RU2786116C2 - Cytokine conjugates for treatment of proliferative and infectious diseases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to interleukin-2 conjugates; it can be used in medicine. The invention discloses a conjugate of an option of IL-2 peptide with water-soluble polymer. Such a conjugate has increased time of action compared to unmodified natural peptide. In addition, a conjugating fragment included in a molecule of the claimed conjugate also weakens or blocks binding of IL-2 to IL-2Ra.

EFFECT: invention can be used in medical practice as an immune-modulating therapeutic agent in the treatment of a number of proliferative diseases.

28 cl, 6 ex, 11 tbl, 12 dwg

Description

Link

[1] This application claims priority under U.S. Provisional Application No. 62/540,781, filed Aug. 3, 2017, which is incorporated herein by reference in its entirety.

Sequence listing

[2] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on July 27, 2018, has the name 46085-710_601_SL.txt and is 3703 bytes in size.

Background of the Invention

[3] Different populations of T cells modulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T cells (Treg) prevent inappropriate responses of the immune system by preventing pathological self-reactivity, while cytotoxic T cells target and destroy infected cells and/or cancer cells. In some cases, modulation of different populations of T cells provides an opportunity to treat a disease or indication.

Brief summary of the present invention

[4] Disclosed herein are cytokine conjugates and their use in the treatment of one or more indications in certain embodiments. In some embodiments, interleukin 2 (IL-2) conjugates and use in the treatment of one or more indications are also described herein. In some cases, one or more indications include cancer or pathogenic infection. In some cases, described herein are methods for modulating the interaction between IL-2 and the IL-2 receptor to stimulate or expand populations of specific T cells, natural killer (NK) cells and/or natural killer T cells (NKT). In additional cases, further described herein are pharmaceutical compositions and kits that contain one or more of the interleukin conjugates described herein (eg, IL-2 conjugates).

[5] Disclosed herein in some embodiments is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide and a conjugating fragment that binds to the isolated and purified IL-2 polypeptide at an amino acid residue position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, with the amino acid residue numbering corresponding to SEQ ID NO 1. In some embodiments, 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. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 further mutates 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. In some embodiments, an 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 into a non-natural amino acid. In some embodiments, the non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylatethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2 -amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m-acetylphenylalanine, 2- amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p- benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl- L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy) -3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoyl or selenium cysteine. In some embodiments, the IL-2 conjugate has reduced affinity for the α IL-2 receptor (IL-2Rα) subunit relative to the wild-type IL-2 polypeptide. In some embodiments, reduced affinity is about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% reduction in binding affinity. to IL-2Rα relative to the wild-type IL-2 polypeptide. In some embodiments, the reduced 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. fold, 50 fold, 100 fold, 200 fold, 300 fold, 500 fold, 1000 fold or more of the wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety reduces or blocks the binding of IL-2 to IL-2Rα. In some embodiments, the conjugating moiety contains a water-soluble polymer. In some embodiments, the additional conjugating moiety contains a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), ethylene glycol-propylene glycol copolymers, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide ), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each of the water-soluble polymers independently contains PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently contains a polysaccharide. In some embodiments, the polysaccharide includes dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently contains a glycan. In some embodiments, each of the water-soluble polymers independently contains a polyamine. In some embodiments, the conjugating fragment contains a protein. In some embodiments, the additional conjugating portion comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently contains an Fc moiety. In some embodiments, each of the proteins independently contains the Fc portion of an IgG. In some embodiments, the conjugating fragment comprises a polypeptide. In some embodiments, the additional conjugating portion comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an 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. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating fragment is directly linked to an isolated and purified IL-2 polypeptide. In some embodiments, the conjugating fragment is indirectly linked to an isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker includes a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobispropionimidate ( DTBP), 1,4-di-(3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), an aryl halide compound (DFDNB) such as, for example, 1,5-difluoro-2,4 -dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde , glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzy din, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker includes a heterobifunctional linker. In some embodiments, 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-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), complex m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MB), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (Sulfo- sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinim ester yes (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl-6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl-6-[6-(((iodoacetyl)amino) hexanoyl)amino]hexanoate (sIAXX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl-6-(((((4-iodoacetyl)amino)methyl)cyclohexane- 1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N- maleimidomethyl)cyclohexane-1-carboxylhydrazide-8 (M ₂ C ₂ H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl 1,3'-dithiopropionate (sAsD), N- hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azide obenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH) , N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl )1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl-4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl- 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate (sulfo-CAMCA), p-nitrophenyldiazopyruvate ( ρNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p- azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG) . In some embodiments, the linker includes a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker includes a non-cleavable linker. In some embodiments, the linker contains a maleimide group optionally containing maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo -sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative or analog thereof. In some embodiments, the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.

[6] In some embodiments, disclosed herein is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide and a conjugating fragment; wherein the IL-2 conjugate has reduced affinity for the a subunit of the IL-2 receptor (IL-2Rα) compared to the wild-type IL-2 polypeptide. In some embodiments, the conjugating fragment is linked to an amino acid residue that interacts with IL-2Rα. In some embodiments, the conjugating fragment is linked to an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, with amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the conjugation moiety contains a water-soluble polymer. In some embodiments, the additional conjugating moiety contains a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), ethylene glycol-propylene glycol copolymers, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide ), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each of the water-soluble polymers independently contains PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently contains a polysaccharide. In some embodiments, the polysaccharide includes dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently contains a glycan. In some embodiments, each of the water-soluble polymers independently contains a polyamine. In some embodiments, the conjugating fragment contains a protein. In some embodiments, the additional conjugating portion comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently contains an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugating fragment comprises a polypeptide. In some embodiments, the additional conjugating portion comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an 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. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating fragment is directly linked to an isolated and purified IL-2 polypeptide. In some embodiments, the conjugating fragment is indirectly linked to an isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker includes a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl suberate) (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST ), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyldimethylpimelimidate (DMP), dimethyl 3,3'-dithiobispropionimidate (DTBP), 1.4 -di-(3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), an aryl halide compound (DFDNB) such as, for example, 1,5-difluoro-2,4-dinitrobenzene or 1, 3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[p-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, diglycidyl ether 1,4-butanediol, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α '-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker includes a heterobifunctional linker. In some embodiments, 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-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), complex m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl(4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo- sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinim ester yes (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl-6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl-6-[6-(((iodoacetyl)amino) hexanoyl)amino]hexanoate (sIAXX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl-6-(((((4-iodoacetyl)amino)methyl)cyclohexane- 1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N- maleimidomethyl)cyclohexane-1-carboxylhydrazide-8 (M ₂ C ₂ H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl 1,3'-dithiopropionate (sAsD), N- hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azide obenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH) , N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl )1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl-4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl- 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyldiazopyruvate ( ρNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p- azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG) . In some embodiments, the linker includes a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker includes a non-cleavable linker. In some embodiments, the linker contains a maleimide group optionally containing maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo -sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer includes p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative or analog thereof. In some embodiments, the conjugating fragment is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.

[7] Disclosed herein in some embodiments is an isolated and modified interleukin 2 (IL-2) polypeptide containing at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and interleukin 2 receptor a ( IL-2Rα), but does not significantly affect binding to the interleukin 2 receptor βγ (IL-2Rβγ) signaling complex to form an IL-2/IL-2Rβγ complex, with reduced binding to IL-2Rα compared to binding between an IL-2 polypeptide wild type and IL-2Rα. In some embodiments, the position of 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, with residue positions corresponding to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105 and 107, as shown in SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, with residue positions corresponding to positions 37, 38 , 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as shown in SEQ ID NO: 1. In some embodiments, the position of at least one non-natural amino acid is selected from K35, K64, V69, N71 , M104, C105, and Y107, with the positions of the residues corresponding to positions 35, 64, 69, 71, 104, 105, and 107, as shown in SEQ ID NO: 1. In some embodiments, the position is at least one non-natural amino acid is selected from T37, R38, T41, Y45, E61, E68 and L72, with residue positions corresponding to positions 37, 38, 41, 45, 61, 68 and 72, as shown in SEQ ID NO: 1 In some embodiments, the at least one unnatural amino acid position is selected from F42, K43, F44, E62, and P65, with residue positions corresponding to positions 42, 43, 44, 62, and 65, as shown in SEQ. ID NO: 1. In some embodiments, at least one non-natural amino acid: is a lysine analog; contains an aromatic side chain; contains an azido group or contains an aldehyde or ketone group. In some embodiments, at least one non-natural amino acid does not contain an aromatic side chain. In some embodiments, at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine , allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m- acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylalanine-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-ClcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3 -(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenyls elanil)propanoyl or selenocysteine. In some embodiments, at least one non-natural amino acid is incorporated into the modified IL-2 polypeptide via an orthogonal tRNA-synthetase/tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair contains at least one non-natural nucleotide base. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugating moiety through at least one non-natural amino acid. In some embodiments, the conjugating moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water-soluble polymer contains a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer contains a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid contains a fatty acid. In some embodiments, the fatty acid has about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms carbon, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 up to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein includes albumin, transferrin, or transthyretin. In some embodiments, the protein comprises a TLR agonist. In some embodiments, the protein includes an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof comprise the Fc portion of the antibody. In some embodiments, the peptide includes an 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. In some embodiments, the conjugating moiety is indirectly linked to at least one non-natural amino acid of modified IL-2 via a linker. In some embodiments, the linker includes a homobifunctional linker, a heterobifunctional linker, a zero length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the reduction in binding affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in binding affinity for IL-2Rα relative to the polypeptide IL-2 wild type. In some embodiments, the reduction 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 with respect to IL-2Rα with respect to wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; recombinant IL-2 polypeptide; or recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide contains an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, an N-terminal deletion includes 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 N-terminus, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion includes the deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, with the positions of the residues referring to positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment contains the IL-2 region 10-133, 20- 133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the positions of the residues refer to those in SEQ ID NO: 1 In some embodiments, the modified IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. According to some implementation options m a modified IL-2 polypeptide with reduced binding affinity for IL-2Rα is capable of causing expansion of populations of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T cells (NKT), or combinations thereof. In some embodiments, the conjugating moiety reduces or blocks the binding of IL-2 to IL-2Rα. In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T (NKT) cells via the IL-2Rβγ complex with a modified IL-2 polypeptide is not significantly different from activation said cell population with a wild-type IL-2 polypeptide, and wherein the activity of the modified IL-2 polypeptide is at least 1-fold higher than the activity of the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide causes expansion of CD4+ T regulatory cells (Treg) by less than 20%, 15%, 10%, 5%, 1%, or 0.1% when said activator is in contact with said population of cells. In some embodiments, the modified IL-2 polypeptide does not cause expansion of Treg cells in said population of cells.

[8] Disclosed herein in some embodiments is an isolated and modified interleukin 2 (IL-2) polypeptide containing at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and the interleukin 2 receptor α ( IL-2Rα), but retains significant binding to the interleukin 2 βγ receptor (IL-2Rβγ) signaling complex to form an IL-2/IL-2Rβγ complex, with reduced binding to IL-2Rα compared to binding between wild-type IL-2 polypeptide and IL-2Ra. In some embodiments, the difference in activity in receptor signaling is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. In some embodiments, the position of 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, with residue positions corresponding to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105 and 107, as shown in SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, with residue positions corresponding to positions 37, 38 , 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as shown in SEQ ID NO: 1. In some embodiments, the position of at least one non-natural amino acid is selected from K35, K64, V69, N71 , M104, C105, and Y107, with the positions of the residues corresponding to positions 35, 64, 69, 71, 104, 105, and 107, as shown in SEQ ID NO: 1. In some embodiments, the position is at least one non-natural amino acid is selected from T37, R38, T41, Y45, E61, E68 and L72, with residue positions corresponding to positions 37, 38, 41, 45, 61, 68 and 72, as shown in SEQ ID NO: 1 In some embodiments, the at least one unnatural amino acid position is selected from F42, K43, F44, E62, and P65, with residue positions corresponding to positions 42, 43, 44, 62, and 65, as shown in SEQ. ID NO: 1. In some embodiments, at least one non-natural amino acid: is a lysine analog; contains an aromatic side chain; contains an azido group; contains an alkyne group or contains an aldehyde or ketone group. In some embodiments, at least one non-natural amino acid does not contain an aromatic side chain. In some embodiments, at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine , allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylalanine- L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L- tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-( (3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(pheny lselanil)propanoyl or selenocysteine. In some embodiments, at least one non-natural amino acid is incorporated into the modified IL-2 polypeptide via an orthogonal tRNA-synthetase/tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair contains at least one non-natural nucleotide base. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugating moiety through at least one non-natural amino acid. In some embodiments, the conjugating moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water-soluble polymer contains a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer contains a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid contains a fatty acid. In some embodiments, the fatty acid has about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms carbon, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 up to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein includes albumin, transferrin, or transthyretin. In some embodiments, the conjugating fragment comprises a TLR agonist. In some embodiments, the protein includes an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof comprise the Fc portion of the antibody. In some embodiments, the peptide includes an 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. In some embodiments, the conjugating moiety is indirectly linked to at least one non-natural amino acid of modified IL-2 via a linker. In some embodiments, the linker includes a homobifunctional linker, a heterobifunctional linker, a zero length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the reduction in binding affinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% reduction in binding affinity for IL-2Rα relative to the IL-2 polypeptide wild type. In some embodiments, the reduction in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more for IL-2Rα relative to the IL- 2 wild type. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; recombinant IL-2 polypeptide; or recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide contains an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, an N-terminal deletion includes 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 N-terminus, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion includes the deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, with the positions of the residues referring to positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment contains the IL-2 region 10-133, 20- 133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the positions of the residues refer to those in SEQ ID NO: 1 In some embodiments, the modified IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. According to some implementation options m a modified IL-2 polypeptide with reduced binding affinity for IL-2Rα is capable of causing expansion of populations of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T cells (NKT), or combinations thereof. In some embodiments, the conjugating moiety or non-natural amino acid reduces or blocks the binding of IL-2 to IL-2Rα. In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T (NKT) cells via the IL-2Rβγ complex with a modified IL-2 polypeptide retains significant activation activity. said cell population relative to the wild-type IL-2 polypeptide. In some embodiments, the activity in receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is greater than the activity in receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the receptor signaling activity of the modified IL-2 polypeptide of the IL-2Rβγ complex is lower than the receptor signaling activity of the wild-type IL-2 polypeptide of the IL-2Rβγ complex. In some embodiments, the modified IL-2 polypeptide exhibits a first IL-2Rβγ receptor signaling activity and a second IL-2Rαβγ receptor signaling activity, wherein the first receptor signaling activity is at least 1-fold, 2- 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 50x, 100x, 500-fold or higher than the second activity in receptor signaling. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is higher than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rβγ, and the second activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of a wild-type IL-2 polypeptide relative to IL-2Rαβγ. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is at least 1-fold lower than the activity in receptor signaling of the IL-2 polypeptide wild type.

[9] Disclosed herein in some embodiments is an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid, wherein the isolated and modified IL-2 polypeptide exhibits a first activity in receptor signaling to the IL signaling complex -2βγ and a second activity in receptor signaling to the IL-2α signaling complex, and wherein the difference between the first activity in receptor signaling and the second activity in receptor signaling is less than 10-fold. In some embodiments, the difference in activity in receptor signaling is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. In some embodiments, the position of 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, with residue positions corresponding to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105 and 107, as shown in SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, with residue positions corresponding to positions 37, 38 , 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as shown in SEQ ID NO: 1. In some embodiments, the position of at least one non-natural amino acid is selected from K35, K64, V69, N71 , M104, C105, and Y107, with the positions of the residues corresponding to positions 35, 64, 69, 71, 104, 105, and 107, as shown in SEQ ID NO: 1. In some embodiments, the position is at least one non-natural amino acid is selected from T37, R38, T41, Y45, E61, E68 and L72, with residue positions corresponding to positions 37, 38, 41, 45, 61, 68 and 72, as shown in SEQ ID NO: 1 In some embodiments, the at least one unnatural amino acid position is selected from F42, K43, F44, E62, and P65, with residue positions corresponding to positions 42, 43, 44, 62, and 65, as shown in SEQ. ID NO: 1. In some embodiments, at least one non-natural amino acid: is a lysine analog; contains an aromatic side chain; contains an azido group; contains an alkyne group or contains an aldehyde or ketone group. In some embodiments, at least one non-natural amino acid does not contain an aromatic side chain. In some embodiments, at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine , allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m- acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylalanine-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3 -(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenyls elanil)propanoyl or selenocysteine. In some embodiments, at least one non-natural amino acid is incorporated into the modified IL-2 polypeptide via an orthogonal tRNA-synthetase/tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair contains at least one non-natural nucleotide base. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugating moiety through at least one non-natural amino acid. In some embodiments, the conjugating moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water-soluble polymer contains a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer contains a polysaccharide. In some embodiments, the polysaccharide includes dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid contains a fatty acid. In some embodiments, the fatty acid has about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms carbon, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 up to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein includes albumin, transferrin, or transthyretin. In some embodiments, the conjugating fragment comprises a TLR agonist. In some embodiments, the protein includes an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof comprise the Fc portion of the antibody. In some embodiments, the peptide includes an 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. In some embodiments, the conjugating moiety is indirectly linked to at least one non-natural amino acid of modified IL-2 via a linker. In some embodiments, the linker includes a homobifunctional linker, a heterobifunctional linker, a zero length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the reduction in binding affinity is a reduction in binding affinity for IL-2Rα of about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% relative to wild-type IL-2 polypeptide. In some embodiments, the reduction in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more for IL-2Rα relative to the IL- 2 wild type. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; recombinant IL-2 polypeptide; or recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide contains an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, an N-terminal deletion includes 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 N-terminus, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion includes the deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, with the positions of the residues referring to positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment contains the IL-2 region 10-133, 20- 133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the positions of the residues refer to those in SEQ ID NO: 1 In some embodiments, the modified IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. According to some implementation options m a modified IL-2 polypeptide with reduced binding affinity for IL-2Rα is capable of causing expansion of populations of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T cells (NKT), or combinations thereof. In some embodiments, the conjugating moiety or non-natural amino acid reduces or blocks the binding of IL-2 to IL-2Rα. In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T (NKT) cells via the IL-2Rβγ complex with a modified IL-2 polypeptide retains the essential activation activity of said cell population relative to the wild-type IL-2 polypeptide. In some embodiments, the activity in receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is greater than the activity in receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the activity in receptor signaling of the modified IL-2 polypeptide of the IL-2Rβγ complex is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the modified IL-2 polypeptide exhibits a first IL-2Rβγ receptor signaling activity and a second IL-2Rαβγ receptor signaling activity, wherein the first receptor signaling activity is at least 1-fold, 2- 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 50x, 100x, 500-fold or higher than the second activity in receptor signaling. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is higher than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rβγ, and the second activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of a wild-type IL-2 polypeptide relative to IL-2Rαβγ. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is at least 1-fold lower than the activity in receptor signaling of the IL-2 polypeptide wild type.

[10] Disclosed herein in some embodiments is an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid, wherein the isolated and modified IL-2 polypeptide provides a first EC50 value for activation of the IL-2βγ signaling complex and a second EC50 value for activating the IL-2αβγ signaling complex, wherein the difference between the first EC50 value and the second EC50 value is less than 10-fold. In some embodiments, the difference is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. In some embodiments, the difference in activity in receptor signaling is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. In some embodiments, the position of 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, with residue positions corresponding to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105 and 107, as shown in SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, with residue positions corresponding to positions 37, 38 , 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as shown in SEQ ID NO: 1. In some embodiments, the position of at least one non-natural amino acid is selected from K35, K64, V69, N71 , M104, C105, and Y107, with the positions of the residues corresponding to positions 35, 64, 69, 71, 104, 105, and 107, as shown in SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is selected from T37, R38, T41, Y45, E61, E68 and L72, with residue positions corresponding to positions 37, 38, 41, 45, 61, 68 and 72, as shown in SEQ ID NO: 1 In some embodiments, the at least one non-natural amino acid position is selected from F42, K43, F44, E62, and P65, with residue positions corresponding to positions 42, 43, 44, 62, and 65, as shown in SEQ. ID NO: 1. In some embodiments, at least one non-natural amino acid: is a lysine analog; contains an aromatic side chain; contains an azido group; contains an alkyne group or contains an aldehyde or ketone group. In some embodiments, at least one non-natural amino acid does not contain an aromatic side chain. In some embodiments, at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine , allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m- acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylalanine-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcHAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3 -(benzyloxy)-3-xopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylse lanyl)propanoyl or selenocysteine. In some embodiments, at least one non-natural amino acid is incorporated into the modified IL-2 polypeptide via an orthogonal tRNA-synthetase/tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair contains at least one non-natural nucleotide base. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugating moiety through at least one non-natural amino acid. In some embodiments, the conjugating moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water-soluble polymer contains a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer contains a polysaccharide. In some embodiments, the polysaccharide includes dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid contains a fatty acid. In some embodiments, the fatty acid has about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms carbon, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 up to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein includes albumin, transferrin, or transthyretin. In some embodiments, the conjugating fragment comprises a TLR agonist. In some embodiments, the protein includes an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof comprise the Fc portion of the antibody. In some embodiments, the peptide includes an 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. In some embodiments, the conjugating moiety is indirectly linked to at least one non-natural amino acid of modified IL-2 via a linker. In some embodiments, the linker includes a homobifunctional linker, a heterobifunctional linker, a zero length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the reduction in binding affinity is a reduction in binding affinity for IL-2Rα of about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% relative to the polypeptide IL-2 wild type. In some embodiments, the reduction in binding affinity is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more for IL-2Rα relative to the IL- 2 wild type. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; recombinant IL-2 polypeptide; or recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide contains an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, an N-terminal deletion includes 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 N-terminus, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion includes the deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, with the positions of the residues referring to positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment contains the IL-2 region 10-133, 20- 133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, where the positions of the residues refer to those in SEQ ID NO: 1 In some embodiments, the modified IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, a modified IL-2 polypeptide with reduced binding affinity for IL-2Rα is capable of causing expansion of populations of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T cells (NKT), or their combinations. In some embodiments, the conjugating moiety or non-natural amino acid reduces or blocks the binding of IL-2 to IL-2Rα. In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T (NKT) cells via the IL-2Rβγ complex with a modified IL-2 polypeptide retains the essential activation activity of said cell population relative to the wild-type IL-2 polypeptide. In some embodiments, the activity in receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is greater than the activity in receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the activity in receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the modified IL-2 polypeptide exhibits a first IL-2Rβγ receptor signaling activity and a second IL-2Rαβγ receptor signaling activity, wherein the first receptor signaling activity is at least 1-fold, 2- 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 50x, 100x, 500-fold or higher than the second activity in receptor signaling. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is higher than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rβγ, and the second activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of a wild-type IL-2 polypeptide to IL-2Rαβγ. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is at least 1-fold lower than the activity in receptor signaling of the wild-type IL-2 polypeptide .

[11] Disclosed herein in some embodiments is a pharmaceutical composition comprising: an IL-2 conjugate as described above and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. [12] Disclosed herein, in some embodiments, is 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 the isolated and modified IL-2 polypeptide described above, the IL-2 conjugate described above, the binding IL described above. -2Rβγ protein, the activator of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, natural killer T cells (NKT) described above, or the pharmaceutical composition described above. In some embodiments, the proliferative disease or condition is cancer. In some embodiments, the cancer is a solid malignancy. In some embodiments, the solid 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, cancer pancreas or prostate cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematologic malignancy is chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma , extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's large B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, B-lymph region lymphoma progenitor cells, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, an isolated and modified IL-2 polypeptide, an IL-2 conjugate, an IL-2Rβ binding protein, an activator of a CD4+ helper cell, a CD8+ effector naive and memory cell, a natural killer (NK) cell, or a natural killer T cell (NKT ) or the pharmaceutical composition and the additional therapeutic agent are administered simultaneously. In some embodiments, an isolated and modified IL-2 polypeptide, an IL-2 conjugate, an IL-2Rβ binding protein, an activator of a CD4+ helper cell, a CD8+ effector naive and memory cell, a natural killer (NK) cell, or a natural killer T cell (NKT ) or the pharmaceutical composition and the additional therapeutic agent are administered sequentially. In some embodiments, an isolated and modified IL-2 polypeptide, an IL-2 conjugate, an IL-2Rβ binding protein, an activator of a CD4+ helper cell, a CD8+ effector naive and memory cell, a natural killer (NK) cell, or a natural killer T cell (NKT ) or the pharmaceutical composition and the additional therapeutic agent are administered prior to the administration of the additional therapeutic agent. In some embodiments, an isolated and modified IL-2 polypeptide, an IL-2 conjugate, an IL-2Rβ binding protein, an activator of a CD4+ helper cell, a CD8+ effector naive and memory cell, a natural killer (NK) cell, or a natural killer T cell (NKT ) or the pharmaceutical composition and the additional therapeutic agent are administered after the administration of the additional therapeutic agent.

