A1 (en) 2022-09-09 2024-03-14 Iovance Biotherapeutics, Inc. Processes for generating til products using pd-1/tigit talen double knockdown

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