[13] Disclosed herein, in some embodiments, is a method for expanding a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T cells (NKT), comprising: contacting the cell population with the described the above isolated and modified IL-2 polypeptide, the above-described IL-2 conjugate, the above-described IL-2Rβγ binding protein, the above-described activator of CD4+ helper cell, CD8+ effector naive cell and memory cell, natural killer (NK) cell or natural killer T cell killer (NKT) or the pharmaceutical composition described above for a time sufficient to induce the formation of a complex with IL-2Rβγ, thereby stimulating the expansion of the Teff and/or NK cell population. In some embodiments, the above-described isolated and modified IL-2 polypeptide, the above-described IL-2 conjugate, the above-described IL-2Rβγ binding protein, the above-described CD4+ helper cell activator, CD8+ effector naive and memory cells, natural killer (NK) cells or natural killer T cells (NKT) or the pharmaceutical composition described above causes expansion of CD4+ T regulatory cells (Treg) by less than 20%, 15%, 10%, 5% or 1% in CD3+ cell population compared to CD4+ expansion Treg cells in a CD3+ cell population in contact with a wild-type IL-2 polypeptide. In some embodiments, the above-described isolated and modified IL-2 polypeptide, the above-described IL-2 conjugate, the above-described IL-2Rβγ binding protein, the above-described CD4+ helper cell activator, CD8+ effector naive and memory cells, natural killer (NK) cells or natural killer T cells (NKT) or the pharmaceutical composition described above does not cause expansion of CD4+ Treg cells in the cell population. In some embodiments, the ratio of 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-2Rβγ binding protein described above, the CD4+ helper cell activator described above , CD8+ effector naive cell and memory cell, natural killer (NK) cell or natural killer T cell (NKT) or 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. In some embodiments, 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.

[14] Disclosed herein in some embodiments is a method for expanding a population of CD4+ helper cells, a population of CD8+ effector naive and memory cells, a population of natural killer (NK) cells, or a population of natural killer T cells (NKT), or a combination thereof, comprising: (a) contacting the cell with the above-described IL-2 conjugate and (b) interaction of IL-2 with IL-2Rβ and IL-2Rγ subunits to form an IL-2/IL-2Rβγ complex; wherein the IL-2 conjugate has reduced affinity for the IL-2Rα subunit, and wherein the IL-2/IL-2Rβγ complex stimulates the expansion of CD4+ helper cells, CD8+ effector naive and/or memory cells, NK cells, NKT cells, or combinations thereof .

[15] Disclosed herein in some embodiments is a kit comprising an isolated and modified IL-2 polypeptide as described above, an IL-2 conjugate as described above, an IL-2Rβγ binding protein as described above, a CD4+ helper cell activator as described above, a CD8+ effector naive cell as described above. and memory cells, natural killer (NK) cells or natural killer T cells (NKT), or a pharmaceutical composition as described above containing an IL-2 conjugate as described above. In some embodiments, the present document also describes a kit containing a polynucleic acid molecule encoding an IL-2 polypeptide as described above.

Brief description of graphic materials

[16] New features of the present invention are set forth with particular attention in the appended claims. A better understanding of the features and advantages of the present invention will be obtained with reference to the following detailed description, which provides illustrative embodiments that use the principles of the present invention, and the accompanying drawings.

[17] FIG. 1 shows illustrative non-natural amino acids. This figure is adapted from Fig. 2 Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry 285(15): 11039-11044 (2010).

[18] FIG. 2A-fig. 2B shows exemplary non-natural amino acids. In FIG. 2A shows exemplary lysine derivatives. In FIG. 2B shows exemplary phenylalanine derivatives.

[19] FIG. 3A-fig. 3D shows illustrative non-natural amino acids. These unnatural amino acids (UAA) 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). Fig. 3A-3D are taken from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69.

[1] FIG. 4A-fig. 4B shows the surface plasmon resonance (SPR) analysis of the P65_30kD, P65_5kD, E62_30kD and E62_5kD PEG conjugates. In FIG. 4A shows SPR analysis of IL-2 binding variants with immobilized IL-2 Rα. In FIG. 4B shows SPR analysis of IL-2 binding variants with immobilized IL-2 Rβ.

[2] FIG. 5A-fig. 5C shows exemplary variant IL-2 dose-response curves for pSTATS signaling in primary human LRS cell populations. Fig. 5A: native IL-2; fig. 5B: E62_30kD and fig. 5C: P65_30kD.

[3] FIG. 6 shows mean (±SD) plasma concentration versus time after a single intravenous bolus dose of aldesleukin (IL-2), E62_5, E62_30 and P65_30 mice C57BL/6.

[4] FIG. 7 shows the percentage of pSTAT5+CD8+ T cells over time in peripheral blood after administration of a single intravenous bolus dose of P65_30 or aldesleukin to C57BL/6 mice.

[5] FIG. 8A-fig. 8C shows the percentage of CD8+ T cells (FIG. 8A), NK cells (FIG. 8B), and CD4+ Treg cells (FIG. 8C) in the PBMC population after administration of a single intravenous bolus dose of P65_30 or aldesleukin (IL-2). Blood was collected by cardiac puncture at the indicated time points and immune cell populations were assessed by flow cytometry. Each data point is the mean of three repetitions at each time point ± SEM.

[6] FIG. 9A - fig. 9B shows the differences between P65_30 and IL-2 (aldesleukin) in promoting CD8+CD44+ memory T cell proliferation in the CD3+ population after administration of a single intravenous bolus dose of P65_30 or aldesleukin (IL-2). Blood was collected by cardiac puncture at the indicated time points and immune cell populations were assessed by flow cytometry. Data were analyzed using unpaired Student's t-test. *** denotes P<0.001 values. In FIG. 9A shows the proliferation of CD8+ CD44+ memory T cells at 72, 96 and 120 hours. In FIG. 9B shows the flow cytometry analysis of these cells at 120 hours.

[7] FIG. 10 shows the increase in tumor infiltrating lymphocytes (TIL) over time in C57B16 mice with syngeneic B16F10 tumors following a single intravenous bolus dose of P65_30. In FIG. 10A shows the percentage of NK, CD8+ T and CD4+ T reg cells in P65_30 treated animals compared to unexposed animals (vehicle) on day 5 of exposure. In FIG. 10B shows the ratio of CD8+/CD4+Treg cells in P6530 treated and control (vehicle) animals. Data were analyzed using unpaired Student's t-test. *** denotes P<0.001 values.

[20] FIG. 11A - fig. 11B shows plasma levels of mouse IL-2, TNF-α, IFNγ, IL-5, and IL-6 following a single intravenous bolus dose of P65_30 or aldesleukin (IL-2) at increasing levels (0.01-5 mg/kg ). Plasma concentration of each cytokine was determined by ELISA (Abcam, Cambridge, UK). For each N=3 dose group, mice were sampled at 4, 34, and 72 hours post-dose. In FIG. 11A shows the content of cytokines for animals treated with aldesleukin, and FIG. 11 B for animals dosed with P65_30.

[21] FIG. 12 shows the number of leukocytes, lymphocytes and eosinophils (mean ± standard deviation) after a single intravenous dose of P65_30kD in male cynomolgus monkeys.

Detailed description of the present disclosure

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

[23] Interleukins are signaling proteins that modulate the development and differentiation of T- and B-lymphocytes, cells of monocytic origin, 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.

[24] Interleukin 2 (IL-2) is a type 1 pleiotropic cytokine whose structure includes four a-helical bundles of 15.5 kDa. The IL-2 precursor form is 153 amino acid residues long, with the first 20 amino acids forming the signal peptide and residues 21-153 forming the mature form. IL-2 is produced primarily by CD4+ T cells upon antigen stimulation, and to a lesser extent by CD8+ cells, natural killer (NK) and natural killer T (NKT) cells, activated dendritic cells (DC), and mast cells. IL-2 signaling occurs through interaction with specific combinations of IL-2 receptor subunits (IL-2R), IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132). ). The interaction of IL-2 with IL-2Rα forms a "low-affinity" IL-2 receptor complex with a K _d of about 10 ^-8 M. The interaction of IL-2 with IL-2Rβ and IL-2Rγ forms an "intermediate affinity" IL-2 receptor complex with K _d about 10 ^-9 M. The interaction of IL-2 with all three subunits, IL-2Rα, IL-2Rβ and IL-2Rγ, forms a "high-affinity" IL-2 receptor complex with K _d about >10 ^-11 M.

[25] In some cases, IL-2 signaling through the "high affinity" IL-2Rαβγ complex modulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells (Treg) mediate the maintenance of immune homeostasis by suppressing effector cells such as CD4 ⁺ T cells, CD8 ⁺ T cells, B cells, NK cells and NKT -cells. In some cases, Treg cells are derived from the thymus (tTreg cells) or induced from naive T cells in the periphery (pTreg cells). In some cases, Treg cells are considered as a mediator of peripheral tolerance. Indeed, in one study, the transfer of peripheral CD25-depleted CD4 ⁺ T cells caused various autoimmune diseases in nude mice, while the co-transfer of CD4 ⁺ CD25 ⁺ T cells suppressed the development of autoimmunity (Sakaguchi, et al., "Immunologic self- tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)," J. Immunol. 155(3): 1151-1164 (1995)). An increase in the population of Treg cells suppresses the proliferation of effector T cells and suppresses autoimmunity and antitumor T cell responses.

[26] IL-2 signaling through the "intermediate affinity" IL-2Rβγ complex modulates the activation and proliferation of CD8 ⁺ effector T cells (Teff), NK cells, and NKT cells. CD8 ⁺ Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTL, killer T cells, cytolytic T cells, Tcon, or killer T cells) are T lymphocytes that recognize and kill damaged cells, cancer cells, and pathogen-infected cells. NK and NKT cells are types of lymphocytes that, like CD8 ⁺ Teff cells, target cancer cells and pathogen-infected cells.

[27] In some cases, IL-2 signaling is used to modulate T cell responses and then to treat cancer. For example, IL-2 is administered in high dose form to induce expansion of Teff cell populations for cancer treatment. However, high doses of IL2 additionally lead to concomitant stimulation of Treg cells, which attenuate antitumor immune responses. IL2 at high doses also induces toxic adverse events due to involvement in the vasculature of cells expressing the alpha chain of IL-2R, including innate immune cells type 2 (ILC-2), eosinophils and endothelial cells. This results in eosinophilia, capillary leak syndrome, and vascular leak syndrome (VLS).

[28] Disclosed herein in some embodiments is a method for selectively activating a distinct population(s) of lymphocytes (e.g., CD4+ helper cells, CD8+ effector naive and memory cells, NK cells, and NKT cells) via cytokine/cytokine receptor signaling . In some cases, the cytokine includes an interleukin, interferon, or tumor necrosis factor. In some cases, the cytokine is a cytokine conjugate, for example, an interleukin conjugate, an interferon conjugate, or a tumor necrosis factor conjugate. In additional cases described herein, pharmaceutical compositions and kits containing one or more of the cytokine conjugates described herein are included.

[29] In some embodiments, the present document also describes a method for selectively activating populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells, and NKT cells through IL-2/IL-2R signaling. In some instances, IL-2 is an IL-2 conjugate that interacts with an "intermediate affinity" IL-2Rαβγ complex, optionally with similar potency as the IL-2Rαβγ complex and with a reduced IL-2Rα interaction relative to wild-type IL-2. In some embodiments, this document further describes methods of treating cancer using the IL-2 conjugate described herein. In additional embodiments, pharmaceutical compositions and kits are described herein that contain one or more of the IL-2 conjugates described herein.

Cytokine conjugates

[30] In some embodiments, cytokine conjugates are described herein. In some cases, the cytokine includes interleukins, tumor necrosis factor, interferon, chemokine, lymphokine, or growth factor. In some cases, the cytokine is an interleukin. In some cases, the cytokine is an interferon. In additional cases, the cytokine is a tumor necrosis factor. In additional cases, the cytokine is a growth factor.

[31] In some embodiments, an interleukin conjugate is described herein. Illustrative interleukins include, without limitation, 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). In some cases, this document describes an 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 cojugates

[32] In some embodiments, IL-2 conjugates modified at the amino acid position are described herein. In some cases, the modification refers to a natural amino acid. In some cases, the modification refers to a non-natural amino acid. In some instances, this document describes an isolated and modified IL-2 polypeptide that contains at least one non-natural amino acid. In some instances, 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. In some instances, the IL-2 polypeptide is a human IL-2 protein. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some instances, the IL polypeptide -2 contains 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 is characterized by a sequence identity of about 80%, 85%, 90%, 95% , 96%, 97%, 98% or 99% with respect to SEQ ID NO: 2. In additional cases, the IL-2 polypeptide contains the sequence of SEQ ID NO: 2. In additional cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 2.

[33] In some cases, the IL-2 polypeptide is a truncated version. In some cases, the truncation is an N-terminal deletion. In other cases, the truncation is a C-terminal deletion. In additional cases, truncation includes both N-terminal and C-terminal deletions. For example, the truncation may 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, either from N- end, either from the C-end, or from both ends. In some cases, the IL-2 polypeptide contains 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 leftovers. In some cases, the IL-2 polypeptide contains 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 contains an N-terminal deletion of at least or about 2 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 3 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 4 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 5 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 6 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 7 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 8 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 9 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 10 residues.

[34] In some embodiments, the IL-2 polypeptide is a functionally active fragment. In some cases, the functionally active fragment contains the IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130 or 1-125, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment contains the region 10-133 of IL-2, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment the active fragment contains the region 20-133 of IL-2, and the positions of the residues refer to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment contains the region 30-133 of IL-2, and the positions of the residues refer to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment contains the region 10-125 IL-2, and the positions of the residues refer to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment contains the region 20-125 IL-2, and the positions of the residues refer to provisions in SEQ ID NO: 1. In some cases, the functional act The active fragment contains the 1-130 region of IL-2, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment contains the region 1-125 of IL-2, with the positions of the residues referring to the positions in SEQ ID NO: one.

[35] In some embodiments, an IL-2 conjugate described herein that comprises an isolated, purified, and modified IL-2 polypeptide and a conjugating fragment. In some instances, the IL-2 conjugate has reduced affinity for the α subunit of the IL-2 receptor (IL-2Rα) compared to the wild-type IL-2 polypeptide. In some instances, the conjugating moiety is linked to an amino acid residue that interacts with IL-2Rα (eg, at the IL-2/IL-2Rα contact). In some cases, the conjugating moiety is linked to an amino acid residue that is proximal to the IL-2/IL-2Rα interface (e.g., about 5Å, about 10Å, about 15Å, or about 20Å from the IL-2/IL-2Rα interface) . As used herein, residues involved in the IL-2/IL-2Rα interface include IL-2 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2Rα subunit.

[36] In some cases, the conjugating fragment is linked to an amino acid residue selected from the 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, and the numbering of amino acid residues corresponds to SEQ ID NO: 1. In some cases, 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. In some cases, 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. In some cases, 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 cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from R38 and K64. In some cases, the amino acid position is selected from E61, E62 and E68. In some cases, the position of the amino acid is at K35. In some cases, the amino acid position is at T37. In some cases, the amino acid position is in R38. In some cases, the amino acid position is at T41. In some cases, the amino acid position is in F42. In some cases, the amino acid position is at K43. In some cases, the amino acid position is in F44. In some cases, the amino acid position is at Y45. In some cases, the position of the amino acid is at E61. In some cases, the amino acid position is at E62. In some cases, the position of the amino acid 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 in 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. [37] In some cases, the IL-2 conjugate additionally contains an additional mutation. In some cases, the additional mutation is an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72 and Y107. In such cases, the amino acid is conjugated to an additional conjugating moiety to increase serum half-life, stability, or a combination of the two. Alternatively, the amino acid is first mutated to a naturally occurring amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine; or to a non-natural amino acid prior to binding to an additional conjugating moiety.

[38] In some embodiments, the reduced affinity of a modified IL-2 polypeptide for the IL-2 receptor a subunit (IL-2Rα) relative to a wild-type IL-2 polypeptide without non-natural amino acid modification (e.g., a wild-type IL-2 polypeptide) is about 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more than 99%. In some cases, the decrease in affinity is about 10%. In some cases, the decrease in affinity is about 20%. In some cases, the decrease in affinity is about 40%. In some cases, the decrease in affinity is about 50%. In some cases, the reduction in affinity is about 60%. In some cases, the reduction in affinity is about 80%. In some cases, the reduction in affinity is about 90%. In some cases, the reduction in affinity is about 99%. In some cases, the reduction in affinity exceeds 99%. In some cases, the reduction in affinity is about 80%. In some cases, the reduction in affinity is about 100%.

[39] In some embodiments, the reduction in affinity of a modified IL-2 polypeptide for the IL-2 receptor a subunit (IL-2Rα) relative to an equivalent IL-2 polypeptide without non-natural amino acid modification (e.g., a wild-type IL-2 polypeptide) is about 1- x, 2 x, 3 x, 4 x, 5 x, 6 x, 7 x, 8 x, 9 x, 10 x, 30 x, 50 x, 100 x, 200x, 300x, 400x, 500x, 1000x or more. In some cases, the decrease in affinity is about 1-fold. In some cases, the decrease in affinity is about 2-fold. In some cases, the decrease in affinity is about 4-fold. In some cases, the decrease in affinity is about 5-fold. In some cases, the decrease in affinity is about 6-fold. In some cases, the decrease in affinity is about 8-fold. In some cases, the decrease in affinity is about 10-fold. In some cases, the decrease in affinity is about 30-fold. In some cases, the reduction in affinity is about 50-fold. In some cases, the reduction in affinity is about 100-fold. In some cases, the decrease in affinity is about 300-fold. In some cases, the decrease in affinity is about 500-fold. In some cases, the reduction in affinity is about 1000-fold. In some cases, the reduction in affinity is more than 1000-fold.

[40] In some cases, the modified IL-2 polypeptide does not interact with IL-2Rα. 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-2Rα.

[41] In some embodiments, the modified IL-2 polypeptide exhibits a first receptor signaling activity for the IL-2βγ signaling complex and a second receptor signaling activity for the IL-2αβγ signaling complex, wherein the difference between the first receptor signaling activity and the second activity in receptor signaling is less than 10-fold. In some embodiments, the modified IL-2 polypeptide exhibits a first activity in receptor signaling to the IL-2βγ signaling complex and a second activity in receptor signaling to the IL-2αβγ signaling complex, wherein the difference between the first activity in receptor signaling and the second activity in receptor signaling 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. In some instances, the modified IL-2 polypeptide is a partial agonist, such as an agonist that activates a receptor (eg, an IL-2βγ signaling complex or an IL-2αβγ signaling complex) but has only partial efficacy at the receptor relative to a full agonist. In some instances, the modified IL-2 polypeptide is a full agonist, eg, an agonist that activates a receptor (eg, IL-2βγ signaling complex or IL-2αβγ signaling complex) in a maximal response.

[42] In some cases, activity in receptor signaling is measured by the EC50 value. In some cases, the modified IL-2 polypeptide provides a first EC50 value for activation of the IL-2βγ signaling complex and a second EC50 value for activation of the IL-2αβ signaling complex, and the difference between the first EC50 value and the second EC50 value is less than 10-fold. In some cases, the modified IL-2 polypeptide provides a first EC50 value for activation of the IL-2βγ signaling complex and a second EC50 value for activation of the IL-2αβ signaling complex, and the difference between the first EC50 value 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.

[43] In some cases, activity in receptor signaling is measured by the ED50 value. In some cases, the modified IL-2 polypeptide provides a first ED50 value for activation of the IL-2βγ signaling complex and a second ED50 value for activation of the IL-2αβγ signaling complex, and the difference between the first ED50 value and the second ED50 value is less than 10-fold. In some cases, the modified IL-2 polypeptide provides a first ED50 value for activation of the IL-2βγ signaling complex and a second ED50 value for activation of the IL-2αβγ signaling complex, and the difference between the first ED50 value 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.

[44] In some embodiments, the conjugating fragment is linked to the N-terminus or C-terminus of the IL-2 polypeptide, either directly or indirectly via a linker peptide. In some instances, the conjugating moiety (eg, polymer, protein, or peptide) is genetically fused to IL-2 at the N-terminus or C-terminus of IL-2, either directly or indirectly through a linker peptide. In some cases, the conjugating fragment is linked to the N-terminus or amino acid residue of the C-terminus. In some cases, the conjugating moiety is linked to a reactive group that is linked to the N-terminus or C-terminus of the amino acid residue.

[45] In some embodiments, an IL-2 conjugate with reduced binding affinity for IL-2Rα is capable of causing expansion of populations of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells. killers (NKT). In some cases, the conjugating fragment weakens or blocks the binding of IL-2 to IL-2Rα.

[46] In some cases, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T cells (NKT) via the IL-2Rβγ complex with a modified IL-2 polypeptide retains significant activity activation of said cell population relative to the wild-type IL-2 polypeptide. In some cases, the activation of the modified IL-2 polypeptide is equivalent to that of the wild-type IL-2 polypeptide. In other cases, the activation of the modified IL-2 polypeptide is greater than that of the wild-type IL-2 polypeptide. In some cases, the activity in receptor signaling of the modified IL-2 polypeptide to the IL-2Rβ complex is higher than the activity in receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some cases, the activity in receptor signaling of the modified IL-2 polypeptide is at least 1-fold higher than the corresponding activity of the wild-type IL-2 polypeptide. In some cases, the activity in receptor signaling of the modified IL-2 polypeptide is about or at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, 300 times, 400 times, 500 times, 1000 times or more above the corresponding wild-type IL-2 polypeptide activity. In such cases, the dose or concentration of the modified IL-2 polypeptide used to achieve a similar level of activation in a population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT) as a wild-type IL-2 polypeptide is lower than the dose or concentration used for wild-type IL-2 polypeptide.

[47] In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT) via the IL-2Rβγ complex with a modified IL polypeptide -2 retains a significant activation activity of the indicated cell population by the wild-type IL-2 polypeptide. In some cases, the activity in receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some instances, the receptor signaling activity 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 corresponding IL polypeptide activity. -2 wild type.

[48] In some embodiments, the modified IL-2 polypeptide exhibits a first IL-2Rβγ receptor signaling activity and a second IL-2Rαβγ receptor signaling activity. In some instances, the first activity in IL-2Rβ receptor signaling is an improved activity compared to the wild-type IL-2 polypeptide. In some instances, the second activity in IL-2Rαβ receptor signaling is an aberrant activity compared to the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide exhibits a first IL-2Rβγ receptor signaling activity and a second IL-2Rαβγ receptor signaling activity, wherein the first receptor signaling activity is at least 1-fold, 2- 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 50x, 100x, 500-fold, 1000-fold or higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 1-fold or greater than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 2 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 5 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 10 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 20 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 50 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 100 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 500 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 1000 times or more higher than the second activity in receptor signaling. In some cases, the first activity in receptor signaling of the modified IL-2 polypeptide is higher than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rβγ, and the second activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some cases, both activities in receptor signaling are lower than their respective activities in the wild-type IL-2 polypeptide. In other cases, both activities in receptor signaling are higher than their respective activities in the wild-type IL-2 polypeptide.

[49] In some embodiments, the IL-2 conjugate reduces a toxic adverse event in a subject to which the IL-2 conjugate is administered. Illustrative toxic adverse events include eosinophilia, capillary leak syndrome, and vascular leak syndrome (VLS). In some cases, the IL-2 conjugate reduces the likelihood of a toxic adverse event in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% relative to the second subject who was injected with wild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugate reduces the severity of a toxic adverse event in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% relative to the second subject who was injected with wild-type IL-2 or aldesleukin.

[50] In some cases, eosinophilia is a toxic adverse event. In some cases, the IL-2 conjugate reduces the likelihood of a subject experiencing eosinophilia by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% relative to the second subject who was injected with wild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugate reduces the severity of eosinophilia in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% % relative to the second subject who was injected with wild-type IL-2 or aldesleukin.

[51] In some cases, increased capillary permeability is a toxic adverse event. In some cases, the IL-2 conjugate reduces the likelihood of increased capillary permeability in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% relative to the second subject who was injected with wild-type IL-2 or aldesleukin. In some cases, an IL-2 conjugate reduces the severity of capillary leak in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% relative to the second subject who was injected with wild-type IL-2 or aldesleukin.

[52] In some cases, VLS is a toxic adverse event. In some cases, the IL-2 conjugate reduces the likelihood of VLS in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% relative to the second subject who was injected with wild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugate reduces the severity of VLS in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% % relative to the second subject who was injected with wild-type IL-2 or aldesleukin.

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

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

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

[56] In some embodiments, the IL-2 conjugate has a plasma half-life of at least about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, from about 2 hours to about 8 hours, 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 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 10 o'clock.

[57] In some embodiments, the IL-2 conjugate has a plasma half-life at which it is capable of leading to the proliferation and/or expansion of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or Natural killer T cells (NKT) or combinations thereof, but no deleterious effects such as apoptosis.

[58] In some embodiments, the IL-2 conjugate has an increased plasma half-life, e.g., 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 wild-type IL-2. In some embodiments, the IL-2 conjugate has an increased plasma half-life, e.g., 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 wild-type IL-2.

[59] In some embodiments, the IL-2 conjugate has an increased plasma half-life, for example, 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 up to about 10 hours relative to wild-type IL-2.

[60] In some embodiments, the IL-2 conjugate has an increased plasma half-life, e.g., 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. In some embodiments, the IL-2 conjugate has an increased plasma half-life, e.g., 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

[61] In some embodiments, the IL-2 conjugate has an increased plasma half-life, for example, 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 up to about 10 hours relative to aldesleukin.

[62] In some embodiments, the IL-2 conjugate has an extended plasma half-life with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours or more with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, 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, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 18 hours, about 8 hours to about 12 hours, or about 8 hours to about 10 hours with reduced toxicity . In some cases, the reduction in 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 relative to wild-type IL2. In some cases, reduced toxicity is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% , 500% or more relative to wild-type IL-2.

[63] In some embodiments, the IL-2 conjugate has an extended plasma half-life with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours or more with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, 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, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 18 hours, about 8 hours to about 12 hours, or about 8 hours to about 10 hours with reduced toxicity . In some cases, the reduction in 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 relative to wild-type IL2. In some cases, reduced toxicity is a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% , 500% or more relative to aldesleukin.

[64] In some embodiments, the IL-2 conjugate comprises a conjugate moiety, wherein the size (eg, volume or length) of the conjugate moiety increases plasma stability but does not decrease activity. In some cases, the size of the conjugating fragment increases the plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours , 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more. In some cases, the size of the conjugating fragment increases the plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours , 18 hours, 24 hours or more. In some cases, the size of the conjugating fragment increases the 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. In some cases, the size of the conjugating fragment reduces the activity by less than 5%, 4%, 3%, 2%, 1% or less relative to aldesleukin.

[65] In some embodiments, the IL-2 conjugate comprises a conjugating moiety, wherein the size (eg, volume or length) of the conjugating moiety enhances plasma stability and activity. In some cases, the size of the conjugating fragment increases the plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more. In some cases, the size of the conjugating fragment increases the plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours , 18 hours, 24 hours or more. In some cases, the size of the conjugating moiety increases the 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. In some cases, the size of the conjugating fragment further increases the activity by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more according to towards aldesleukin.

[66] According to some embodiments, an IL-2 conjugate is described herein comprising a non-natural amino acid covalently attached to the conjugating moiety, wherein the non-natural amino acid is located in the 35-107 region and wherein the 35-107 region corresponds to residues K35-Y107 in SEQ ID NO : one.

[67] In some embodiments, the present document describes a protein that binds the interleukin 2 βγ receptor (IL-2Rβγ), wherein the binding affinity for the interleukin 2 receptor α (IL-2Rα) of said binding protein is less than wild-type human IL-2. (hIL-2), wherein the binding affinity for the interleukin 2 receptor α (IL-2Rα) of said binding protein is less than wild-type human IL-2 (hIL-2). In some embodiments, a protein is described herein that binds an interleukin 2 βγ receptor (IL-2Rβγ), wherein the binding affinity for the interleukin 2 receptor α (IL-2Rα) of said binding protein is less than wild-type human IL-2 (hIL- 2), and moreover, the specified binding protein contains at least one non-natural amino acid. In some instances, said binding protein is a modified IL-2 polypeptide or functionally active fragment thereof, wherein the modified IL-2 polypeptide contains at least one non-natural amino acid. In some cases, at least one non-natural amino acid is located in the 35-107 region, with the 35-107 region corresponding to residues K35-Y107 in SEQIDNO: 1.

[68] In some embodiments, an IL-2/IL-2Rβγ complex is described herein comprising a modified IL-2 polypeptide containing a mutation and IL-2Rβγ, wherein the modified IL-2 polypeptide has reduced binding affinity for IL-2Rα and wherein the reduced binding affinity is compared to the binding affinity between the wild-type IL-2 polypeptide and IL-2Rα. In some cases, the modified IL-2 polypeptide further comprises a conjugating moiety covalently linked to the mutation site. In some cases, the mutation site contains an amino acid mutated to a naturally occurring amino acid. In some cases, the mutation site contains an amino acid mutated to a cysteine residue. In other cases, the mutation site contains an amino acid mutated to a lysine residue.

[69] In some embodiments, an IL-2/IL-2Rβγ complex is described herein comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rβγ, wherein the modified IL-2 polypeptide has reduced binding affinity for IL-2Rα, and moreover, the reduced binding affinity is compared with the binding affinity between the wild-type IL-2 polypeptide and IL-2Rα. In some instances, the modified IL-2 polypeptide further comprises a conjugating moiety covalently linked to a non-natural amino acid.

[70] In some embodiments, an IL-2/IL-2Rβγ complex is described herein comprising a modified IL-2 polypeptide containing a non-natural amino acid and IL-2Rβγ, wherein the modified IL-2 polypeptide has reduced activity in receptor signaling at towards IL-2Rα, and wherein the reduced activity in receptor signaling is compared to the activity in receptor signaling between the wild-type IL-2 polypeptide and IL-2Rα. In some instances, the modified IL-2 polypeptide further comprises a conjugating moiety covalently linked to a non-natural amino acid.

[71] In some embodiments, described herein is an activator of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT) that selectively causes expansion of CD4+ helper cells. cells, CD8+ effector naive and memory T cells, NK cells, NKT cells, or combinations thereof in a cell population, said activator comprising a modified interleukin 2 (IL-2) polypeptide containing at least one mutation. In some cases, the mutation refers to a naturally occurring amino acid. In other cases, the mutation refers to a non-natural amino acid. In some embodiments, an activator of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT) is described herein that selectively causes expansion of CD4+ helper cells. , CD8+ effector naive and memory T cells, NK cells, NKT cells, or combinations thereof in a cell population, wherein said activator comprises a modified interleukin 2 (IL-2) polypeptide containing at least one unnatural amino acid. In some instances, said activator causes expansion of 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+ population. cells compared with the expansion of CD4+ Treg cells in the population of CD3+ cells in contact with the wild-type IL-2 polypeptide. In some cases, said activator does not cause expansion of Treg cells in said cell population. In some instances, 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.

[72] In some cases, this document also describes a method for expanding a population of CD4+ helper cells, CD8+ efficient naive and memory T cells, natural killer (NK) cells or natural killer T cells (NKT), involving contact said cell population with a therapeutically effective amount of an activator of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T (NKT) cells, said activator comprising a modified interleukin 2 polypeptide (IL-2) containing at least one mutation, thereby causing expansion of the population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T (NKT) cells. In some cases, the mutation occurs in a naturally occurring amino acid. In other cases, the mutation occurs to an unnatural amino acid. In some cases, this document also describes a method for expanding a population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT), which involves contacting said cell population with a therapeutically an effective amount of an activator for CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells or natural killer T cells (NKT), said activator comprising a modified interleukin 2 (IL-2) polypeptide containing at least one non-natural amino acid, thereby causing an expansion of the population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T (NKT) cells.

IL-15 conjugates

[73] In some embodiments, described herein are IL-15 conjugates modified at an amino acid position. IL-15 regulates the activation and proliferation of T cells and NK cells. In some cases, the IL-15 conjugate contains an isolated and purified IL-15 polypeptide and a conjugating fragment. In some instances, the IL-15 conjugate has reduced affinity for the IL-15 receptor compared to the wild-type IL-15 polypeptide. In some instances, the conjugating moiety is linked to an amino acid residue that interacts with the IL-15 receptor (eg, at the IL-15/IL-15Rα interface) or with IL-2Rβγ subunits. In some cases, the conjugating moiety is linked to an amino acid residue that is proximal to the IL-15/IL-15Ra interface (e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å from the IL-15/IL-15Rα interface ) or proximal to IL-2Rβγ subunits. As used herein, residues involved in the IL-15/IL-15R interface include IL-15 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from IL-15R. Similarly, residues involved in the IL-15/IL-2Rβγ interface include IL-15 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from IL-2Rβγ subunits. In some instances, the conjugating moiety is linked to the N-terminus or C-terminus of an IL-15 polypeptide (e.g., to the amino acid residue of the N-terminus or C-terminus, or to a reactive group linked to the terminal amino acid residue), either directly or indirectly via a linker peptide . In additional cases, the conjugating fragment modulates the interaction between IL-15 and IL-15R. In some cases, the IL-15 conjugate activates different populations of tumor infiltrating lymphocytes through IL-15/IL-15R signaling. In some cases, the IL-15 conjugate reduces the proliferation and/or expansion of tumor-associated lymphocytes. In some cases, the IL-15 conjugate modulates immune activity.

Additional cytokine conjugates

[74] In some embodiments, one or more additional cytokine conjugates modified at an amino acid position are described herein. Illustrative cytokines include, without limitation, IL-1β, IL-7, IL-10, IL-12, IL-18, IL-21, INF (e.g., IFN-α, IFN-β, IFN-κ, IFN -δ, IFN-ε, IFN-τ, IFN-ω, or IFN-ζ, and TNF (e.g., TNFα or CD40L).In some cases, the cytokine conjugate contains an isolated and purified cytokine polypeptide and a conjugating fragment.In some cases, the cytokine conjugate has a reduced affinity for its respective receptor compared to the wild-type cytokine.In some cases, the conjugating moiety is bound to an amino acid residue that is proximal to the receptor interface (e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å from the receptor junction ).In some cases, the conjugating fragment is connected to the N-terminus or C-terminus of the cytokine directly or indirectly through a linker peptide.In additional cases, the conjugating fragment modulates the interaction between the cytokine and its corresponding receptor.In some cases, the cytokine conjugate activates various local populations of tumor-infiltrating lymphocytes. In some cases, the cytokine conjugate reduces the proliferation and/or expansion of tumor-associated lymphocytes. In some cases, the cytokine conjugate modulates immune activity.

Precursors of cytokine conjugates

[75] Described herein are cytokine conjugate precursors containing a mutant cytokine (such as IL-2) in which one or more amino acids have been mutated from a wild-type amino acid. Such precursors are often used in the methods described herein for the treatment of diseases or conditions. In some embodiments, the cytokine precursor is not conjugated. Such mutations variously include additions, deletions or substitutions. In some embodiments, the mutation comprises a substitution of another naturally occurring amino acid. In some cases, the mutant cytokine contains a mutation at the 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, with amino acid residue numbering corresponding to SEQ ID NO: 1. In some cases 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. In some cases, 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. In some cases, 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 cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from R38 and K64. In some cases, the amino acid position is selected from E61, E62 and E68. In some cases, the amino acid position is in K35. In some cases, the amino acid position is at T37. In some cases, the amino acid position is in R38. In some cases, the amino acid position is at T41. In some cases, the amino acid position is in F42. In some cases, the amino acid position is in K43. In some cases, the amino acid position is in F44. In some cases, the amino acid position is at Y45. In some cases, the position of the amino acid is at E61. In some cases, the amino acid position is at E62. In some cases, the amino acid position is in 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 in 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. In some embodiments, the mutant cytokine contains a conjugating moiety, wherein the conjugating moiety is attached to a mutated site in the mutant cytokine.

[76] The cytokine mutants described herein often contain one or more mutations in natural amino acids. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and at least one mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62K mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62C mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and the E62A mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62I mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62L mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62Y mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62W mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62N mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62R mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62D mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62Q mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62G mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and an E62H mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and the E62M mutation. In some embodiments, the cytokine mutant contains 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 contains SEQ ID NO: 1 and an E62S mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62T mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and an E62V mutation.

[77] In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and at least one mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65K mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65C mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65A mutation. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65I mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and a P65L mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and a P65Y mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and a P65W mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and a P65N mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and a P65R mutation. In some embodiments, the cytokine mutant contains SEQ ID NO: 1 and the 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. In some embodiments, the cytokine mutant comprises SEQ ID NO: 1 and a P65M mutation. In some embodiments, the cytokine mutant contains 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 contains 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 contains SEQ ID NO: 1 and a P65V mutation.

Fusions of proteins or peptides

[78] In some embodiments, the cytokine conjugate described herein comprises a cytokine (eg, IL-2 or other cytokine) that is fused to a peptide or protein (fusion). In some embodiments, the peptide or protein is an antibody or antibody fragment. In some embodiments, a cytokine conjugate described herein comprises a cytokine (eg, IL-2 or other cytokine) that is fused to an antibody or binding fragments thereof. In some embodiments, a cytokine described herein is fused to multiple proteins or peptides. In some embodiments, a cytokine conjugate comprises a fusion of a cytokine to a protein or peptide and at least one conjugating fragment. In some instances, an antibody or binding fragment thereof includes a humanized antibody or binding fragment thereof, a mouse antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab', divalent Fab ₂ , F( ab)' ₃ , single chain variable fragment (scFv), bis-scFv, (scFv) ₂ , diabody, minibody, nanobody, tribody, tetrabody, humabody, disulfide-stabilized protein Fv (dsFv), single domain antibody (sdAb), Ig NAR , a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof or a chemically modified derivative thereof. Such fusion proteins are in some cases produced directly by translation. In some embodiments, fusions are obtained using a chemical or other enzymatic ligation method. In some embodiments, the cytokine conjugate comprises a fusion peptide or protein attached by a linker. In some embodiments, the linker is a peptide. In some embodiments, the cytokine conjugate comprises an N-terminal peptide or fusion protein. In some embodiments, the cytokine conjugate comprises a C-terminal fusion peptide or protein. In some instances, a cytokine fused to a peptide or protein is further conjugated to one or more of the conjugating moieties described below.

[79] In some cases, the cytokine conjugate includes a fusion with the fusion of scFv, bis-scFv, (scFv) ₂ , dsFv or sdAb. In some cases, the merge includes scFv. In some cases, the cytokine conjugate includes a fusion with bis-scFv. In some cases, the cytokine conjugate includes a fusion with (scFv) ₂ . In some cases, the cytokine conjugate includes a fusion with dsFv. In some cases, the cytokine conjugate includes a fusion with sdAb. In some cases, a cytokine fused to scFv, bis-scFv, (scFv) ₂ , dsFv, or sdAb is additionally conjugated to one or more of the conjugating moieties described below.

[80] In some cases, the cytokine conjugate includes a fusion with the Fc portion of the antibody, for example, IgG, IgA, IgM, IgE or IgD. In some cases, the cytokine conjugate includes a fusion to the Fc portion of an IgG (eg, IgG1, IgG3, or IgG4). In some cases, the cytokine fused to the Fc portion is additionally conjugated to one or more of the conjugating moieties described below.

[81] In some cases, a cytokine (eg, interleukin, IFN, or TNF) polypeptide is fused to an antibody or binding fragments thereof. In some cases, a cytokine polypeptide is fused to a humanized antibody or binding fragment thereof, a mouse antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab' fragments, divalent Fab ₂ , F(ab)' ₃ , single-chain variable fragment (scFv), bis-scFv, (scFv) ₂ , diabody, mini-antibody, nanobody, triabody, tetrabody, humabody, disulfide-stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camel antibody or its a binding fragment, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the cytokine polypeptide is fused to the Fc portion of the antibody. In additional cases, the cytokine polypeptide is fused to the Fc portion of an IgG (eg, IgG1, IgG3, or IgG4). In some cases, a cytokine fused to an antibody or binding fragments thereof is additionally conjugated to one or more of the conjugating fragments described below.

[82] In some cases, the IL-2 polypeptide is fused to the antibody or its binding fragments. In some cases, an IL-2 polypeptide is fused to a humanized antibody or binding fragment thereof, a mouse antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab' fragments, divalent Fab ₂ , F(ab) ' ₃ , 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, camel antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the IL-2 polypeptide is fused to the Fc portion of the antibody. In additional instances, the IL-2 polypeptide is fused to the Fc portion of an IgG (eg, IgG1, IgG3, or IgG4). In some instances, an IL-2 polypeptide fused to an antibody or binding fragments thereof is additionally conjugated to one or more of the conjugating fragments described below.

Natural and non-natural amino acids

[83] In some embodiments, the amino acid residue described herein (e.g., in a cytokine such as IL-2) is mutated to lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine for binding (or interactions) with the conjugating moiety. For example, a lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine side chain can be linked to the conjugating moiety described herein. In some cases, the amino acid residue mutates to cysteine, lysine, or histidine. In some cases, the amino acid residue mutates into a cysteine. In some cases, the amino acid residue mutates to lysine. In some cases, the amino acid residue mutates to histidine. In some cases, the amino acid residue mutates into tyrosine. In some cases, the amino acid residue mutates into tryptophan. In some embodiments, the non-natural amino acid does not conjugate to the conjugating moiety. In some embodiments, the cytokine described herein comprises a non-natural amino acid, wherein the cytokine is conjugated to a protein, and the point of attachment is not a non-natural amino acid.

[84] In some embodiments, the amino acid residue described herein (eg, in a cytokine such as IL-2) is mutated to a non-natural amino acid prior to binding to the conjugating moiety. In some cases, a mutation to a non-natural amino acid prevents or minimizes the response to the immune system's autoantigen. As used herein, the term "non-natural amino acid" refers to an amino acid other than the 20 amino acids that are naturally present in a protein. Non-limiting examples of non-natural amino acids include: p-acetyl-L-phenylalanine, p-iodine-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargylphenylalanine, L-3-(2 -naphthyl)alanine, 3-methylphenylalanine, 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-borophophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonothyrosine, p-bromophenylalanine, selenocysteine, p-amino- L-phenylalanine, isopropyl-L-phenylalanine, azido-lysine (AzK), a non-natural analogue of the amino acid tyrosine; non-natural analogue of the amino acid glutamine; non-natural analogue of the amino acid phenylalanine; non-natural analogue of the amino acid series; non-natural analogue of the amino acid threonine; alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine , aldehyde, hydroxylamine, keto or amino-substituted amino acid, or a combination thereof; amino acid with a photoactivated crosslinker; spin-labeled amino acid; fluorescent amino acid; metal-binding amino acid; a metal-containing amino acid; radioactive amino acid; photocell and/or photoisomerizable amino acid; biotin or a biotin analogue containing an amino acid; a ketogenic amino acid; amino acid containing polyethylene glycol or polyether; an amino acid substituted by a heavy atom; a chemically cleavable or photo-cleavable amino acid; an amino acid with an extended side chain; an amino acid containing a toxic group; sugar-substituted amino acid; a carbon-linked sugar-containing amino acid; redox amino acid; a-hydroxy acid; aminothioacid; α,α-disubstituted amino acid; β-amino acid; a cyclic amino acid other than proline or histidine; and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

[85] In some embodiments, the non-natural amino acid contains a selective reactive group or a reactive group for site-selective labeling of the target polypeptide. In some cases, the chemistry is a biorthogonal reaction (eg, biocompatible and selective reactions). In some cases, the chemistry is Cu(I) catalyzed or "copper-free" triazole alkynazide formation reaction, Staudinger ligation, inverse electronic demand Diels Alder (IEDDA) reaction, "photo-click" chemistry, or a metal-mediated process, such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.

[86] In some embodiments, the non-natural amino acid contains a photoreactive group that crosslinks upon irradiation, such as UV radiation.

[87] In some embodiments, the non-natural amino acid comprises a photo-crosslinked amino acid.

[88] In some instances, the non-natural amino acid is a para-substituted, meta-substituted, or ortho-substituted amino acid derivative.

[89] In some cases, the non-natural amino acid includes p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, o-methyl-L-tyrosine, p-methoxyphenylalanine, p-propargyloxyphenylalanine, p-propargylphenylalanine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine , L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonothyrosine, p-bromophenylalanine, p-amino-L-phenylalanine or isopropyl-L-phenylalanine.

[90] In some cases, the non-natural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, or 3-iodotyrosine.

[91] In some cases, the non-natural amino acid is phenylselenocysteine.

[92] In some cases, the non-natural amino acid is a phenylalanine derivative containing a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide.

[93] In some cases, the non-natural amino acid is a lysine derivative containing a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide.

[94] In some cases, the non-natural amino acid contains an aromatic side chain.

[95] In some cases, the non-natural amino acid does not contain an aromatic side chain.

[96] In some cases, the non-natural amino acid contains an azido group.

[97] In some cases, the non-natural amino acid contains a Michael acceptor group. In some cases, Michael acceptor groups contain an unsaturated moiety capable of forming a covalent bond through a 1,2-addition reaction. In some cases, Michael acceptor groups include electron-deficient alkenes or alkynes. In some cases, Michael acceptor groups include, without limitation, alpha, beta unsaturated: ketones, aldehydes, sulfoxides, sulfones, nitriles, imines or aromatics.

[98] In some cases, the non-natural amino acid is dehydroalanine.

[99] In some cases, the non-natural amino acid contains an aldehyde or ketone group.

[100] In some cases, the non-natural amino acid is a lysine derivative containing an aldehyde or ketone group.

[101] In some cases, the non-natural amino acid is a lysine derivative containing one or more O, N, Se, or S atoms at the beta, gamma, or delta position. In some cases, the unnatural amino acid is a lysine derivative containing O, N, Se, or S atoms at the gamma position.

[102] In some cases, the non-natural amino acid is a lysine derivative in which the epsilon N-atom is replaced by an oxygen atom.

[103] In some cases, the non-natural amino acid is a lysine derivative that is not a natural post-translationally modified lysine.

[104] In some cases, a non-natural amino acid is an amino acid containing a side chain in which the sixth atom from the alpha position contains a carbonyl group. In some cases, the non-natural amino acid is an amino acid containing a side chain in which the sixth atom from the alpha position contains a carbonyl group and the fifth atom from the alpha position is nitrogen. In some cases, a non-natural amino acid is an amino acid containing a side chain in which the seventh atom from the alpha position is an oxygen atom.

[105] In some cases, the non-natural amino acid is a serine derivative containing selenium. In some cases, the non-natural amino acid is selenoserine (2-amino-3-hydroselenopropanoic acid). In some cases, the non-natural amino acid is 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid. In some cases, the non-natural amino acid is 2-amino-3-(phenylselanyl)propanoic acid. In some cases, the non-natural amino acid includes selenium, and the oxidation of selenium leads to the formation of a non-natural amino acid containing an alkene.

[106] In some cases, the non-natural amino acid contains a cyclooctynyl group.

[107] In some cases, the non-natural amino acid contains a transcycloctenyl group.

[108] In some cases, the non-natural amino acid contains a norbornenyl group.

[109] In some cases, the non-natural amino acid contains a cyclopropenyl group.

[110] In some cases, the non-natural amino acid contains a diazirine group.

[111] In some cases, the non-natural amino acid contains a tetrazine group.

[112] In some cases, the non-natural amino acid is a lysine derivative in which the side chain nitrogen is carbamylated. In some cases, the non-natural amino acid is a lysine derivative in which the side chain nitrogen is acylated. In some cases, the non-natural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some cases, the non-natural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some cases, the non-natural amino acid is N6-Boc-N6-methyllysine. In some cases, the non-natural amino acid is N6-acetyllisine. In some cases, the non-natural amino acid is pyrrolysine. In some cases, the non-natural amino acid is N6-trifluoroacetyl lysine. In some cases, the non-natural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some instances, the non-natural amino acid is 2-amino-6-{[(p-iodobenzyloxy)carbonyl]amino}hexanoic acid. In some cases, the non-natural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some cases, the non-natural amino acid is N6-prolyllysin. In some cases, the non-natural amino acid is 2-amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some cases, the non-natural amino acid is H6-(cyclopentanecarbonyl)lysine. In some cases, the non-natural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some cases, the non-natural amino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some cases, the non-natural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some cases, the non-natural amino acid is 2-amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some cases, the non-natural amino acid is N6-((2-azidoethoxy)carbonyl)lysine. In some cases, the non-natural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some instances, the non-natural amino acid is 2-amino-6-{[(2-cyclooctinyloxy)carbonyl]amino}hexanoic acid. In some cases, the non-natural amino acid is N6-(2-aminobut-3-inoyl)lysine. In some cases, the non-natural amino acid is 2-amino-6-((2-aminobut-3-inoyl)oxy)hexanoic acid. In some cases, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some cases, the non-natural amino acid is N6-(butenyl-4-hydroxycarbonyl)lysine. In some cases, the unnatural amino acid is K6-(pentenyl-5-hydroxycarbonyl)lysine. In some cases, the non-natural amino acid is N6-((but-3-yn-1-yloxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-((pent-4-yn-1-yloxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-(thiazolidine-4-carbonyl)lysine. In some cases, the non-natural amino acid is 2-amino-8-oxononanoic acid. In some cases, the non-natural amino acid is 2-amino-8-oxooctanoic acid. In some cases, the non-natural amino acid is N6-(2-oxoacetyl)lysine.

[113] In some cases, the non-natural amino acid is N6-propionyllysin. In some cases, the non-natural amino acid is N6-butyrillisine. In some cases, the non-natural amino acid is N6-(but-2-enoyl)lysine. In some cases, the non-natural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-((spiro[2,3]hex-1-en-5-ylmethoxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)lysine. In some cases, the non-natural amino acid is cysteinyl lysine. In some cases, the non-natural amino acid is N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-((methanitrobenyloxy)N6-methylcarbonyl)lysine. In some cases, the non-natural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)lysine. In some cases, the non-natural amino acid is N6-((cyclohept-3-en-1-yloxy)carbonyl)-L-lysine.

[114] In some cases, the non-natural amino acid is 2-amino-3-(((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.

[115] In some embodiments, the non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) with a rearranged amber, opal, or ocher stop codon.

[116] In some embodiments, the non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) with a 4 base codon.

[117] In some embodiments, the non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) with a rearranged rare sense codon.

[118] In some embodiments, the non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) via a synthetic codon containing the non-natural nucleic acid. In some cases, the non-natural amino acid is incorporated into the cytokine via an orthogonal modified synthetase/tRNA pair. Such orthogonal pairs contain a non-natural synthetase that is capable of charging the non-natural tRNA with the non-natural amino acid while minimizing the charging of a) other endogenous amino acids onto the non-natural tRNA and b) non-natural amino acids onto other endogenous tRNAs. Such orthogonal pairs contain tRNAs that are able to be charged by non-natural synthetase, while avoiding charging a) other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacterial, yeast, archaeal, or human sources. In some embodiments, the orthogonal synthetase/tRNA pair contains components from the same organism. In some embodiments, the orthogonal synthetase/tRNA pair contains components from two different organisms. In some embodiments, the orthogonal synthetase/tRNA pair contains components that promote translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthetase is an alanine-modified synthetase. In some embodiments, the orthogonal synthetase is an arginine-modified synthetase. In some embodiments, the orthogonal synthetase is an asparagine-modified synthetase. In some embodiments, the orthogonal synthetase is an aspartic acid modified synthetase. In some embodiments, the orthogonal synthetase is a cysteine-modified synthetase. In some embodiments, the orthogonal synthetase is a glutamine-modified synthetase. In some embodiments, the orthogonal synthetase is a glutamic acid modified synthetase. In some embodiments, the orthogonal synthetase is alanine and glycine modified. In some embodiments, the orthogonal synthetase is a histidine-modified synthetase. In some embodiments, the orthogonal synthetase is a leucine-modified synthetase. In some embodiments, the orthogonal synthetase is an isoleucine-modified synthetase. In some embodiments, the orthogonal synthetase is a lysine-modified synthetase. In some embodiments, the orthogonal synthetase is a methionine-modified synthetase. In some embodiments, the orthogonal synthetase is a phenylalanine-modified synthetase. In some embodiments, the orthogonal synthetase is a proline-modified synthetase. In some embodiments, the orthogonal synthetase is a serine-modified synthetase. In some embodiments, the orthogonal synthetase is a threonine-modified synthetase. In some embodiments, the orthogonal synthetase is a tryptophan-modified synthetase. In some embodiments, the orthogonal synthetase is a tyrosine-modified synthetase. In some embodiments, the orthogonal synthetase is a valine-modified synthetase. In some embodiments, the orthogonal synthetase is a phosphoserine-modified synthetase. In some embodiments, the orthogonal tRNA is an alanine-modified tRNA. In some embodiments, the orthogonal tRNA is an arginine-modified tRNA. In some embodiments, the orthogonal tRNA is an asparagine-modified tRNA. In some embodiments, the orthogonal tRNA is an aspartic acid-modified tRNA. In some embodiments, the orthogonal tRNA is a cysteine-modified tRNA. In some embodiments, the orthogonal tRNA is a glutamine-modified tRNA. In some embodiments, the orthogonal tRNA is a glutamic acid-modified tRNA. In some embodiments, the orthogonal tRNA is alanine and glycine modified. In some embodiments, the orthogonal tRNA is a histidine-modified tRNA. In some embodiments, the orthogonal tRNA is a leucine-modified tRNA. In some embodiments, the orthogonal tRNA is an isoleucine-modified tRNA. In some embodiments, the orthogonal tRNA is a lysine-modified tRNA. In some embodiments, the orthogonal tRNA is a methionine-modified tRNA. In some embodiments, the orthogonal tRNA is a phenylalanine-modified tRNA. In some embodiments, the orthogonal tRNA is a proline-modified tRNA. In some embodiments, the orthogonal tRNA is a serine-modified tRNA. In some embodiments, the orthogonal tRNA is a threonine-modified tRNA. In some embodiments, the orthogonal tRNA is a tryptophan-modified tRNA. In some embodiments, the orthogonal tRNA is a tyrosine-modified tRNA. In some embodiments, the orthogonal tRNA is a valine-modified tRNA. In some embodiments, the orthogonal tRNA is a phosphoserine-modified tRNA.

[119] In some embodiments, the non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) by an aminoacyl (aaRS or RS)-tRNA-synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, without limitation, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/tRNA _CUA B. stearothermophilus pairs, E. coli (Ec-Leu )/tRNA _CUA B. stearothermophilus and pyrrolysyl-tRNA pairs. In some cases, a non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) by a Mj-TyrRS/tRNA pair. Exemplary UAAs that can be included with the Mj-TyrRS/tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methophenylalanine; metasubstituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine and 3-iodotyrosine; phenylselencytosine; p-boronphenylalanine and o-nitrobenzyltyrosine.

[120] In some cases, a non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) with an Ec-Tyr/tRNA _CUA or Ec-Leu/tRNA _CUA pair. Exemplary UAAs that can be incorporated with an Ec-Tyr/tRNA _CUA pair or an Ec-Leu/tRNA _CUA pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O-propargyltyrosine; α-aminocaprylic acid, O-methyltyrosine, O-nitrobenzylcysteine and 3-(naphthalene-2-ylamino)-2-aminopropanoic acid.

[121] In some cases, a non-natural amino acid is incorporated into a cytokine (eg, an IL polypeptide) via a pyrrolysyl-tRNA pair. In some cases, PylRS is derived from archaebacteria, such as methanogenic archaebacteria. In some cases, PylRS is derived from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated with a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N-ε -D-prolyl-L-lysine and N-ε-cyclopentyloxycarbonyl-L-lysine; N-ε-acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine; and N-ε-(1-methylcyclopro-2-encarboxamido)lysine.

[122] In some cases, a non-natural amino acid is incorporated into a cytokine described herein (eg, an IL polypeptide) using the synthetase disclosed in US Pat. Nos. 9,888,819 and 9,938,516. Exemplary UAAs that can be incorporated by such synthetases include p-methylazido-L-phenylalanine, aralkyl, heterocyclyl, heteroaralkyl, unnatural amino acids, and others. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycle. Such amino acids, in some embodiments, include azides, tetrazines, or other chemical groups capable of conjugation with a binding partner, such as a water-soluble moiety. In some embodiments, such synthetases are expressed and used to incorporate UAA into cytokines in vivo. In some embodiments, such synthetases are used to incorporate UAA into cytokines using a cell-free translation system.

[123] In some cases, a non-natural amino acid is incorporated into a cytokine described herein (eg, an IL polypeptide) via a naturally occurring synthetase. In some embodiments, the non-natural amino acid is incorporated into a cytokine by an organism that is auxotrophic for one or more amino acids. In some embodiments, synthetases corresponding to an auxotrophic amino acid are capable of charging the corresponding tRNA with a non-natural amino acid. In some embodiments, the non-natural amino acid is selenocysteine or a derivative thereof. In some embodiments, the non-natural amino acid is selenomethionine or a derivative thereof. In some embodiments, the non-natural amino acid is an aromatic amino acid, wherein the aromatic amino acid contains an aryl halide, such as iodide. In embodiments, the non-natural amino acid is structurally similar to the auxotrophic amino acid.

[124] In some cases, the non-natural amino acid includes the non-natural amino acid shown in FIG. one.

[125] In some cases, the non-natural amino acid includes a derivative or analogue of lysine or phenylalanine. In some instances, the non-natural amino acid includes a lysine derivative or lysine analogue. In some cases, the non-natural amino acid includes pyrrolysine (Pyl). In some instances, the non-natural amino acid includes a phenylalanine derivative or phenylalanine analogue. In some cases, the non-natural amino acid is the non-natural 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 non-natural amino acid comprises the non-natural amino acid shown in FIG. 2 (for example, Fig. 2A and Fig. 2B).

[126] In some embodiments, the non-natural amino acid includes the non-natural amino acid depicted in FIG. 3A-fig. 3D (taken from Table 1 Dumas et al., Chemical Science 2015, 6, 50-69).

[127] In some embodiments, a non-natural amino acid included in a cytokine described herein (eg, an IL polypeptide) is disclosed in US 9840493; US 9682934; US 2017/0260137; US 9938516 or US 2018/0086734. Exemplary UAAs that can be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl, and non-natural amino acid lysine derivatives. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycle. Such amino acids, in some embodiments, include azides, tetrazines, or other chemical groups capable of conjugation with a binding partner, such as a water-soluble moiety. In some embodiments, the UAA contains an azide attached to the aromatic moiety via an alkyl linker. In some embodiments, the alkyl linker is a C ₁ -C ₁₀ linker. In some embodiments, the UAA contains a tetrazine attached to an aromatic moiety via an alkyl linker. In some embodiments, the UAA contains a tetrazine attached to an aromatic moiety via an amino group. In some embodiments, the UAA contains a tetrazine attached to the aromatic moiety via an alkylamino group. In some embodiments, the UAA contains an azide attached to the terminal nitrogen (eg, N6 of a lysine derivative or N5, N4, or N3 of a derivative containing a shorter alkyl side chain) of the amino acid side chain via an alkyl chain. In some embodiments, the UAA comprises a tetrazine attached to the amino acid side chain nitrogen terminal via an alkyl chain. In some embodiments, the UAA contains an azide or tetrazine attached to the amide via an alkyl linker. In some embodiments, UAA is an azide or a tetrazine-containing carbamate or an amide of 3-aminoalanine, serine, lysine, or a derivative thereof. In some embodiments, such UAAs are incorporated into cytokines in vivo. In some embodiments, such UAAs are incorporated into cytokines in a cell-free system.

Conjugating fragments

[128] In certain embodiments, disclosed herein are conjugating fragments that are associated with one or more of the cytokines (eg, interleukins, IFN, or TNF) described above. In some cases, the conjugating moiety is a molecule that interferes with the interaction of a cytokine with its receptor. In some cases, the conjugating moiety is any molecule that, by binding to a cytokine, allows the cytokine conjugate to modulate an immune response. In some cases, the conjugating moiety is linked to the cytokine via a covalent bond. In some cases, the cytokine described herein is attached to a conjugating fragment with a triazole group. In some cases, the cytokine described herein is attached to a conjugating moiety with a dihydropyridazine or pyridazine group. In some cases, the conjugating fragment contains a water-soluble polymer. In other cases, the conjugating fragment contains a protein or a binding fragment. In additional cases, the conjugating fragment contains a peptide. In additional cases, the conjugating fragment contains a nucleic acid. In additional cases, the conjugating fragment contains a small molecule. In additional instances, 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, CFA, CFA, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or flagellin). In some cases, the conjugating fragment increases the serum half-life and/or improves stability. In some cases, the conjugating fragment reduces the interaction of cytokines with one or more receptor domains or cytokine subunits. In additional cases, the conjugating fragment blocks the cytokine interaction of one or more cytokine domains or subunits with its cognate receptor(s). In some embodiments, the cytokine conjugates described herein comprise a plurality of conjugating moieties. In some embodiments, the conjugating moiety is attached to a non-natural or naturally occurring amino acid in a cytokine peptide. In some embodiments, the cytokine conjugate comprises a conjugating moiety attached to a natural amino acid. In some embodiments, the cytokine conjugate is attached to a non-natural amino acid in the cytokine peptide. In some embodiments, the conjugating moiety is attached to the N- or C-terminal amino acid of the cytokine peptide. Various combination sites are disclosed herein, for example, the first conjugation moiety is attached to a non-natural or naturally occurring amino acid in the cytokine peptide, and the second conjugation moiety is attached to the N or C-terminal amino acid of the cytokine peptide. In some embodiments, one conjugating fragment is attached to a plurality of cytokine peptide residues (eg, brackets). In some embodiments, the conjugating moiety is attached to both the N- and C-terminal amino acids of the cytokine peptide.

Water soluble polymers

[129] In some embodiments, the conjugating moiety described herein is a water-soluble polymer. In some instances, the water-soluble polymer is non-peptide, non-toxic, and biocompatible. As used herein, a substance is considered to be biocompatible if the beneficial effects associated with the use of the substance alone or with another substance (e.g., an active agent such as a cytokine moiety) in association with living tissues (e.g., administration to a patient) outweigh any detrimental effects. evaluated by a clinician, such as a physician, toxicologist, or clinical development specialist. In some cases, the water-soluble polymer is also non-immunogenic. In some cases, a substance is considered non-immunogenic if the intended use of the substance in vivo does not elicit an undesirable immune response (e.g., antibody formation) or, if an immune response occurs, such a response is not considered clinically relevant or important in the judgment of a clinician, such as a physician, toxicologist, or specialist for clinical development.

[130] In some cases, the water-soluble polymer is characterized by the presence of from about 2 to about 300 ends. Exemplary water-soluble polymers include, without limitation, poly(alkylene glycols) such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), ethylene glycol-propylene glycol copolymers, and the like, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), polysaccharides, poly(α-hydroxy acid), polyvinyl 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 above.

[131] In some cases, the water-soluble polymer is not limited to a specific structure. In some instances, the water-soluble polymer is linear (eg, terminated, such as alkoxy-PEG or bifunctional PEG), branched or multi-armed (eg, branched PEG or PEG attached to a polyol core), characterized by a dendritic (or star) architecture, each with one or more decomposable bonds or without them. In addition, the internal structure of the water-soluble polymer can be arranged in any number of different repeating structures and can be selected from the group consisting of homopolymer, variable copolymer, random copolymer, block copolymer, variable tripolymer, random tripolymer, and block tripolymer.

[132] In some embodiments, the weight average molecular weight of the water-soluble polymer in the IL-2 conjugate is from about 100 daltons to about 150,000 daltons. Illustrative ranges include, for example, weight average molecular weights ranging from greater than 5000 Daltons to about 100,000 Daltons, ranging from about 6000 Daltons to about 90,000 Daltons, ranging from about 10,000 Daltons to about 85,000 Daltons, ranging from greater than 10,000 Daltons to about 85,000 Daltons, in the range of about 20,000 Daltons to about 85,000 Daltons, in the range of about 53,000 Daltons to about 85,000 Daltons, in the range of about 25,000 Daltons to about 120,000 Daltons, in the range of about 29,000 Daltons to about 120,000 Daltons , in the range from about 35,000 Daltons to about 120,000 Daltons, and in the range from about 40,000 Daltons to about 120,000 Daltons.

[133] 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, approx. 1000 daltons, approx. 1500 daltons, approx. 2000 daltons, approx. 2200 daltons, approx. 2500 daltons, approx. , about 7000 daltons, about 7500 daltons, about 8000 daltons, about 9000 daltons, about 10000 daltons, about 11000 daltons, about 12000 daltons, about 13000 daltons, about 14000 daltons, about 15000 daltons, about 20000 daltons, about 22500 daltons 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons and about 75,000 dalton. Branched versions of the water-soluble polymer (eg, a 40,000 dalton branched water-soluble polymer composed of two 20,000 dalton molecular weight polymers) having a total molecular weight of any of the above factors can also be used. In one or more embodiments, the conjugate will not have any PEG moieties associated, directly or indirectly, with a PEG having a weight average molecular weight of less than about 6000 daltons.

[134] PEGs typically contain a range of (OCH ₂ CH ₂ ) monomers [or (CH ₂ CH ₂ O) monomers, depending on how PEG is defined]. As used herein, the number of repeating blocks is identified by the subscript "n" in "(OCH ₂ CH ₂ )n". Thus, the value of (n) typically falls within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, 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 1200 to about 1900. For any given polymer whose molecular weight is known, the number of repeating units (i.e. "n") can be determined by dividing the total weight average molecular weight of the polymer by the molecular weight of the repeating monomer.

[135] In some cases, the water-soluble polymer is a polymer with an end group, i.e. a polymer containing at least one end coated with a relatively inert group such as a C _1-6 lower alkoxy group or a hydroxyl group. When the polymer is, for example, PEG, methoxy-PEG (commonly referred to as mPEG) can be used, which is a linear form of PEG where one end of the polymer is a methoxy (-OCH ₃ ) group and the other end is a hydroxyl or another functional group, which may optionally be chemically modified.

[136] In some embodiments, exemplary water-soluble polymers include, without limitation, linear or branched discrete PEG (dPEG) from Quanta Biodesign, Ltd.; linear, branched or bifurcated PEGs from Nektar Therapeutics and Y-shaped PEG derivatives from JenKem Technology.

[137] In some embodiments, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a water-soluble polymer selected from poly(alkylene glycols) such as polyethylene glycol (“PEG”), poly(propylene glycol) (“PPG "), copolymers of ethylene glycol and propylene glycol, etc., poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids ), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polyphosphazene, polyoxazolines ("POZ"), poly(N-acryloylmorpholine ) and their combinations. In some instances, the cytokine polypeptide is PEG-conjugated (eg, pegylated). In some cases, the cytokine polypeptide is conjugated to PPG. In some cases, the cytokine polypeptide is conjugated to POZ. In some cases, the cytokine polypeptide is conjugated to PVP.

[138] In some embodiments, 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(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polyphosphazene, polyoxazolines ("POZ"), poly(N-acryloylmorpholine), and combinations thereof. In some instances, the IL-2 polypeptide is PEG-conjugated (eg, pegylated). In some cases, the IL-2 polypeptide is conjugated to PPG. In some cases, the IL-2 polypeptide is conjugated to POZ. In some cases, the IL-2 polypeptide is conjugated to PVP.

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

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

[141] In some embodiments, the water-soluble polymer is a degradable synthetic alternative to PEG. Illustrative degradable synthetic alternatives to PEG include, without limitation, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone-modified PEG derivatives obtained by polymerization of telechelic or di-terminal PEG-based functionalized macromonomers; PEG derivatives containing comonomers containing a degradable bond, such as poly[(ethyleneoxy)-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-(ε-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

[142] In some instances, a cytokine polypeptide (eg, interleukin, IFN, or TNF) described herein is conjugated to a degradable synthetic alternative to PEG, such as, for example, POEGM; backbone-modified PEG derivatives obtained by polymerization of telechelic or di-terminal PEG-based functionalized macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO and MPDL copolymerized with OEGMA or PCL-g-PEO. In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a degradable synthetic alternative to PEG, such as, for example, POEGM; backbone-modified PEG derivatives obtained by polymerization of telechelic or di-terminal PEG-based functionalized macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO and MPDL copolymerized with OEGMA or PCL-g-PEO.

[143] In some embodiments, the water-soluble polymer contains poly(zwitterions). Exemplary poly(zwitterions) include, without limitation, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methacryloyloxyethylphosphorylcholine) (PMPC). In some instances, a cytokine polypeptide (eg, interleukin, IFN, or TNF) described herein is conjugated to a poly(zwitterion) such as PSBMA, PCBMA, or PMPC. In some cases, the cytokine is an IL-2 polypeptide. In some instances, the IL-2 polypeptide is conjugated to a poly(zwitterion) such as PSBMA, PCBMA, or PMPC.

[144] In some embodiments, the water-soluble polymer comprises a polycarbonate. Illustrative polycarbonates include, without limitation, pentafluorophenyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC ₆ F ₅ ). In some cases, a cytokine polypeptide (eg, interleukin, IFN, or TNF) described herein is conjugated to a polycarbonate, such as MTC-OC ₆ F ₅ . In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a polycarbonate such as MTC-OC ₆ F ₅ .

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

[146] In some cases, the water-soluble polymer contains a polysaccharide. Exemplary polysaccharides include, without limitation, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some instances, a cytokine (eg, interleukin, IFN, or TNF) polypeptide is conjugated to a polysaccharide. In some cases, the IL-2 polypeptide is conjugated to dextran. In some cases, the IL-2 polypeptide is conjugated to PSA. In some cases, the IL-2 polypeptide is conjugated to HA. In some cases, the IL-2 polypeptide is conjugated to amylose. In some cases, the IL-2 polypeptide is conjugated to heparin. In some cases, the IL-2 polypeptide is conjugated to HS. In some cases, the IL-2 polypeptide is conjugated to dextrin. In some cases, the IL-2 polypeptide is conjugated to HES.

[147] In some cases, the water-soluble polymer contains a glycan. Illustrative classes of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans. In some instances, a cytokine (eg, interleukin, IFN, or TNF) polypeptide is conjugated to a glycan. In some cases, the IL-2 polypeptide is conjugated to N-linked glycans. In some cases, the IL-2 polypeptide is conjugated to O-linked glycans. In some cases, the IL-2 polypeptide is conjugated to glycolipids. In some cases, the IL-2 polypeptide is conjugated to O-GlcNAc. In some cases, the IL-2 polypeptide is conjugated to glycosaminoglycans.

[148] In some embodiments, the water-soluble polymer includes a polyoxazoline polymer. The polyoxazoline polymer is a linear synthetic polymer and, like PEG, has a low polydispersity. In some instances, the polyoxazoline polymer is a medium molecular weight polydisperse polyoxazoline polymer. In some cases, the average molecular weight of the polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10000, 20000, 35000, 40000, 50000, 60000, 100000, 200000, 300000, 400000 or 500000 Yes. In some cases, the polyoxazoline polymer includes poly(2-methyl 2-oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), or poly(2-propyl 2-oxazoline) (PPOZ). In some instances, a cytokine (eg, interleukin, IFN, or TNF) polypeptide is conjugated to a polyoxazoline polymer. In some cases, the IL-2 polypeptide is conjugated to a polyoxazoline polymer. In some cases, the IL-2 polypeptide is conjugated to PMOZ. In some cases, the IL-2 polypeptide is conjugated to PEOZ. In some cases, the IL-2 polypeptide is conjugated to PPOZ.

[149] In some cases, the water-soluble polymer includes a polyacrylic acid polymer. In some instances, a cytokine (eg, interleukin, IFN, or TNF) polypeptide is conjugated to a polyacrylic acid polymer. In some cases, the IL-2 polypeptide is conjugated to a polyacrylic acid polymer.

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

[151] In some instances, a water-soluble polymer is described in US Pat. Nos. 7,744,861, 8,273,833, and 7,803,777. cases, the IL-2 polypeptide is conjugated to a linker described in US Pat.

Lipids

[152] In some embodiments, the conjugating moiety described herein is a lipid. In some cases, the lipid is a fatty acid. In some cases, the fatty acid is a saturated fatty acid. In other cases, the fatty acid is an unsaturated fatty acid. Exemplary fatty acids include, but are not limited to, fatty acids having about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms or about 12 to about 18 carbon atoms. In some cases, the lipid binds to one or more serum proteins, thereby increasing serum stability and/or serum half-life.

[153] In some embodiments, the lipid is conjugated to IL-2. In some cases, the lipid is a fatty acid, such as a saturated fatty acid or an unsaturated fatty acid. In some cases, the fatty acid has about 6 to about 26 carbons, about 6 to about 24 carbons, about 6 to about 22 carbons, about 6 to about 20 carbons, about 6 to about 18 carbons , about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some cases, the fatty acid is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 carbons in length. In some cases, the fatty acid includes caproic acid (hexanoic acid), enanthanoic 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 (pentadecanic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanic acid), stearic acid (octadecanoic acid), non-adecylic acid (nonadecanoic acid), arachidonic acid (eicosanoic acid), geneicosic acid (geneicosanoic acid), behenic acid (docosanoic acid), tricosylic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosyl acid (pentacosanoic acid) or cerotinic acid (hexacosanoic acid)

[154] In some embodiments, the IL-2 lipid conjugate improves serum stability and/or serum half-life.

Squirrels

[155] In some embodiments, the conjugating fragment described herein is a protein or a binding fragment thereof. Illustrative proteins include albumin, transferrin, or transthyretin. In some cases, the protein or its binding fragment contains the antibody or its binding fragments. In some cases, the cytokine conjugate contains a protein or a binding fragment thereof. In some cases, an IL-2 conjugate containing a protein or a binding fragment thereof is characterized by an increased serum half-life and/or stability. In some cases, an IL-2 conjugate containing a protein or a binding fragment thereof is characterized by a reduced interaction of IL-2 with one or more IL-2R subunits. In additional cases, the protein or its binding fragment blocks the interaction of IL-2 with one or more IL-2R subunits.

[156] In some embodiments, the conjugating moiety is albumin. Albumin is a family of water-soluble globular proteins. It is usually found in blood plasma, which makes up 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 contains a binding site that can either reversibly or irreversibly interact with endogenous ligands such as long and medium chain fatty acids, bilirubin or hemin, or with exogenous compounds such as heterocyclic or aromatic compounds.

[157] In some cases, a cytokine polypeptide (eg, interleukin, IFN or TNF) is conjugated to albumin. In some cases, the cytokine polypeptide is conjugated to human serum albumin (HSA). In additional instances, the cytokine polypeptide is conjugated to a functional albumin moiety.

[158] In some cases, the IL-2 polypeptide is conjugated to albumin. In some cases, the IL-2 polypeptide is conjugated to human serum albumin (HSA). In additional instances, the IL-2 polypeptide is conjugated to a functional albumin moiety.

[159] In some embodiments, the conjugating moiety is transferrin. Transferrin is a 679 amino acid polypeptide of about 80 kDa and contains two Fe ³⁺ binding sites, one located at the N-terminal domain and the other located at the C-terminal domain. In some cases, human transferrin has a half-life of about 7-12 days.

[160] In some cases, a cytokine polypeptide (eg, interleukin, IFN, or TNF) is conjugated to transferrin. In some cases, the cytokine polypeptide is conjugated to human transferrin. In additional instances, the cytokine polypeptide is conjugated to a functional transferrin moiety.

[161] In some cases, the IL-2 polypeptide is conjugated to transferrin. In some cases, the IL-2 polypeptide is conjugated to human transferrin. In additional instances, the IL-2 polypeptide is conjugated to a functional transferrin moiety.

[162] In some embodiments, the conjugating moiety is transthyretin (TTR). Transthyretin is a transport protein located in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine (T ₄ ) and retinol-associated retinol-binding protein.

[163] In some cases, a cytokine polypeptide (eg, interleukin, IFN or TNF) is conjugated to transthyretin (through one of its ends or through the inner hinge region). In some cases, the cytokine polypeptide is conjugated to a functional transthyretin fragment.

[164] In some cases, the IL-2 polypeptide is conjugated to transthyretin (through one of its ends or through the inner hinge region). In some cases, the IL-2 polypeptide is conjugated to a functional transthyretin fragment.

[165] In some embodiments, the conjugating fragment is an antibody or binding fragments thereof. In some instances, the antibody or binding fragments thereof include a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab' fragments, divalent Fab ₂ , F( ab)' ₃ , single chain variable fragment (scFv), bis-scFv, (scFv) ₂ , diabody, minibody, nanobody, tribody, tetrabody, humabody, disulfide-stabilized protein Fv (dsFv), single domain antibody (sdAb), Ig NAR , a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof or a chemically modified derivative thereof.

[166] In some cases, the conjugating fragment contains scFv, bis-scFv, (scFv) ₂ , dsFv or sdAb. In some cases, the conjugating fragment contains scFv. In some cases, the conjugating fragment contains bis-scFv. In some cases, the conjugating fragment contains (scFv) ₂ . In some cases, the conjugating fragment contains dsFv. In some cases, the conjugating fragment contains sdAb.

[167] In some cases, the conjugating fragment contains the Fc portion of the antibody, for example, IgG, IgA, IgM, IgE or IgD. In some cases, the fragment contains the Fc portion of an IgG (eg, IgG ₁ , IgG ₃ or IgG ₄ ).

[168] In some cases, a cytokine polypeptide (eg, interleukin, IFN, or TNF) is conjugated to an antibody or binding fragments thereof. In some instances, the cytokine polypeptide is conjugated to a humanized antibody or binding fragment thereof, a mouse antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab' fragments, divalent Fab ₂ , F(ab) ^' ₃ , 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, camel antibody, or its binding fragment, the bispecific antibody or its binding fragment or its chemically modified derivative. In additional instances, the cytokine polypeptide is conjugated to the Fc portion of the antibody. In additional instances, the cytokine polypeptide is conjugated to the Fc portion of an IgG (eg, IgG ₁ , IgG ₃ or IgG ₄ ).

[169] In some cases, the IL-2 polypeptide is conjugated to the antibody or its binding fragments. In some instances, the IL-2 polypeptide is conjugated to a humanized antibody or binding fragment thereof, a mouse antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab' fragments, divalent Fab ₂ , F(ab) ^' ₃ , single chain variable fragment (scFv), bis-scFv, (scFv) ₂ , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide-stabilized protein Fv (dsFv), single domain antibody (sdAb), Ig NAR, antibody camelid or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the IL-2 polypeptide is conjugated to the Fc portion of the antibody. In additional instances, the IL-2 polypeptide is conjugated to the Fc portion of an IgG (eg, IgG ₁ , IgG ₃ or IgG ₄ ).

[170] In some embodiments, the IL-2 polypeptide is conjugated to a water-soluble polymer (eg, PEG) and an antibody or binding fragment thereof. In some instances, the antibody or binding fragments thereof include a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab' fragments, divalent Fab ₂ , F( ab) ^' ₃ , single chain variable fragment (scFv), bis-scFv, (scFv) ₂ , diabody, minibody, nanobody, tribody, tetrabody, humabody, disulfide-stabilized protein Fv (dsFv), single domain antibody (sdAb), Ig NAR , a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof or a chemically modified derivative thereof. In some cases, the antibody or its binding fragments contain scFv, bis-scFv, (scFv) ₂ dsFv or sdAb. In some cases, the antibody or its binding fragments contain scFv. In some cases, the antibody or binding fragment directs the IL-2 conjugate to the target cell of interest, and the water-soluble polymer increases stability and/or serum half-life.

[171] In some instances, one or more IL-2 polypeptide-water-soluble polymer (eg, PEG) conjugates are further linked to an antibody or binding fragments thereof. In some cases, the ratio of IL-2 conjugate to antibody is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10: 1, 11:1 or 12:1. In some cases, the ratio of IL-2 conjugate to antibody is about 1:1. In other cases, the ratio of IL-2 conjugate to antibody is about 2:1, 3:1, or 4:1. In additional instances, the ratio of IL-2 conjugate to antibody is about 6:1 or greater.

[172] In some embodiments, one or more IL-2 polypeptide water-soluble polymer (eg, PEG) conjugates are directly linked to an antibody or binding fragments thereof. In other cases, the IL-2 conjugate is indirectly linked to the antibody or its binding fragments by a linker. Exemplary linkers include homobifunctional linkers, heterobifunctional linkers, maleimide-based linkers, zero-trace linkers, self-cleaving linkers, spacers, and the like.

[173] In some embodiments, the antibody or binding fragments thereof are linked directly or indirectly to the polypeptide portion of an IL-2 polypeptide-IL-2-water-soluble polymer (eg, PEG) conjugate. In such cases, the site of conjugation of the antibody to the IL-2 polypeptide is at a site that will not interfere with the binding of the IL-2 polypeptide to IL-2Rβγ. In additional instances, the site of conjugation of the antibody to the IL-2 polypeptide is at a site that partially blocks the binding of the IL-2 polypeptide to IL-2Rβγ. In additional instances, the site of conjugation of the antibody to the IL-2 polypeptide is located at a site that would prevent or further prevent the binding of the IL-2 polypeptide to IL-2Rα. In other embodiments, the antibody or binding fragments thereof are linked directly or indirectly to the water-soluble polymer portion of an IL-2 polypeptide-water-soluble polymer (eg, PEG) conjugate.

Peptides

[174] In some embodiments, the conjugating fragment described herein is a peptide. In some cases, the peptide is an unstructured peptide. In some instances, a cytokine polypeptide (eg, interleukin, IFN, or TNF) is conjugated to the peptide. In some cases, the IL-2 conjugate containing the peptide is characterized by increased serum half-life and/or stability. In some cases, the IL-2 conjugate containing the peptide is characterized by reduced interaction of IL-2 with one or more IL-2R subunits. In additional cases, the peptide blocks the interaction of IL-2 with one or more IL-2R subunits.

[175] In some cases, the conjugating fragment is an XTEN™ peptide (Amunix Operating Inc.) and this modification is called XTENylation. XTENylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding an XTEN™ peptide (Amunix Operating Inc.), a long, unstructured, hydrophilic peptide containing varying percentages of six amino acids: Ala, Glu, Gly, Ser, and Thr. In some instances, the XTEN™ peptide is selected based on properties such as expression, genetic stability, solubility, resistance to aggregation, extended half-life, increased activity, and/or increased activity in vitro when combined with the polypeptide of interest. In some instances, a cytokine polypeptide (eg, interleukin, IFN, or TNF) is conjugated to an XTEN peptide. In some instances, the IL-2 polypeptide is conjugated to an XTEN peptide.

[176] In some cases, the conjugating fragment is a glycine-rich homo amino acid polymer (HAP), and this modification is called 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). In some cases, the HAP polymer contains a repeating motif (Gly ₄ Ser) _n (SEQ ID NO: 3) and is sometimes about 50, 100, 150, 200, 250, 300 or more residues in length. In some instances, a cytokine polypeptide (eg, interleukin, IFN, or TNF) is conjugated to a HAP. In some cases, the IL-2 polypeptide is conjugated to HAP.

[177] In some embodiments, the conjugating fragment is a PAS polypeptide, and this modification is called PASylation. PASylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a PAS polypeptide. The PAS polypeptide is a hydrophilic uncharged polypeptide consisting of Pro, Ala and Ser residues. In some instances, the PAS polypeptide is at least about 100, 200, 300, 400, 500, or 600 amino acids long. In some instances, a cytokine polypeptide (eg, interleukin, IFN, or TNF) is conjugated to a PAS polypeptide. In some cases, the IL-2 polypeptide is conjugated to a PAS polypeptide.

[178] In some embodiments, the conjugating fragment is an elastin-like polypeptide (ELP) and the modification is called ELPylation. ELPylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding an elastin-like polypeptide (ELP). The ELP contains the VPGxG repeat motif (SEQ ID NO: 4) in which x is any amino acid other than proline. In some instances, a cytokine polypeptide (eg, interleukin, IFN, or TNF) is conjugated to an ELP. In some cases, the IL-2 polypeptide is conjugated to an ELP.

[179] In some embodiments, the conjugating fragment is a CTP peptide. The CTP peptide contains the 31 amino acid residue peptide FQSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 5) in which S* denotes O-glycosylation sites (OPKO). In some cases, the CTP peptide is genetically fused to a cytokine polypeptide (eg, an IL-2 polypeptide). In some instances, a cytokine polypeptide (eg, an IL-2 polypeptide) is conjugated to a CTP peptide.

[180] In some embodiments, the cytokine (eg, IL-2 polypeptide) is modified by glutamylation. Glutamylation (or polyglutamylation) is a reversible post-translational modification of glutamate, in which the γ-carboxy group of glutamate forms a peptide-like bond with the amino group of free glutamate, in which the α-carboxy group extends into the polyglutamate chain.

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

Additional conjugating fragments

[182] In some cases, the conjugating fragment contains an extracellular biomarker. In some cases, the extracellular biomarker is a tumor antigen. In some instances, an exemplary extracellular biomarker comprises CD 19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y , mesothelin, Mucl, Muc 16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8 and B220. In some instances, the conjugating moiety is linked to or conjugated to a cytokine (eg, IL-2). In some cases, the conjugating fragment is genetically fused, for example, at the N-terminus or C-terminus of a cytokine (eg, IL-2).

[183] In some cases, the conjugating fragment contains a post-translational modification molecule. In some cases, examples of 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 (eg with additional alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, addition of nucleotides, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation or deamidation. In some cases, a cytokine (eg, IL-2) is modified by post-translational modification such as myristoylation, palmitoylation, isoprenylation (or prenylation) (eg, farnesylation or geranylgeranylation), glypiation, acylation (eg, O-acylation, N-acylation, S- acylation), alkylation (eg with additional alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, addition of nucleotides, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation or deamidation.

Conjugation

Linkers

[184] In some embodiments, useful functional reactive groups for conjugating or linking a conjugating moiety to a cytokine polypeptide described herein (e.g., an IL-2 polypeptide) include, for example, zero or higher order linkers. In some cases, the non-natural amino acid included in the interleukin described herein contains a functional reactive group. In some cases, the linker contains a functional reactive group that reacts with a non-natural amino acid included in the interleukin described herein. In some cases, the conjugating fragment contains a functional reactive group that reacts with a non-natural amino acid included in the interleukin described herein. In some cases, the conjugating fragment contains a functional reactive group that reacts with the linker described herein (optionally pre-attached to the cytokine peptide). In some embodiments, the linker contains a reactive group that reacts with a natural amino acid in the cytokine peptide described herein. In some instances, higher order linkers include difunctional linkers such as homobifunctional linkers or heterobifunctional linkers. Illustrative homobifunctional linkers include, without limitation, Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (Sulfo DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyladipimidimidate (DMA), dimethylpimelimidate (DMP), dimethylsuberimidate (DMS), dimethyl-3,3' -dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide compound (DFDNB), e.g. 1,5-difluoro-2,4 -dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde , glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benz idine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide).

[185] In some embodiments, the bifunctional linker includes a heterobifunctional linker. Illustrative heterobifunctional linkers include, without limitation, amine-reactive and sulfhydryl crosslinkers such as N-succinimidyl-3-(2-pyridyldithio)propionate (sPDP), long chain N-succinimidyl-3-(2-pyridyldithio)propionate (LC -sPDP), water soluble long chain N-succinimidyl-3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α -methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate (Sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBs), N-succinimidyl(4-iodacteyl)aminobenzoate (sIAB), sulfosuccinimidyl( 4-iodacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SIAB), complex e γ-maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl-6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl-6-[6-(( iodoacetyl)amino)amino]hexanoate (sIAXX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl-6-(((((4-iodoacetyl)amino)methyl) cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-hydrazide-(4-N-maleimidophenyl)butyric acid (MPBH), 4- (N-maleimidomethyl)cyclohexane-1-carboxylhydrazide-8 (M ₂ C ₂ H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), amine-reactive and photoreactive crosslinkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid ( NHs-AsA), X-hydroxysulfosuccinimidyl-4-azidosalicylic acid (Sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamide)hexanoate (Sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionate (sAsD), N-g hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB) -azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'- dithiopropionate (sAND), N-succinimidyl-4-(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl -4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl-7-azido- 4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyldiazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinkers such as 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), b enzophenone-4-iodoacetamide, benzophenone-4-maleimide, carbonyl-reactive and photoreactive crosslinkers such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers such as 4-(p-azidosalicylamido)butylamine (AsBA) and arginine -reactive and photoreactive crosslinkers such as p-azidophenylglyoxal (APG).

[186] In some cases, the reactive functional group contains a nucleophilic group that is responsive to an electrophilic group present in the linking moiety (eg, in the conjugating moiety or in IL-2). Exemplary electrophilic groups include carbonyl groups such as an aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide, or acid anhydride. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In some embodiments, the non-natural amino acid included in the interleukin described herein contains an electrophilic group.

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

[188] In some embodiments, the linker is a peptide linker containing, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids. In some cases, the peptide linker contains no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or fewer amino acids. In additional cases, the peptide linker contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[189] In some embodiments, the linker includes a self-cleaving linker moiety. In some embodiments, the self-cleaving linker moiety includes p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker includes a dipeptide linker moiety and a self-cleaving linker moiety. In some embodiments, the self-cleaving linker fragment is as described in US Pat. No. 9,089,614 and WIPO Application No. WO 2015038426.

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

[191] In some embodiments, the linker contains a maleimide group. In some cases, the maleimide group is also referred to as a maleimide spacer. In some cases, the maleimide group additionally contains caproic acid, forming maleimidocaproyl (mc). In some cases, the linker contains maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other instances, the maleimide group includes a maleimidomethyl group such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo- scc).

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

Chemistry of conjugation

[193] Various conjugation reactions are used to conjugate linkers, conjugating fragments, and non-natural amino acids included in the cytokine peptides described herein. Such conjugation reactions are often compatible with aqueous conditions such as "biorthogonal" reactions. In some embodiments, the conjugation reactions are mediated by chemicals such as catalysts, light, or reactive chemical groups found on linkers, conjugating moieties, or unnatural amino acids. In some embodiments, the conjugation reactions are mediated by enzymes. In some embodiments, the conjugation reaction described herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, the conjugation reaction described herein is described in Chen, X.; wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417.

[194] In some embodiments described herein, the conjugation reaction comprises the reaction of a ketone or aldehyde with a nucleophile. In some embodiments, the conjugation reaction comprises reacting a ketone with an amino group to form an oxime. In some embodiments, the conjugation reaction comprises reacting a ketone with an aryl or heteroarylamino group to form an imine. In some embodiments, the conjugation reaction comprises reacting an aldehyde with an aryl or heteroarylamino group to form an imine. In some embodiments, the conjugation reaction described herein results in a cytokine peptide containing a linker or conjugating moiety attached via an oxime. In some embodiments, the conjugation reaction comprises a Pictet-Spengler reaction of an aldehyde or ketone with a tryptamine nucleophile. In some embodiments, the conjugation reaction includes a hydrazino-Pictet-Spengler reaction. In some embodiments, the conjugation reaction includes a Pictet-Spengler ligation.

[195] In some embodiments described herein, the conjugation reaction described herein comprises the reaction of an azide and a phosphine (Staudinger ligation). In some embodiments, the phosphine is an arylphosphine. In some embodiments, the arylphosphine contains an orthoester group. In some embodiments, the phosphine is characterized by the structure methyl-2-(diphenylphosphanenyl)benzoate. In some embodiments, the conjugation reaction described herein results in a cytokine peptide containing a linker or conjugating moiety attached via an arylamide. In some embodiments, the conjugation reaction described herein results in a cytokine peptide containing a linker or conjugating moiety attached via an amide.

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

[197] According to some embodiments described herein, the conjugation reaction described herein includes a reverse electron-demand cycloaddition reaction containing a diene and a dienophile. In some embodiments, the diene contains a tetrazine. In some embodiments, the dienophile contains an alkene. In some embodiments, the dienophile contains an alkyne. In some embodiments, the alkyne is a strained alkyne. In some embodiments, the alkene is a stressed diene. In some embodiments, the alkyne is transcyclooctyne. In some embodiments, the alkyne is cyclooctene. In some embodiments, the alkene is cyclopropene. In some embodiments, the alkene is a fluorocyclopropene. In some embodiments, the conjugation reaction described herein results in the formation of a cytokine peptide attached to a linker or conjugating moiety via a 6-membered ring heterocycle containing two ring nitrogen atoms.

[198] In some embodiments described herein, the conjugation reaction described herein includes an olefin metathesis reaction. In some embodiments, the conjugation reaction described herein comprises reacting an alkene and an alkyne with a ruthenium catalyst. In some embodiments, the conjugation reaction described herein comprises the reaction of two alkenes with a ruthenium catalyst. In some embodiments, the conjugation reaction described herein comprises reacting two alkynes with a ruthenium catalyst. In some embodiments, the conjugation reaction described herein comprises reacting an alkene or alkyne with a ruthenium catalyst and an amino acid containing an allyl group. In some embodiments, the conjugation reaction described herein comprises reacting an alkene or alkyne with a ruthenium catalyst and an allyl sulfide or selenide containing amino acid. In some embodiments, the ruthenium catalyst is a 2nd generation Hoved-Grubbs catalyst. In some embodiments, the olefin metathesis reaction comprises the reaction of one or more strained alkenes or alkynes.

[199] In some embodiments described herein, the conjugation reaction described herein includes a cross-coupling reaction. In some embodiments, the cross-coupling reactions include transition metal catalysts such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or another transition metal catalyst, and one or more ligands. In some embodiments, the transition metal catalysts are water soluble. In some embodiments described herein, the conjugation reaction described herein includes a Suzuki-Miyaura cross-coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises the reaction of an aryl halide (or triflate or tosylate), an aryl or alkenylboronic acid, and a palladium catalyst. In some embodiments described herein, the conjugation reaction described herein includes a Sonogashira cross-coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises the reaction of an aryl halide (or triflate or tosylate), an alkyne, and a palladium catalyst. In some embodiments, cross-coupling reactions result in the attachment of a linker or conjugating moiety to the cytokine peptide via a carbon-carbon bond.

[200] In some embodiments described herein, the conjugation reaction described herein includes a deprotection or "release" reaction of a reactive group prior to conjugation. In some embodiments, the conjugation reaction described herein comprises light cleavage of a reactive group followed by a conjugation reaction. In some embodiments, the reactive group is protected by an aralkyl group containing one or more nitro groups. In some embodiments, cleavage of a reactive group results in the formation of a free amine, sulfide, or other reactive group. In some embodiments, the conjugation reaction described herein comprises cleavage of a reactive group with a transition metal catalyst followed by a conjugation reaction. In some embodiments, the transition metal catalyst comprises palladium and one or more ligands. In some embodiments, the reactive group is protected by an allyl group. In some embodiments, the reactive group is protected with an allyl carbamate. In some embodiments, the reactive group is protected by a propargyl group. In some embodiments, the reactive group is protected with propargyl carbamate. In some embodiments, the reactive group is protected with a dienophile, wherein exposure to the diene (such as tetrazine) deprotects the reactive group.

[201] According to some embodiments described herein, the conjugation reaction described herein includes a ligand-directed reaction in which a ligand (optionally attached to a reactive group) facilitates a conjugation site between the reactive group and the cytokine peptide. In some embodiments, the ligand is cleaved during or after the reaction of the cytokine peptide with the reactive group. In some embodiments, the cytokine peptide conjugation site is a naturally occurring amino acid. In some embodiments, the cytokine peptide conjugation site is lysine, cysteine, or serine. In some embodiments, the cytokine peptide conjugation site is a non-natural amino acid as described herein. In some embodiments, the reactive group includes a substitutable group, such as an electron-poor aryl or heteroaryl group. In some embodiments, the reactive group includes a substitutable group, such as an electron-poor alkyl group, that is substituted by a cytokine peptide. In some embodiments, the conjugation reaction described herein comprises reacting a radical scavenging agent with radical compounds. In some embodiments, the conjugation reaction described herein includes an addition reaction of an oxidizing radical. In some embodiments, the radical scavenging agent is an arylamine. In some embodiments, the radical moiety is a tyrosyl radical. In some embodiments, radical species are produced by a ruthenium catalyst (such as [Ru(bpy) ₃ ]) and light.

[202] Enzymatic reactions are optionally used for the conjugation reactions described herein. Exemplary enzymatic conjugations include SortA-mediated conjugation, TGs-mediated conjugation, or FGE-mediated conjugation. In some embodiments, the conjugation reaction described herein comprises ligating a native protein (NPL) terminal 1-amino-2-thio group with a thioether to form an amide bond.

[203] Various conjugation reactions are described herein for the interaction of a linker or conjugating fragment with a cytokine peptide, wherein the reaction occurs with a natural ("canonical") amino acid in the cytokine peptide. In some embodiments, the naturally occurring amino acid found at the conjugation position is found in the wild-type sequence, or alternatively, the position has been mutated. In some embodiments, the conjugation reaction involves the formation of a disulfide bond at a cysteine residue. In some embodiments, the conjugation reaction includes a Michael 1,4 addition reaction of cysteine or lysine. In some embodiments, the conjugation reaction includes cyanobenzothiazole ligation of cysteine. In some embodiments, the conjugation reaction includes crosslinking with an acetone moiety, such as 1,3-dichloro-2-propionone. In some embodiments, the conjugation reaction comprises a 1,4-Michael addition to dehydroalanine formed by the reaction of cysteine with O-mesitylenesulfonylhydroxylamine. In some embodiments, the conjugation reaction comprises reacting tyrosine with triazolinedione (TAD) or a TAD derivative. In some embodiments, the conjugation reaction comprises the reaction of tryptophan with a rhodium carbenoid.

Methods of application

Proliferative diseases or conditions

[204] In some embodiments, provided herein is a method of treating a proliferative disease or condition in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) herein. In some instances, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment, wherein the IL-2 conjugate has reduced affinity for the α subunit of the IL-2 receptor (IL-2Rα) relative to the wild-type IL-2 polypeptide. In some cases, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment 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, with the amino acid residue numbering corresponding to SEQ ID NO 1. In some cases, the IL-2 conjugate preferentially interacts with IL-2Rβ subunits and IL-2Rβγ to form an IL-2/IL-2Rβγ complex. In some cases, the IL-2/IL-2Rαβγ complex stimulates and/or enhances the expansion of CD4+ helper cells, CD8+ effector naive and memory T cells, NK cells and/or NKT cells. In additional cases, the expansion of Teff cells shifts the Teff : Treg ratio to the Teff population.

[205] In some embodiments, the proliferative disease or condition is cancer. In some cases, the cancer is a solid tumor. Illustrative solid tumors include, without limitation, 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. In some cases, a solid tumor is a metastatic cancer. In some cases, a solid tumor is a recurrent or refractory cancer.

[206] In some instances, a cytokine (eg, interleukin, IFN, or TNF) conjugate described herein is administered to a subject in need thereof for the treatment of a solid tumor. In such cases, the subject is characterized by the presence of 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. In some cases, a solid tumor is a metastatic cancer. In some cases, a solid tumor is a recurrent or refractory cancer.

[207] In some cases, the IL-2 conjugate described herein is administered to a subject in need of it for the treatment of a solid tumor. In such cases, the subject is characterized by the presence of 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. In some instances, the IL-2 conjugate is administered to a subject for the treatment of bladder cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of breast cancer. In some instances, the IL-2 conjugate is administered to a subject for the treatment of colorectal cancer. In some instances, the IL-2 conjugate is administered to a subject for the treatment of esophageal cancer. In some instances, the IL-2 conjugate is administered to a subject for the treatment of head and neck cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of kidney cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of lung cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of melanoma. In some cases, the IL-2 conjugate is administered to a subject for the treatment of ovarian cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of pancreatic cancer. In some instances, the IL-2 conjugate is administered to a subject for the treatment of prostate cancer.

[208] In some embodiments, the IL-2 conjugate is administered to a subject for the treatment of metastatic cancer. In some cases, metastatic cancer includes 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. In some cases, the IL-2 conjugate is administered to a subject to treat 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 cancer kidney, metastatic lung cancer, metastatic melanoma, metastatic ovarian cancer, metastatic pancreatic cancer, or metastatic prostate cancer.

[209] In some cases, the IL-2 conjugate is administered to a subject for the treatment of relapsed or refractory cancer. In some cases, relapsed or refractory cancer includes 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, recurrent 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. In some cases, the IL-2 conjugate is administered to a subject for the treatment of 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 cancer of the esophagus, 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 .

[210] In some embodiments, the cancer is untreated cancer. In such cases, untreated cancer is cancer that has not been treated with therapy. In some cases, untreated cancer is a solid tumor such as 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. In some embodiments, provided herein is a method of treating an untreated solid tumor in a subject in need thereof, comprising administering to the subject a cytokine conjugate (eg, an IL-2 conjugate) as described herein.

[211] In some embodiments, the cancer is a hematological malignancy. In some cases, the hematologic malignancy includes leukemia, lymphoma, or myeloma. In some cases, the hematologic malignancy is a T-cell malignancy. In other cases, the hematologic malignancy is a B-cell malignancy. Illustrative hematologic malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's large B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, B-lymphoblastic lymphoma progenitor cells, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[212] In some cases, the hematological malignancy is a metastatic cancer. In some cases, the metastatic cancer is a metastatic T-cell malignancy or a metastatic B-cell malignancy.

[213] In some cases, hematological malignancy is a recurrent or refractory cancer. In some cases, a relapsed or refractory cancer is a relapsed or refractory T-cell malignancy or a relapsed or refractory B-cell malignancy.

[214] In some instances, a cytokine (eg, interleukin, IFN, or TNF) described herein is administered to a subject in need thereof for the treatment of a hematologic malignancy. In some cases, the subject has a T-cell malignancy. In some cases, the subject has a B-cell malignancy. In some cases, the subject has chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal B-cell marginal zone cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's large B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[215] In some instances, an IL-2 conjugate described herein is administered to a subject in need thereof for the treatment of a hematologic malignancy. In some cases, the subject has a T-cell malignancy. In some cases, the subject has a B-cell malignancy. In some cases, the subject has chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal B-cell marginal zone cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's large B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some instances, the IL-2 conjugate is administered to a subject to treat CLL. In some instances, the IL-2 conjugate is administered to a subject to treat SLL. In some instances, the IL-2 conjugate is administered to a subject to treat FL. In some instances, the IL-2 conjugate is administered to a subject to treat DLBCL. In some instances, the IL-2 conjugate is administered to a subject to treat MCL. In some instances, the IL-2 conjugate is administered to a subject to treat Waldenström's macroglobulinemia. In some instances, 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 to treat Burkitt's lymphoma.

[216] In some instances, an IL-2 conjugate is administered to a subject for the treatment of a metastatic hematologic malignancy. In some instances, the IL-2 conjugate is administered to a subject for the treatment of metastatic T-cell cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of metastatic B-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of metastatic chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's large B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, B-lymphoblastic progenitor cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[217] In some instances, an IL-2 conjugate is administered to a subject for the treatment of recurrent or refractory hematologic malignancy. In some instances, the IL-2 conjugate is administered to a subject for the treatment of relapsed or refractory T-cell cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of relapsed or refractory B-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), macroglobulinemia Waldenström, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's large B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, B progenitor lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal large B-cell lymphoma (thymus), intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

Additional therapeutic agents

[218] In some embodiments, the additional therapeutic agent is additionally administered to the subject. In some instances, the additional therapeutic agent is administered simultaneously with the cytokine conjugate (eg, IL-2 conjugate). In other cases, the additional therapeutic agent and IL-2 conjugate are administered sequentially, for example, the cytokine conjugate (eg, IL-2 conjugate) is administered before the administration of the additional therapeutic agent, or the cytokine conjugate (eg, IL-2 conjugate) is administered after the administration of the additional therapeutic agent.

[219] In some cases, the additional therapeutic agent includes a chemotherapeutic agent, an immunotherapeutic agent, targeted therapy, radiation therapy, or a combination thereof. Illustrative additional therapeutic agents include, without limitation, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxaplatin, 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 or irinotecan (CPT-11); topoisomerase II inhibitors such as etoposide (VP-16), teniposide or mitoxantrone; mitosis inhibitors such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine or vinorelbine; or corticosteroids such as prednisone, methylprednisolone, or dexamethasone.

[220] In some cases, an additional therapeutic agent includes first-line therapy. As used herein, the term "first line therapy" includes primary treatment for a subject with cancer. In some cases, the cancer is a primary cancer. In other cases, the cancer is a metastatic or recurrent cancer. In some cases, first-line therapy includes chemotherapy. In other cases, first-line therapy includes radiation therapy. One skilled in the art will readily appreciate that different first line therapies may be applicable to different types of cancer.

[221] In some cases, a cytokine conjugate (e.g., an 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, oxaplatin , 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 topoisomerase II inhibitor such as etoposide (VP-16), teniposide or mitoxantrone; a mitosis inhibitor such as docetaxel, estr amustine, ixabepilone, paclitaxel, vinblastine, vincristine or vinorelbine; or a corticosteroid such as prednisone, methylprednisolone, or dexamethasone.

[222] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) described herein is administered with an inhibitor of the poly(ADP-ribose) polymerase (PARP) enzyme. Exemplary PARP inhibitors include, without limitation, 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 Abb Vie), CK-102 (formerly CEP 9722 from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201 from Sanofi), and pamiparib (BGB-290 from BeiGene). In some instances, a cytokine conjugate (eg, an IL-2 conjugate) is administered in combination with a PARP inhibitor such as olaparib, rucaparib, niraparib, talazoparib, veliparib, CK-102, E7016, iniparib, or pamiparib.

[223] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) described herein is administered with an immune checkpoint inhibitor. Exemplary checkpoint inhibitors include:

[224] PD-L1 inhibitors such as MPDL3280A (RG7446) Genentech, anti-mouse PD-L1 clone 10F.9G2 (cat. no. BE0101) from BioXcell, anti-PD-Ll monoclonal antibody MDX-1105 (BMS-936559), and BMS-935559 from Bristol-Meyer Squibb, MSB0010718C, mouse clone 29E.2A3 to PD-L1 and AstraZeneca MEDI4736;

[225] PD-L2 inhibitors such as AMP-224 (Amplimmune) from GlaxoSmithKline and rHIgM12B7;

[226] PD-1 inhibitors such as anti-mouse PD-1 antibody clone J43 (cat. no. BE0033-2) from BioXcell, anti-mouse PD-1 antibody clone RMP1-14 (cat. no. BE0146) from BioXcell, clone EH12 mouse anti-PD-1 antibody, Merk's MK-3475 mouse anti-PD-1 antibody (Keytruda, pembrolizumab, lambrolizumab), AnaptysBio anti-PD-1 antibody known as ANB011, MDX-1 106 antibody (ONO-4538), human monoclonal antibody IgG4 from Bristol-Myers Squibb's - nivolumab (Opdivo®, BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224, and pidilizumab (CT-011) from Cure Tech Ltd;

[227] CTLA-4 inhibitors such as Bristol-Myers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101), Millipore anti-CTLA4 clone 9H10, Pfizer tremelimumab (CP-675,206, ticilimumab) and anti-CTLA4 antibody clone BNI3 from Abcam;

[228] LAG3 inhibitors such as eBioscience anti-Lag-3 antibody (C9B7W) clone eBioC9B7W, Lifespan Biosciences anti-Lag3 antibody LS-B2237, Immutep IMP321 (LnmuFact), BMS-986016 anti-Lag3 antibody, and LAG-3 chimeric antibody A9H12;

[229] B7-H3 inhibitors such as MGA271;

[230] KIR inhibitors such as lirilumab (IPH2101);

[231] CD137 inhibitors such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer) or XmAb-5592 (Xencor);

[232] PS inhibitors such as bavituximab;

[233] and inhibitors such as antibody or fragments thereof (e.g. monoclonal antibody, human, humanized or chimeric antibody), RNAi molecules or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD 160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2 or SLAM.

[234] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is administered in combination with pembrolizumab, nivolumab, tremelimumab, or ipilimumab.

[235] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) described herein is administered with an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, adotrastuzumab emtansine, or blinatumomab.

[236] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is administered with an additional therapeutic agent selected from an additional cytokine. In some cases, an additional cytokine enhances and/or synergizes the expansion and/or proliferation of T-effector cells. In some cases, the additional cytokine includes IL-1β, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNFα. In some cases, the additional cytokine is IL-7. In some cases, the additional cytokine is IL-15. In some cases, the additional cytokine is IL-21. In some cases, the additional cytokine is TNFα.

[237] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is administered with an additional therapeutic agent selected from a receptor agonist. In some cases, the receptor agonist contains a Toll-like receptor (TLR) ligand. In some cases, the TLR ligand includes TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some cases, the TLR ligand includes 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 , MDF2β, CFA, or flagellin. In some instances, a cytokine conjugate (eg, an IL-2 conjugate) is administered with one or more TLR agonists selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some cases, a cytokine conjugate (eg, 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, MDF2β, CFA and flagellin.

[238] In some embodiments, a cytokine conjugate (eg, an IL-2 conjugate) is used in combination with adaptive T lymphocyte transfer (ACT) therapy. In one embodiment, ACT includes identifying autologous T lymphocytes in the subject, eg, with antitumor activity, expanding the autologous T lymphocytes in vitro, and then reinfusing the expanded T lymphocytes into the subject. In another embodiment, ACT includes the use of allogeneic T lymphocytes, eg, with antitumor activity, expansion of the T lymphocytes in vitro, and subsequent infusion of the expanded allogeneic T lymphocytes into a subject in need thereof. In some cases, a cytokine conjugate (eg, an IL-2 conjugate) described herein is used in combination with autologous T lymphocytes as part of ACT therapy. In other instances, a cytokine conjugate (eg, IL-2 conjugate) described herein is used in combination with allogeneic T lymphocytes as part of ACT therapy. In some instances, a cytokine conjugate (eg, an IL-2 conjugate) is administered concurrently with ACT therapy to a subject in need thereof. In other instances, a cytokine conjugate (eg, an IL-2 conjugate) is administered sequentially with ACT therapy to a subject in need thereof.

[239] In some embodiments, a cytokine conjugate (eg, an IL-2 conjugate) is used to activate ex-vivo and/or expand autologous and/or allogeneic T cell transfer. In such cases, a cytokine conjugate (eg, IL-2 conjugate) is used to activate and/or expand the sample containing autologous and/or allogeneic T cells, and the cytokine conjugate (eg, IL-2 conjugate) is optionally removed from the sample prior to sample administration. subject in need.

[240] In some embodiments, the cytokine conjugate (eg, IL-2 conjugate) is administered with the vaccine. In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is used in combination with an oncolytic virus. In such cases, the cytokine conjugate (eg, IL-2 conjugate) acts as a stimulant to modulate the immune response. In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is used with an oncolytic virus as part of adjuvant therapy. Exemplary oncolytic viruses include T-Vec (Amgen), G47Δ (Todo et al.), JX-594 (Sillajen), CG0070 (Cold Genesys), and Reolysin (Oncolytics Biotech). In some instances, a cytokine conjugate (eg, an IL-2 conjugate) is used in combination with an oncolytic virus such as T-Vec, G47A, JX-594, CG0070, or reolysin.

[241] In some embodiments, a cytokine conjugate (eg, an IL-2 conjugate) is administered in conjunction with radiation therapy.

[242] In some embodiments, a cytokine conjugate (eg, an IL-2 conjugate) is administered in combination with surgery.

Pathogenic infections

[243] In some embodiments, provided herein is a method of treating a pathogenic infection in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a cytokine conjugate (eg, an IL-2 conjugate) described herein. In some instances, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment, wherein the IL-2 conjugate has reduced affinity for the IL-2 receptor subunit (IL-2Rα) relative to the wild-type IL-2 polypeptide. In some cases, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment 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, and the numbering of amino acid residues corresponds to SEQ ID NO:l. In some cases, the IL-2 conjugate preferentially interacts with the IL-2Rβ and IL-2Rβγ subunits to form the IL-2/IL-2Rβγ complex, which stimulates and/or enhances the expansion of CD4+ helper cells, CD8+ effector naive and memory cells, NK- cells and/or NKT cells. In additional cases, the IL-2 conjugate facilitates recognition of the pathogenic reservoir by CD8+ T cells.

[244] In some embodiments, the pathogenic infection is a viral infection in which, after treatment with antiviral therapy, a viral reservoir (eg, resting CD4+ T cells) persists in the treated host. In such cases, the cytokine conjugate (eg, IL-2 conjugate) described herein induces recognition of the viral reservoir by CD8+ T cells (or cytotoxic T cells). In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is used as monotherapy to redirect CD8+ T cells to infected quiescent cells for elimination. In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is used in combination with adjunctive therapy to redirect CD8+ T cells to infected quiescent cells for elimination. Exemplary adjunctive therapies include antiviral treatments such as acyclovir, brivudine, docosanol, famciclovir, foscarnet, idoxuridine, penciclovir, trifluridine, valaciclovir, and pritelivir.

[245] In some embodiments, the virus is a DNA virus or an RNA virus. DNA viruses include single-stranded (ss) DNA viruses, double-stranded (ds) DNA viruses, or DNA viruses that contain both ss and ds DNA regions. RNA viruses include single-stranded (ss) RNA viruses or double-stranded (ds) RNA viruses. In some cases, ssRNA viruses are further classified into positive strand RNA viruses or negative strand RNA viruses.

[246] Exemplary dsDNA viruses include those 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, Sphaerolipoviridae and Tectiviridae.

[247] Exemplary ssDNA viruses include those from the family: Anelloviridae, Bacillariodnaviridae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, and Spiraviridae.

[248] Exemplary DNA viruses that contain both ss and ds regions of DNA include viruses from the group of pleolipoviruses. In some cases, 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.

[249] Exemplary dsRNA viruses include those from the family: Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megavirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Rotavirus, and Totiviridae.

[250] Exemplary ssRNA viruses include those from the family: Alphaflexiviridae, Alphatetraviridae, Alvernaviridae, Arteriviridae, Astroviridae, Barnaviridae, Betaflexiviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Coronaviridae, Dicistroviridae, Flaviviridae, Gammaflexiviridae, Inlaviridae, Leviviridae, Luteoviridae, Marnaviridae, Mesoniviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Picornaviridae, Potyviridae, Roniviridae, Retroviridae, Secoviridae, Togaviridae, Tombusviridae, Tymoviridae and Virgaviridae.

[251] Exemplary negative strand ssRNA viruses include viruses from the family: Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Nyamiviridae, Ophioviridae, Orthomyxoviridae, Paramyxoviridae, and Rhabdoviridae.

[252] In some embodiments, the pathogenic infection is caused by Abelson leukemia virus, Abelson murine leukemia virus, Abelson virus, acute laryngotracheobronchitis virus, Adelaide river virus, adeno-associated virus group, adenovirus, African horse sickness virus, African swine fever virus, AIDS virus, Aleutian parvovirus mink disease, alpha retrovirus, alpha virus associated with ALV virus, Amapari virus, aphthovirus, Aquareovirus virus, arbovirus, arbovirus C, arbovirus group A, arbovirus group B, arenavirus group, Argentine hemorrhagic fever virus, arterivirus, astrovirus, group Ateline herpesviruses, Aujezky's disease virus, Auravirus, Ausduk's disease virus, Australian bat lyssavirus, aviadenovirus, avian erythroblastosis virus, avian infectious bronchitis virus, avian leukemia virus, avian lymphomatosis virus, avian myeloblastosis virus, avian paramyxovirus om, avian pseudodistemper virus, avian influenza virus, paramyxovirus, avian pneumoencephalitis virus, avian reticuloendotheliosis virus, avian sarcoma virus, avian retrovirus type C group, avichepadnavirus, avipoxvirus, B-virus, B19 virus, Babanki virus, baboon herpes virus, bakulo om virus, Barmah Forest virus, Bebaru virus, Berrimah virus, beta retrovirus, Bogna virus, Bittner virus, BK virus, Black Creek Canal virus, Sheep blue tongue virus, Bolivian hemorrhagic fever virus, Bohm disease virus, Sheep border disease virus, bovine virus, bovine alpha herpes virus 1, bovine alpha herpes virus 2, bovine coronavirus, bovine ephemeral fever virus, bovine immunodeficiency virus, bovine leukemia virus, porcine leukemia virus, mammillitis virus cattle, bovine papilloma virus, bovine papular stomatitis virus, bovine parvovirus, bovine syncytial virus, bovine oncovirus type C, bovine viral diarrhea virus, Buggy Creek virus, rod-shaped group of viruses, Bunyamwera virus supergroup, bunyavirus, Burkitt's lymphoma virus , Bwamba fever virus, SA virus, calicivirus, Californian encephalitis virus, camelpox virus, canaripox virus, canine herpesvirus, canine coronavirus, canine distemper virus, canine herpesvirus, canine minute virus, canine parvovirus, Sapo Delgadito virus, goat arthritis virus and ovine encephalitis virus, goat herpes virus, sheep pox virus, cardiovirus, mumps herpesvirus 1, marmoset herpesvirus 1, marmoset herpesvirus 1, marmoset herpesvirus 2, Chandipur virus, Changwinol virus, American channel catfish virus, Charleville virus, in varicella virus, chikungunya virus, chimpanzee herpes virus, chub reovirus, chum salmon virus, Cocala virus, coho reovirus, coital rash virus, Colorado tick fever virus, coltivirus, Columbia SK virus, acute respiratory infection virus, contagious pustular dermatitis virus, contagious pustular pustular virus dermatitis virus, coronavirus, Corriparta virus, acute rhinitis virus, vaccinia virus, Coxsackie virus, CPV (cytoplasmic polyhedrosis virus), cricket paralysis virus, Crimean Congo hemorrhagic fever virus, pseudocroup virus, cryptovirus, Cypovirus virus, cytomegalovirus, cytomegalovirus group, virus cytoplasmic polyhedrosis, deer papillomavirus, deltaretrovirus, dengue virus, densovirus, dependdovirus, dhori virus, diploma virus, Drosophila C virus, duck hepatitis B virus, duck hepatitis 1 virus, duck hepatitis 2 virus, duovirus, Duvenhage virus, deformed wing virus DWV, Eastern equine encephalitis virus, Eastern equine encephalomyelitis virus, EB virus, Ebola virus, Ebola-like virus, echovirus, echovirus, echovirus 10, echovirus 28, echovirus 9, ectromelia virus, EEE virus, EIA virus, EIA virus, encephalitis virus , encephalomyocarditis virus, encephalomyocarditis virus, enterovirus, enzyme-increasing enzyme-increasing virus (LDH), epidemic hemorrhagic fever virus, epizootic hemorrhagic disease virus, Epstein-Barr virus, equine alpha herpesvirus 1, alpha- equine herpesvirus 4, equine herpesvirus 2, equine abortion virus, equine arteritis virus, equine encephalosis virus, equine infectious anemia virus, equine morbillivirus, equine rhinopneumonitis virus, equine rhinovirus, Eubenangu virus, European elk papilloma virus, European distemper virus pigs, Everglades virus, virus Eyach, feline herpesvirus 1, feline calicivirus, feline fibrosarcoma virus, feline herpesvirus, feline immunodeficiency virus, feline infectious peritonitis virus, feline leukemia/sarcoma virus, feline leukemia virus, feline panleukopenia virus, feline parvovirus, feline sarcoma virus, syncytial virus feline virus, filovirus, Flanders virus, flavivirus, foot-and-mouth disease virus, Fort Morgan virus, Four Corners hantavirus, poultry virus 1, chickenpox virus, Friend virus, gammaretrovirus, hepatitis GB virus, GB virus, German measles virus, Goeta virus, leukemia virus gibbon monkey virus, infectious mononucleosis virus, goatpox virus, golden notemigonus virus, Gonometa virus, geese parvovirus, granulosa virus, Gross virus, gopher hepatitis B virus, group A arbovirus, Guanarito virus, guinea pig cytomegalovirus, guinea pig type C virus , Hantaan virus, hantavirus, hard shellfish reovirus , hare fibroma virus, HCMV (human cytomegalovirus), haemadsorption virus 2, haemagglutinating virus of Japan, hemorrhagic fever virus, Hendra virus, Nipah virus, feline hepatitis virus, hepatitis A virus, hepatitis B group, hepatitis C virus, hepatitis D virus, Hepatitis delta virus, Hepatitis E virus, Hepatitis F virus, Hepatitis G virus, Hepatitis nonA virus, Hepatitis nonB virus, Hepatitis virus (non-human), Hepatoencephalomyelitis 3 reovirus, Hepatovirus, Heron hepatitis B virus, Herpes B virus, Herpes simplex virus , herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 2, herpesvirus, herpesvirus 7, spider monkey herpesvirus, herpes simplex virus causing herpesvirus infection, Saimiri herpesvirus, Aujeszky's disease virus, varicella zoster virus, Highlands J virus, Asian paralicht rhabdovirus, porcine cholera virus, human adenovirus 2, alpha herp human esvirus 1, human alpha herpesvirus 2, human alpha herpesvirus 3, human B lymphotropic virus, human beta herpesvirus 5, human coronavirus, human cytomegalovirus group, human foaming virus, human gamma herpesvirus 4, human gamma herpesvirus 6 , human hepatitis A virus, human herpesvirus 1 group, human herpesvirus 2 group, human herpesvirus 3 group, human herpesvirus 4 group, human herpesvirus 6, human herpesvirus 8, human immunodeficiency virus, human immunodeficiency virus 1, human immunodeficiency virus 2, papillomavirus human T-cell leukemia virus, human T-cell leukemia virus I, human T-cell leukemia virus II, human T-cell leukemia virus III, human T-cell lymphoma virus I, human T-cell lymphoma virus II, lymphotropic human T-cell type 1 virus, human T-cell lymphotropic virus type 2, T-lymphotropic virus human T-lymphotropic virus II, human T-lymphotropic virus III, ichnovirus, childhood gastroenteritis virus, infectious bovine rhinotracheitis virus, infectious hematopoietic pancreosis virus, necrosis virus, influenza A virus, influenza B virus, influenza C virus, influenza D, influenza pr8 virus, insect iridescent virus, insect virus, iridovirus, Japanese B virus, Japanese encephalitis virus, JC virus, Junin virus, Kaposi's sarcoma-associated herpesvirus, Kemerovo virus, Kilham rat virus, Klamath virus, Kolongo virus, virus Korean Hemorrhagic Fever Virus, Kumba Virus, Kyasanur Forest Disease Virus, Kyzylagach Virus, La Crosse Virus, Lactate Dehydrogenase-Elevating Virus, Lactate Dehydrogenase Virus, Bat Lagos Virus, Langur Virus, Rabbit Parvovirus, Lassa Hemorrhagic Fever Virus, Lassa Virus, Latent Rat Virus, Virus LCM, Leaky virus, Lentivirus, Lepori poxvirus, leukemia virus, leukovirus, lumpy skin disease virus, lymphadenopathy-associated virus, lymphocryptovirus, lymphocytic choriomeningitis virus, lymphoproliferative virus group, Machupo hemorrhagic fever virus, infectious bulbar palsy virus, mammalian type B oncovirus group, mammalian type B retrovirus, mammalian type retrovirus group Mammalian, Mammalian retrovirus type D, Mammary tumor virus, Mapuera virus, Marburg virus, Marburg-like virus, Mason-Pfizer simian virus, Mastadenovirus, Mayaro virus, ME virus, Measles virus, Menangle virus, Mengo virus, Mengovirus, Middelburg virus, milker's nodule disease virus, mink enteritis virus, small mouse virus associated with MLV virus, MM virus, Mokola virus, Molluscum virus, Molluscum contagiosum virus, Monkey B virus, Monkeypox virus, Mononegavirales viruses, Morbillivirus, Elgo virus n bats, murine cytomegalovirus, murine encephalomyelitis virus, murine hepatitis virus, mouse K virus, murine leukemia virus, murine mammary tumor virus, murine minute virus, murine pneumonia virus, murine poliomyelitis virus, murine polyomavirus, murine sarcoma virus, murine pox, Mozambique virus, Mucambo virus, mucosal disease virus, mumps virus, murine beta-herpesvirus 1, murine cytomegalovirus 2, murine cytomegalovirus group, murine encephalomyelitis virus, murine hepatitis virus, murine leukemia virus, murine nodule-inducing virus, murine polyomavirus , murine sarcoma virus, muromegalovirus, Murray Valley encephalitis virus, myxoma virus, myxovirus, myxovirus multiforme, Myxovirus parotitidis virus, ovine Nairobi virus, Nairovirus, Nanimavirus, Nariva virus, Ndumo virus, Neethling virus, Nelson Bay virus, neurotropic virus , New World arenavirus, neonatal pneumonitis virus, Newcastle disease virus, Nipah virus, non-cytopathogenic virus, Norwalk virus, nuclear polyhedrosis virus (NPV), nipple neck virus, O'nyong'nyong fever virus, Okkelbo virus, oncogenic virus, oncogenic virus-like particle , oncornavirus, orbivirus, Orff virus, Oropush virus, orthohepadnavirus, orthomyxovirus om, orthopoxvirus, orthoreovirus, Orungo, ovine papillomavirus, sheep bluetongue virus, owl monkey herpesvirus, Palyam virus, papillomavirus, sylvilagi papillomavirus, parainfluenza virus, papovavirus, papillomavirus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, paramyxovirus, parapoxvirus, paravaccinia virus, parvovirus, parvovirus B19, parvovirus group, pestivirus, phlebovirus, swine fever virus, picodnavirus, picornavirus, porcine cytomegalovirus, virus swine pox, Piry virus, Pixuna virus, murine pneumonia virus, pneumovirus, poliomyelitis virus, poliovirus, polydnavirus, nuclear polyhedrosis virus, polyomavirus, polyomavirus, Polyomavirus bovis, Polyomavirus cercopitheci, human polyomavirus 2, macaque polyomavirus 1, murine polyomavirus 1, murine polyomavirus 2, Polyomavirus papionis 1, Polyomavirus papionis 2, Polyomavirus sylvilagi, Orangutan herpesvirus 1, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine parvovirus, porcine transmissible gastroenteritis virus, porcine type C virus, poxvirus, poxvirus, poxvirus variolae virus, Prospect Hill virus, provirus, bovine pseudopox virus, Aujeszky's disease virus, parrotpox virus, quailpox virus, rabbit fibroma virus, vacuolating rabbit virus, rabbit papillomavirus, rabies virus, raccoon parvovirus, raccoon pox virus, Ranikhet virus, cytomegalovirus om rat, rat parvovirus, rat virus, Rauscher virus, recombinant vaccinia virus, recombinant virus, reovirus, reovirus 1, reovirus 2, reovirus 3, reptile type C virus, respiratory infection virus, respiratory syncytial virus, respiratory virus, reticuloendotheliosis virus , rhabdovirus, Rhabdovirus carpia virus, Rhadinovirus, rhinovirus, rhizidiovirus, Rift Valley fever virus, Riley virus, rinderpest virus, RNA tumor virus, Ross river virus, rotavirus, rougeole virus, Rous sarcoma virus, rubella virus, rubivirus, Russian autumn encephalitis virus, simian virus SA 11, SA2 virus, Sabia virus, Sagiyama virus, Saimirine herpesvirus 1, salivary gland virus, phlebotomy fever virus group, Sandjimba virus, SARS virus, SDAV (sialodacryadenitis virus), smallpox virus, Semliki Forest Virus, Seoul Virus, Sheeppox Virus, Shopa Fibroma Virus, Papilla Virus simian vacuolating virus, simian hepatitis A virus, simian human immunodeficiency virus, simian immunodeficiency virus, simian parainfluenza virus, simian T-cell lymphotrophic virus, simian virus, simian virus 40, Simplexvirus, Sin Nombre virus, Sindbis fever virus, variola virus, South American hemorrhagic fever virus, passerine-pox virus, spumavirus, squirrel fibroma virus, squirrel monkey retrovirus, SSV group 1, STLV (simian T-lymphotropic virus) type I, STLV (simian T-lymphotropic virus) type II , type III STLV (simian T-lymphotropic virus), papular stomatitis virus, submaxillary virus, porcine herpes virus 1, porcine herpes virus 2, Suipoxvirus, swamp fever virus, swine pox virus, Swiss murine leukemia virus, TAC virus, virus Tacaribe complex, Tacaribe virus, tanapoxvirus, gerbilpox virus, Tench reovirus, virus Tayler encephalomyelitis, Tayler virus, Togoto virus, Thottapalayam virus, TBE virus, Tioman virus, Togavirus, Torovirus, Tumor virus, Tupaia virus, Turkey rhinotracheitis virus, Turkey pox virus, Type C retrovirus, Type D oncovirus, Type D group of retroviruses, ulcer rhabdovirus, Una virus, Uukuniemi group of viruses, vaccinia virus, vacuolating virus, varicella zoster virus, Varicellovirus, Varicola virus, variola major virus, variola virus, Wasingishu disease virus, VEE virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus, vesicle o virus, Vilyui encephalomyelitis virus, viper retrovirus, hemorrhagic septicemia virus, Visna Maedi virus, Visna virus, volepox virus, VSV (vesicular stomatitis virus), Wallal virus, Warrego virus, virus m of warts, WEE virus, West Nile virus, Western equine encephalitis virus, Western equine encephalomyelitis virus, Whataroa virus, winter vomiting virus, groundhog hepatitis B virus, woolly monkey sarcoma virus, wound tumor virus, WRSV virus, Yaba monkey tumor virus, virus Yaby, Yatapoxvirus, yellow fever virus, or Yug Bogdanovac virus.

[253] In some embodiments, the pathogenic infection is caused by a retrovirus. Illustrative retroviruses include, without limitation, human immunodeficiency virus (HIV), human T-cell leukemia viruses (HTLV), Moloney mouse leukemia virus (MuLV), mouse mammary tumor virus (MMTV), avian leukemia and sarcoma viruses, or Mason-Pfizer monkeys.

[254] In some embodiments, 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 dormant. In some cases, the retrovirus includes human immunodeficiency virus (HIV), human T-cell leukemia viruses (HTLV), Moloney mouse leukemia virus (MuLV), mouse mammary tumor virus (MMTV), avian leukemia and sarcoma viruses, or Mason monkey virus -Pfizer. In some cases, the cytokine conjugate redirects CD8+ T cells to recognize and kill infected cells in a quiescent state.

[255] In some cases, the cytokine conjugate is an IL-2 conjugate. In some instances, the IL-2 conjugate is administered to a subject with a retroviral infection or during a latency period to reduce and/or eliminate infected dormant cells. In some cases, the retrovirus includes human immunodeficiency virus (HIV), human T-cell leukemia viruses (HTLV), Moloney mouse leukemia virus (MuLV), mouse mammary tumor virus (MMTV), avian leukemia and sarcoma viruses, or Mason monkey virus -Pfizer. In some cases, the IL-2 conjugate redirects CD8+ T cells to recognize and kill infected cells that are at rest. In additional cases, the IL-2 conjugate is administered to the subject in combination with antiretroviral therapy.

[256] In some embodiments, the retrovirus is HIV. In some instances, a cytokine conjugate (e.g., IL-2 conjugate) described herein is administered to a subject with acquired immunodeficiency syndrome (AIDS) or during a latency period to reduce and/or eliminate HIV-infected cells (e.g., CD4+ T cells) that are are at rest. In some cases, the cytokine conjugate is an IL-2 conjugate. In some cases, the IL-2 conjugate is administered to a subject in combination with antiretroviral therapy. Illustrative antiretroviral therapy for HIV includes:

[257] nucleoside reverse transcriptase inhibitors (NRTIs) such as abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate and zidovudine;

[258] non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as efavirenz, etravirine, nevirapine or rilpivirine;

[259] protease inhibitors (PIs) such as atazanavir, darunavir, fosamprenavir, ritonavir, saquinavir and tipranavir;

[260] fusion inhibitors such as enfuvirtide;

[261] CCR5 antagonists such as maraviroc;

[262] integrase inhibitors such as dolutegravir and raltegravir;

[263] post-attachment inhibitors such as ibalizumab;

[264] pharmacokinetic enhancers such as cobicistat; and

[265] cocktails 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; elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumarate; emtricitabine, rilpivirine and tenofovir alafenamide; emtricitabine, rilpivirine and tenofovir disoproxil fumarate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumarate; lamivudine and tenofovir disoproxil fumarate; lamivudine and zidovudine; and lopinavir and ritonavir.

[266] In some cases, the IL-2 conjugate is administered to the subject in combination with 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 such as cobicistat; or cocktails 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; elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumarate; emtricitabine, rilpivirine and tenofovir alafenamide; emtricitabine, rilpivirine and tenofovir disoproxil fumarate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumarate; lamivudine and tenofovir disoproxil fumarate; lamivudine and zidovudine and lopinavir and ritonavir.

[267] In some embodiments, the virus is a hepatitis virus, such as hepatitis A, B, C, D, or E. latent period to reduce and/or eliminate infected cells in a dormant period. In some cases, the cytokine conjugate redirects CD8+ T cells to recognize and kill infected cells that are at rest.

[268] In some cases, the cytokine conjugate is an IL-2 conjugate. In some instances, an IL-2 conjugate is administered to a subject with hepatitis infection or during a latency period to reduce and/or eliminate quiescent infected cells. In some cases, the IL-2 conjugate redirects CD8+ T cells to recognize and kill infected cells that are at rest. In some instances, the IL-2 conjugate is administered to a subject in combination with antiviral therapy. Exemplary antiviral therapy for hepatitis includes ribavirin; NS3/4A protease inhibitors such as paritaprevir, simeprevir and grazoprevir; NS5A protease inhibitors such as ledipasvir, ombitasvir, elbasvir and daclatasvir; nucleotide/nucleoside and non-nucleoside inhibitors of NS5B polymerases 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 glycaprevir-pibrentasvir and interferons such as peginterferon alfa-2a, peginterferon alfa-2b and interferon alfa-2b. In some instances, the IL-2 conjugate is administered to a 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; nucleotide/nucleoside and non-nucleoside inhibitors of NS5B polymerases 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 glycaprevir-pibrentasvir; and interferons such as peginterferon alfa-2a, peginterferon alfa-2b and interferon alfa-2b.

Cell population expansion methods

[269] In some embodiments, methods for expanding populations of lymphocytes, such as populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells, are further described herein. In some instances, the method involves contacting a cell with a cytokine conjugate as described herein and interacting the cytokine with the cytokine receptor to form a complex, the complex stimulating the expansion of a distinct population of lymphocytes.

[270] In some embodiments, a method for expanding a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, and/or natural killer T cells (NKT) involves contacting the cell with the isolated and modified IL polypeptide described above. -2 for a time sufficient to induce the formation of a complex with IL-2Rβ, thereby stimulating the expansion of the TefF and/or NK cell population. In some cases, a method for expanding populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells involves (a) contacting the cell population with an IL-2 conjugate described herein and (b) interacting with IL- 2 with IL-2Rβ and IL-2βγ subunits to form an IL-2/IL-2Rβγ complex; moreover, the IL-2 conjugate has a reduced affinity for the IL-2Rα subunit, and wherein the IL-2/IL-2Rβγ complex stimulates the expansion of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells. As described above, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment 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, with amino acid residue numbering corresponding to SEQ ID NO:1. In some cases, 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 cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72 and Y107. In some cases, the amino acid position is selected from R38 and K64. In some cases, the amino acid position is selected from E61, E62 and E68. In some cases, the amino acid position is at E62.

[271] In some cases, the IL-2 conjugate causes expansion of CD4+ T regulatory (Treg) cells by less than 20%, 15%, 10%, 5%, or 1% of the cell population. In some cases, the IL-2 conjugate does not cause the expansion of CD4+ Treg cells in the cell population. In some cases, the ratio of 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. In some cases, the ratio of 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.

[272] In some cases, the time sufficient to induce the formation of a complex with IL-2Rβ 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 cases, the time sufficient to induce complex formation with IL-2Rβ 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.

[273] In some cases, the method is an in vivo method.

[274] In some cases, the method is an in vitro method.

[275] In some cases, the method is an ex vivo method.

Production of cytokine polypeptides

[276] In some cases, cytokine polypeptides (eg, interleukin, IFN, or TNF) containing either a natural amino acid mutation or a non-natural amino acid mutation described herein are recombinantly produced or chemically synthesized. In some instances, the cytokine (eg, IL-2) polypeptides described herein are produced recombinantly, for example, either in a host cell system or in a cell-free system.

[277] In some cases, cytokine polypeptides (eg, IL-2) are produced recombinantly through the host cell system. In some instances, the host cell is a eukaryotic cell (eg, a mammalian cell, insect cell, yeast cell, or plant cell) or a prokaryotic cell (eg, a Gram-positive bacterium or a Gram-negative bacterium). In some instances, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is a stable cell line or a cell line that has incorporated the genetic material of interest into its own genome and has the ability to express the product of the genetic material after many generations of cell division. In other cases, the mammalian host cell is a transient cell line or a cell line that has not incorporated the genetic material of interest into its own genome and lacks the ability to express the product of the genetic material after many generations of cell division.

[278] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293H cells, A549 cells, MDCK cells, DG44 CHO cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™- cell line CHO, Flp-In™-CV-l cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cell line, FreeStyle™ CHO-S cells, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line and T-REx™-HeLa cell line.

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

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

[281] In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, plant cells include cells from algae. Exemplary plant cell lines include strains of Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[282] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Machl™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stb12™, Stb13™ or Stbl4™.

[283] In some instances, suitable polynucleic acid molecules or vectors for the production of an IL-2 polypeptide described herein include any suitable vectors derived from a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors from bacteria (eg, E. coli), insects, yeasts (eg, Pichia pastoris), algae, or mammals. Bacterial vectors include, for example, pACYC177 vector series, pASK75, pBAD, 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.

[284] Векторы насекомых включают в себя, например, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac М30а, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12 , FLAG vectors such as pPolh-FLAGl or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[285] Yeast vectors include, for example, pDEST™ 14 Gateway® vector, pDEST™ 15 Gateway® vector, pDEST™ 17 Gateway® vector, pDEST™ 24 Gateway® vector, pYES-DEST52 Gateway® vector, pBAD- DEST49 Gateway®, PA0815 Pichia vector, pFLDl Pichi pastoris vector, pGAPZA vector, B and C Pichia pastoris, pPIC3.5K Pichia vector, pPIC6 A, B and C Pichia vector, pPIC9K Pichia vector, pTEFl/Zeo yeast vector, pYES2 vector pYES2/CT yeast, pYES2/NT A, B and C yeast vector or pYES3/CT yeast vector.

[286] Algal vectors include, for example, the pChlamy-4 vector or the MCS vector.

[287] 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 includes 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.

[288] In some cases, a cell-free system is used to produce the cytokine polypeptide described herein (eg, IL-2). In some cases, the cell-free system contains a mixture of cytoplasmic and/or nuclear components of the cell and is suitable for in vitro nucleic acid synthesis. In some cases, components of prokaryotic cells are used in the cell-free system. In other cases, eukaryotic cell components are used in a cell-free system. Synthesis of nucleic acids is obtained in a cell-free system based on, for example, Drosophila, Xenopus egg, Archaea or HeLa cells. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.

[289] Cell-free translation systems variously include components such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and extension factors, natural and/or non-natural amino acids and/or other components used for protein expression. Such components are optionally modified to improve yield, increase the rate of synthesis, improve the fidelity of the protein product, or include non-natural amino acids. In some embodiments, the cytokines described herein are synthesized using the cell-free translation systems described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245 or US 8787831. In some embodiments, cell-free translation systems contain modified release factors or even removal of one or more release factors from the system. In some embodiments, cell-free translation systems contain a reduced concentration of protease. In some embodiments, the cell-free translation systems contain modified tRNAs with reassigned codons used to encode non-natural amino acids. In some embodiments, the synthetases described herein for incorporating non-natural amino acids are used in cell-free translation systems. In some embodiments, the tRNA is preloaded with non-natural amino acids using enzymatic or chemical methods prior to being added to the cell-free translation system. In some embodiments, the components for the cell-free translation system are derived from modified organisms, such as modified bacteria, yeast, or other organisms.

[290] In some embodiments, a cytokine (eg, IL-2) polypeptide is produced in a cyclic form, either via a host-based expression system or via a cell-free system.

Obtaining a cytokine polypeptide containing a non-natural amino acid

[291] An orthogonal or extended genetic code may be used herein, in which one or more specific codons present in the nucleic acid sequence of a cytokine (e.g., IL-2) polypeptide are isolated to encode a non-natural amino acid such that it can be genetically incorporated into a cytokine (eg, IL-2) using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA-synthetase/tRNA pair is capable of charging the tRNA with a non-natural amino acid and is capable of incorporating that non-natural amino acid into the polypeptide chain in response to a codon.

[292] In some cases, the codon is an amber codon, an ocher codon, an opal codon, or a quadruplet codon. In some cases, the codon corresponds to an orthogonal tRNA that will be used to transfer the non-natural amino acid. In some cases, the codon is an amber codon. In other cases, the codon is an orthogonal codon.

[293] In some cases, the codon is a quadruplet codon that can be decoded using the orthogonal ribo-Q1 ribosome. In some cases, the quadruplet codon is as shown in Neumann, et at., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature, 464(7287): 441-444 (2010).

[294] In some cases, the codon used in the present disclosure is a recoded codon, such as a synonymous codon or a rare codon that has been replaced by an alternative codon. In some cases, the transcoded 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). In some cases, the recoded codon is as described in Ostrov et al., "Design, synthesis, and testing towards a 57-codon genome," Science 353(6301): 819-822 (2016).

[295] In some cases, non-natural nucleic acids are used, leading to the incorporation of one or more non-natural amino acids into the cytokine (eg, IL-2). Illustrative non-natural nucleic acids include, without limitation, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine , hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenin. Certain non-natural 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, dissimilar nucleic acids, oversized 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-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl adenine and guanine derivatives, 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-C ₃ ) uracil, 5-propynylcytosine, other alkynyl pyrimidine nucleino derivatives acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and others 8 -substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine(1H -pyrimido[5,4]-b][1,4]benzothiazin-2(3H)-one), G-fixers, fenoxazinecytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b ][1,4]benzoxazin-2(3H)-one), carbazolcytidine(2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine(H-pyrido[3',2':4.5 ]pyrrolo[2,3-d]pyrimidin-2-one), in which the purine or pyrimidine base is replaced by other heterocycles, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocyte osine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, ioduracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-ioduracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2 '-deoxyadenosine and those described in US Pat. No. 3,687,808; 4845205; 4910300; 4948882; 5093232; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121; 5596091; 5614617; 5645985; 5681941; 5750692; 5763588; 5830653 and 6005096; W099/62923; Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613 and Sanghvi, Chapter 15, Antisense Research and Applications, Crookeand Lebleu Eds., CRC Press, 1993, 273-288 USA No. 3687808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613 and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993.

[296] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and nucleic acids in some cases include one or more heterocyclic bases other than the basic five base components found in nature of nucleic acids. For example, a heterocyclic base includes, in some cases, the groups uracil-5-yl, cytosin-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4 -aminopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidine -3-yl, where purines are attached to the nucleic acid sugar fragment through position 9, pyrimidines through position 1, pyrrolopyrimidines through position 7, and pyrazolopyrimidines through position 1.

[297] In some embodiments, the nucleotide analogs are also modified in the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that have a modification at the bond between two nucleotides and contain, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl, and other alkylphosphonates, including 3'-alkylenephosphonate and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates. It is understood that these phosphate or modified phosphate bonds between two nucleotides pass through a 3'-5' bond or 2'-5' bond, and this bond contains a reversed polarity such as from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Numerous US patents describe how to make and use modified phosphate containing nucleotides and include, without limitation, 3,687,808; 4469863; 4476301; 5023243; 5177196; 5188897; 5264423; 5276019; 5278302; 5286717; 5321131; 5399676; 5405939; 5453496; 5455233; 5466677; 5476925; 5519126; 5536821; 5541306; 5550111; 5563253; 5571799; 5587361 and 5625050.

[298] In some embodiments, non-natural nucleic acids include 2,3'-dideoxy-2',3,-didehydro-nucleosides (PCT/US 2002/006460), 5'-substituted DNA and RNA derivatives (PCT/US 2011/033961); Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890 and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020; PCT/JP 2000/004720; PCT/JP 2003/002342; PCT/JP 2004/013216; PCT/JP 2005/020435; PCT/JP 2006/315479; PCT/JP 2006/324484; PCT/JP 2009/056718; PCT/JP 2010/067560) or 5'-substituted monomers prepared as base-modified monophosphate (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

[299] In some embodiments, non-natural nucleic acids include modifications at the 5'-position and 2'-position of the sugar ring (PCT/US 94/02993), 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). In some cases, non-natural nucleic acids include amide-linked nucleoside dimers prepared for inclusion in oligonucleotides, where the 3' linked nucleoside in the dimer (from 5' to 3') contains 2'-OCH ₃ and 5'-(S)-CH ₃ (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids may include 2'-substituted 5'-CH ₂ (or O) modified nucleosides (PCT/US 92/01020). Non-natural nucleic acids may include 5'-methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703- 705 and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids may include 5'-phosphonate monomers containing a 2'-substitution (US 2006/0074035) and other modified 5'-phosphonate monomers (WO 1997/35869). Non-natural nucleic acids may include 5'-modified methylene phosphonate monomers (EP 614907 and EP 629633). Non-natural nucleic acids may include analogues of 5' or 6'-phosphonate ribonucleosides containing 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). Non-natural nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers containing a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids may include nucleosides containing a 6'-phosphonate group in which the 5' or/and 6' position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH ₃ ) ₃ ) (and its analogs); methyleneamino group (CH ₂ NH ₂ ) (and its analogs) or cyano group (CN) (and its analogs) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640 and Hampton et al., J. Am Chem Soc, 1973, 95(13), 4404-4414).

[300] In some embodiments, non-natural nucleic acids also include sugar moiety modifications. In some cases, nucleic acids contain one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer increased nuclease stability, increased binding affinity, or some other beneficial biological property. In some embodiments, the nucleic acids comprise a chemically modified ribofuranose ring fragment. Examples of chemically modified ribofuranose rings include, but are not limited to, addition of substituent groups (including 5' and/or 2' substituent groups, bridge of two ring atoms to form bicyclic nucleic acids (BNA), replacement of the oxygen atom of the ribosyl ring with S, N(R) or C(R ₁ )(R ₂ ) (R=H, C ₁ -C ₁₂ alkyl or protecting group) and combinations thereof Examples of chemically modified Sugars can be found in WO 2008/101157, US 2005/0130923 and WO 2007/134181.

[301] In some cases, the modified nucleic acid contains modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar group may be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or an "analogue" of the cyclopentyl sugar group. The sugar may be in the pyranosyl or furanosyl form. The sugar moiety may be a ribose, deoxyribose, arabinose, or 2'-O-alkylribose furanoside, and the sugar may be attached to the appropriate heterocyclic bases in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, without limitation, 2'-alkoxy RNA analogs, 2'-amino RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy or amino RNA/DNA chimeras. For example, the sugar modification may include 2'-O-methyluridine or 2'-O-methylcytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs and the corresponding "nucleosides" in which such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known. Sugar modifications can also be made and combined with other modifications.

[302] Sugar moiety modifications include natural modifications of ribose and deoxyribose, as well as non-natural modifications. Sugar modifications include, without limitation, 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 alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl and alkynyl. 2' sugar modifications also include, without limitation, -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.

[303] Other modifications at the 2' position include, without limitation: C ₁ -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, RNA cleavage group, reporter group, intercalator, group to improve the pharmacokinetic properties of the oligonucleotide or a group to improve the pharmacodynamic properties of the oligonucleotide and other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, in particular at the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5'-position of the 5'-terminal nucleotide. Modified sugars also include those containing modifications in the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also include sugar mimetics, such as cyclobutyl moieties, instead of pentofuranosyl sugar. There are numerous United States patents that describe the preparation of such modified sugar structures and detail a number of base modifications, such as US Pat. Nos. 4,981,957; 5118800; 5319080; 5359044; 5393878; 5446137; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; 4845205; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121, 5596091; 5614617; 5,681,941 and 5,700,920, each of which is incorporated herein by reference in its entirety.

[304] Examples of nucleic acids containing modified sugar moieties include, without limitation, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ₃ and 2'-O(CH ₂ ) ₂ OCH ₃ . The substituent at position 2' may 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 -ON(R _m )(R _n ) and O-CH ₂ -C(=O)-N(R _m )(R _n ), where each R _m and R _n independently represents H or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

[305] In certain embodiments, the nucleic acids described herein include one or more bicyclic nucleic acids. In some such embodiments, the bicyclic nucleic acid contains a bridge between the 4' and 2' atoms of the ribosyl ring. In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, wherein the bridge contains a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, without limitation, one of the formulas: 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(CH ₂ OCH ₃ )-O-2' and their analogs (see US patent No. 7399845); 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see WO 2009/006478, WO 2008/150729, US 2004/0171570, US Patent No. 7427672, Chattopadhyaya et al., J. Org Chem., 209, 74, 118-134 and WO 2008/154401). See also, for example: Singh et al., Chem. Commun, 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc, 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; US patents No. 4849513; 5015733; 5118800; 5118802; 7053207; 6268490; 6770748; 6794499; 7034133; 6525191; 6670461 and 7399845; International Publication Nos. WO 2004/106356, WO 1994/14226, WO 2005/021570, WO 2007/090071 and WO 2007/134181; US Patent Nos. US 2004/0171570, US 2007/0287831 and US 2008/0039618; U.S. Provisional Applications Nos. 60/989574, 61/026995, 61/026998, 61/056564, 61/086231, 61/097787 and 61/099844 and International Applications No. PCT/US 2008/064591, PCT US 2008/066154, PCT US 2008/068922 and PCT/DK 98/00393.

[306] In certain embodiments, nucleic acids include linked nucleic acids. Nucleic acids can be linked together using any bond between nucleic acids. The two main classes of nucleic acid binding groups are defined by the presence or absence of a phosphorus atom. Exemplary phosphorus-containing bonds between nucleic acids include, without limitation, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Illustrative non-phosphorus-containing linking groups between nucleic acids include, without limitation, methylenemethylimino (-CH ₂ -N(CH ₃ )-O-CH ₂ -), thiodiester (-OC(O)-S-), thionocarbamate (-OC( O)(NH)-S-); siloxane (-O-Si(H) ₂ -O-) and N,N*-dimethylhydrazine (-CH ₂ -N(CH ₃ )-N(CH ₃ )). In some embodiments, bonds between nucleic acids containing a chiral atom can be obtained as a racemic mixture, as separate enantiomers, for example, alkyl phosphonates and phosphorothioates. Non-natural nucleic acids may contain one modification. Non-natural nucleic acids may contain several modifications within one of the groups or between different fragments.

[307] Nucleic acid backbone phosphate modifications include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridged or non-bridged), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in any combination. Other non-phosphate bonds may also be used.

[308] In some embodiments, backbone modifications (eg, methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and/or increase their in vivo stability.

[309] In some cases, a phosphorus derivative (or a modified phosphate group) is attached to a sugar or sugar analog and may be a monophosphate, diphosphate, triphosphate, alkyl phosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like. Illustrative polynucleotides containing modified phosphate bonds or non-phosphate bonds can be found in Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323 and Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, "Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; US patent No. 5453496 and Micklefield, 2001, Curr. Med. Chem. 8:1157-1179.

[310] In some cases, the modification of the main chain includes replacing the phosphodiester bond with an alternative moiety, such as an anionic, neutral, or cationic group. Examples of such modifications include: an anionic internucleoside bond; modification of phosphoramidate from N3' to P5'; boranophosphate DNA; prooligonucleotides; neutral internucleoside bonds such as methylphosphonates; DNA with an amide bond; methylene (methylimino) bonds; formacetal and thioformacetal bonds; backbones containing sulfonyl groups; morpholino oligonucleotides; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic guanidine (DNG) oligonucleotides (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). The modified nucleic acid may contain a chimeric or mixed backbone containing one or more modifications, for example, a combination of phosphate bonds, such as a combination of phosphodiester and phosphorothioate bonds.

[311] Phosphate substituents include, for example, short chain alkyl or cycloalkyl internucleoside bonds, mixed heteroatomic and alkyl or cycloalkyl internucleoside bonds, or one or more short chain heteroatomic or heterocyclic internucleoside bonds. These include those containing morpholino bonds (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; foracetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino main chains; sulfonate and sulfonamide backbones; amide backbones and others containing mixed N, O, S and CH ₃ constituents. Numerous US patents disclose how to make and use these types of phosphate substitutes and include, without limitation, US Pat. Nos. 5,034,506; 5166315; 5185444; 5214134; 5216141; 5235033; 5264562; 5264564; 5405938; 5434257; 5466677; 5470967; 5489677; 5541307; 5561225; 5596086; 5602240; 5610289; 5602240; 5608046; 5610289; 5618704; 5623070; 5663312; 5633360; 5,677,437 and 5,677,439. It is also understood that when a nucleotide is replaced, both the sugar and phosphate portions of the nucleotide can be replaced, for example by an amide type (aminoethylglycine) (PNA) bond. US Patents No. 5539082; 5714331 and 5719262 describe how to create and use PNA molecules, each of which is incorporated herein 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 may be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as cholesterol group (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), bile acid (Manoharan et al., Bioorg Med. Chem. Let., 1994, 4, 1053-1060), a thioether such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chain, e.g. dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49 -54), a phospholipid such as dihexadecyl-rac-glycerol or triethylammonium-1-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237) or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous US patents describe the preparation of such conjugates and include, without limitation, US Pat. Nos. 4,828,979; 4948882; 5218105; 5525465; 5541313; 5545730; 5552538; 5578717, 5580731; 5580731; 5591584; 5109124; 5118802; 5138045; 5414077; 5486603; 5512439; 5578718; 5608046; 4587044; 4605735; 4667025; 4762779; 4789737; 4824941; 4835263; 4876335; 4904582; 4958013; 5082830; 5112963; 5214136; 5082830; 5112963; 5214136; 5245022; 5254469; 5258506; 5262536; 5272250; 5292873; 5317098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5510475; 5512667; 5514785; 5565552; 5567810; 5574142; 5585481; 5587371; 5595726; 5597696; 5599923; 5599928 and 5688941.

[312] In some cases, non-natural nucleic acids additionally form non-natural base pairs. Illustrative non-natural nucleotides capable of forming a non-natural DNA or RNA base pair (UBP) under in vivo conditions include, without limitation, 5SICS, d5SICS, NAM, dNaM, and combinations thereof. In some embodiments, non-natural nucleotides include:

[313] In some embodiments, the non-natural base pair produces a non-natural amino acid as described in Dumas et al., "Designing logical codon reassignment - Expanding the chemistry in biology," Chemical Science, 6: 50-69 (2015).

[314] The host cell into which the constructs or vectors disclosed herein are introduced is cultured or maintained in a suitable medium such that tRNA, tRNA synthetase, and the protein of interest are obtained. The medium also contains non-natural amino acid(s), such that the protein of interest includes non-natural amino acid(s).

[315] The orthogonal tRNA synthetase/tRNA pair charges the tRNA with a non-natural amino acid and inserts the non-natural amino acid into the polypeptide chain in response to a codon. Exemplary aaRS-tRNA pairs include, without limitation, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/B pairs. stearothermophilus tRNA _CUA , pairs of E. coli LeuRS (Ec-Leu)/B. stearothermophilus tRNA _CUA and pyrrolysyl-tRNA pairs.

[316] A cytokine (e.g., IL-2) polypeptide containing unnatural amino acid(s) is prepared by introducing the nucleic acid constructs described herein containing tRNA and tRNA synthetase and containing a nucleic acid sequence of interest with one or more orthogonal (stop ) by in-frame codons, into the host cell. The host cell is exposed to saline containing the non-natural amino acid(s) and the host cells are then maintained under conditions that allow expression of the coding sequence of the protein of interest. The non-natural amino acid(s) are inserted into the polypeptide chain in response to a codon. For example, one or more non-natural amino acids are incorporated into a cytokine (eg, IL-2) polypeptide. Alternatively, two or more unnatural amino acids may be incorporated into a cytokine (eg, IL-2) polypeptide at two or more sites in the protein.

[317] When it is desired to include multiple non-natural amino acids in a cytokine (e.g., IL-2) polypeptide, it should be understood that multiple codons at desired positions should be included in the nucleic acid coding sequence so that tRNA synthetase/tRNA pairs can direct the incorporation of non-natural amino acids in response to codon(s). At least 1, 2, 3, 4 or more nucleic acid coding codons may be included in a nucleic acid sequence of interest.

[318] When it is desired to include more than one type of non-natural amino acid in a protein of interest, in one protein, a second or additional orthogonal pair of tRNA-tRNA synthetase can be used to include a second or additional non-natural amino acid; accordingly, said second or additional orthogonal tRNA-tRNA synthetase pair recognizes a different codon in the nucleic acid encoding the protein of interest, so that two or more unnatural amino acids can be specifically incorporated into different defined sites in the protein in a single manufacturing step. In some embodiments, two or more orthogonal tRNA-tRNA synthetase pairs can be used.

[319] Once a cytokine (e.g., IL-2) polypeptide comprising a non-naturally occurring amino acid(s) is produced in a host cell, it can be extracted from it by various methods known in the art, including enzymatic, chemical and/or osmotic lysis and physical destruction. A cytokine polypeptide (eg, IL-2) can be purified by standard methods known in the art, such as preparative chromatography, affinity purification, or any other suitable method.

[320] Suitable host cells may include bacterial cells (e.g., E. coli), but most suitable host cells are eukaryotic cells, e.g., 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 (CHO) cells or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells) or human or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Accordingly, the host cell is a mammalian cell, such as a human cell or an insect cell.

[321] Other suitable host cells that can generally be used in embodiments of the present invention are those indicated in the examples section. Vector DNA can be introduced into host cells using conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of well-established methods for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

[322] When creating cell lines, as a rule, it is preferable that stable cell lines are obtained. For example, for stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection method used, only a small proportion of cells can integrate foreign DNA into their genome. To identify and select these integrants, a gene that encodes a selectable marker (eg, for antibiotic resistance) is typically introduced into host cells along with the gene of interest. Preferred selectable markers include those that confer drug resistance such as G418, hygromycin or methotrexate. Nucleic acid molecules encoding a selectable marker may be introduced into the host cell on the same vector or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (eg, cells that have turned on the selectable marker gene will survive while other cells will die).

[323] In one embodiment, the constructs described herein are integrated into the genome of a host cell. The advantage of stable integration is that homogeneity is achieved between individual cells or clones. Another advantage is that a selection of the best manufacturers can be made. Accordingly, it is desirable to create stable cell lines. In another embodiment, the constructs described herein are transfected into a host cell. The advantage of transfecting the constructs into the host cell is that protein yield can be maximized. According to one aspect, a cell is described that contains a nucleic acid construct or a vector as described herein.

Pharmaceutical compositions and formulations

[324] In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject by multiple routes of administration, including, but not limited to, parenteral, oral, buccal, rectal, sublingual, or transdermal routes of administration. In some cases, parenteral administration includes intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration. In some cases, the pharmaceutical composition is formulated for topical use. In other cases, the pharmaceutical composition is formulated for systemic administration.

[325] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, hot melt formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations (eg, nanoparticulate formulations), and mixed immediate and controlled release formulations.

[326] In some embodiments, the pharmaceutical compositions include a carrier or carrier materials selected based on compatibility with the composition disclosed herein and properties of the release profile of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carriers include, but are not limited to, gum, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid , phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sodium stearoyl lactylate sugars, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980 and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999).

[327] In some cases, the pharmaceutical composition is formulated as an immunoliposome that contains a plurality of IL-2 conjugates associated directly or indirectly with the lipid bilayer of the liposomes. Illustrative lipids include, without limitation, fatty acids; phospholipids; sterols such as cholesterol; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, gloocides and cerebrosides; surfactant amines such as stearyl, oleyl and linoleylamines. In some cases, the lipid contains a cationic lipid. In some cases, the lipid contains a phospholipid. Exemplary phospholipids include, without limitation, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phosphatidylethanolamine ("PE"), phosphatidylinositol ("PI"), and phosphatidylserine ("PS") ), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-alpha-phosphatidylcholine ("DDPC"), dieladoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine (" DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleoylphosphatidylcholine ("DOPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-palmitoyl-2-oleoylphosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol (" DAPG"), didecanoyl-L-alpha-phosphatidylglycerol ("DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DMPG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoylphospho atidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), 1-palmitoyl-2-oleoylphosphatidylglycerol ("POPG"), diarachidoylphosphatidylethanolamine ("DAPE"), didecanoyl-L-alpha-phosphatidylethanolamine ("DDPE"), dielaidoylphosphatidylethanolamine (" DEPE"), dilauroylphosphatidylethanolamine ("DLPE"), dilinoleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine ("DMPE"), dioleoylphosphatidylethanolamine ("DOPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), distearoylphosphatidylethanolamine ("DSPE"), 1-palmitoyl-2-oleoylphosphatidylethanolamine (" POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl-L-alpha-phosphatidylinositol ("DDPI"), dialaidoylphosphatidylinositol ("DEPI"), dilauroylphosphatidylinositol ("DLPI"), dilinoleoylphosphatidylinositol, dimyristoylphosphatidylinositol ("DMPI"), dioleoylphosphatidylinositol (" DOPI"), dipalmitoylphosphatidylinositol ("DPPI"), distearoylphosphatidylinositol ("DSPI"), 1-palmitoyl-2-oleoylphosphatidylinositol ("POPI"), diarachidoylphosphatidylserine ("DAPS"), didecanoyl-L-alpha-pho sphatidylserine ("DDPS"), dielaidoylphosphatidylserine ("DEPS"), dilauroylphosphatidylserine ("DLPS"), dilinoleoylphosphatidylserine, dimyristoylphosphatidylserine ("DMPS"), dioleoylphosphatidylserine ("DOPS"), dipalmitoylphosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS") , 1-пальмитоил-2-олеоилфосфатидилсерин («POPS»), диарахидоилсфингомиелин, дидеканоилсфингомиелин, диэлаидоилсфингомиелин, дилауроилсфингомиелин, дилинолеоилсфингомиелин, димиристоилсфингомиелин, сфингомиелин, диолеоилсфингомиелин, дипальмитоилсфингомиелин, дистеароилсфингомиелин и 1-пальмитоил-2-олеоилсфингомиелин.

[328] In some cases, pharmaceutical formulations further include pH adjusters or buffering agents, which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, borate sodium, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount necessary to maintain the pH of the composition within an acceptable range.

[329] In some cases, the pharmaceutical composition includes one or more salts in an amount necessary to bring the osmolality of the composition to an acceptable range. Such salts include salts containing sodium, potassium or ammonium cations and anions of chlorides, citrates, ascorbates, borates, phosphates, bicarbonates, sulfates, thiosulfates or bisulfites, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[330] In some embodiments, the pharmaceutical compositions include, without limitation, sugars such as trehalose, sucrose, mannitol, maltose, glucose, or salts such as potassium phosphate, sodium citrate, ammonium sulfate, and/or other such agents. like heparin to increase the solubility and stability of polypeptides in vivo.

[331] In some cases, pharmaceutical formulations further include a diluent that is used to stabilize the compounds, as they can provide a more stable environment. Salts dissolved in buffered solutions (which may also provide pH control or maintenance) are used in the art as diluents, including, but not limited to, phosphate buffered saline. In some cases, diluents increase the volume of the composition for ease of compression or create sufficient volume for a homogeneous mixture to be filled into capsules. Such compounds may include, for example, 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, eg latinized starch , compressed sugar such as Di- ^Pac® (Amstar), mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectionery sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal whey, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.

[332] In some cases, pharmaceutical compositions include disintegrating agents or disintegrants to facilitate the destruction or disintegration of the substance. The term "disintegration" includes both dissolution and dispersion of the dosage form upon contact with the gastrointestinal fluid. Examples of disintegrants include starch, such as natural starch such as corn starch or potato starch, pregelatinized starch such as National 1551 or ^Amijel® or sodium starch glycolate such as ^Promogel® or ^Explotab® , cellulose such as wood, methylcrystalline cellulose such as ^Avicel® , ^Avicel® PH101, ^Avicel® PH102, ^Avicel® PH105, ^Elcema® P100, ^Emcocel® , ^Vivacel® , Ming ^Tia® and Solka- ^Floc® , methyl cellulose, croscarmellose or cross-linked cellulose such as crosslinked sodium carboxymethylcellulose (Ac-Di- ^Sol® ), crosslinked carboxymethylcellulose or crosslinked croscarmellose, crosslinked starch such as sodium starch glycolate, crosslinked polymer such as crospovidone, crosslinked polyvinylpyrrolidone, alginate such as alginic acid, or salt alginic acid such as sodium alginate, clay such as ^Veegum® HV (magnesium aluminum silicate), gum such as agar, guar, locust bean, karaya , pectin or tragacanth, sodium starch glycolate, bentonite, natural sponge, surfactant, resin such as cation exchanger, citrus resin, sodium lauryl sulfate, sodium lauryl sulfate combined with starch, and the like.

[333] In some cases, pharmaceutical formulations include excipients 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, etc.[334] Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein to prevent, reduce, or inhibit adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, a hydrocarbon such as mineral oil, or a hydrogenated vegetable oil such as hydrogenated soybean oil ( ^Sterotex® ), higher fatty acids, and their alkali metal salts. and alkaline earth metals such as aluminium, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerin, talc, waxes, ^Stearowet® , boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g. PEG-4000 ) or methoxy polyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silicon dioxide such as ^Syloid ™, Cab-O-Sil®, starch such as corn starch, silicone oil, surfactant and the like.

[335] Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350 and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. Plasticizers can also act as dispersants or wetting agents.

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

[337] Stabilizers include compounds such as any antioxidants, buffers, acids, preservatives, and the like. Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium diphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, Polysorbate-20 or Tween® 20, or trometamol.

[338] Suspension agents include compounds such as polyvinylpyrrolidone, for example, polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, for example, polyethylene glycol may have a molecular weight of from 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 gum tragacanth and gum arabic , guar gum, xanthans, including xanthan gum, sugars, celluloses such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, povidone, etc. .

[339] 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, for example , Pluronic® (BASF), etc. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, for example, polyoxyethylene (60) hydrogenated castor oil and polyoxyethylene alkyl ethers and alkylphenyl ethers, for example, octoxynol 10, octoxynol 40. Sometimes surfactants are included to increase physical stability or for other purposes.

[340] Viscosifying agents include, for example, methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate stearate, hydroxypropyl methyl cellulose, phthalate, carbomer, polyvinyl alcohol, alginates, gums, chitosans, and combinations thereof.

[341] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolaminooleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts etc.

Treatment regimens

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

[343] In the case where the patient's condition does improve, at the discretion of the physician, the composition is administered continuously, alternatively, the dose of the administered composition is temporarily reduced or temporarily suspended for a certain period of time (i.e., "drug rest"). In some cases, the duration of drug rest varies from 2 days to 1 year, including by way of example only: 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days or 365 days. Dose reduction during drug rest ranges from 10% to 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%.

[344] After the improvement of the patient's condition, if necessary, appoint a maintenance dose. Subsequently, the dosage or frequency of administration, or both, may be reduced, depending on the symptoms, to a level at which an improved disease, disorder, or condition is maintained.

[345] In some embodiments, the amount of a given agent that corresponds to that amount varies depending on 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, how usually determined in a manner known in the present technical field, in accordance with the specific circumstances associated with this case, including, for example, the specific administered agent, the route of administration and the subject or host being treated. In some instances, the desired dose may conveniently be presented as a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, such as two, three, four or more sub-doses per day.

[346] The above ranges are merely suggestive, since the number of variables in relation to an individual treatment regimen is large, and significant deviations from these recommended values are not uncommon. Such dosages will vary depending on a number of variables, not limited to the potency of the compound used, the disease or condition being treated, the route of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the attending physician.

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

Kits/product

[348] Disclosed herein in some embodiments are kits and articles for use with one or more of the methods and compositions described herein. Such kits include a carrier, package or container that is divided into parts to receive one or more containers, such as vials, test tubes, etc., each of which contains one or more separate elements to be used in the described in this document method. Suitable containers include, for example, bottles, vials, syringes and test tubes. According to one embodiment, the containers are made from various materials such as glass or plastic.

[349] The products presented herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, pouches, containers, vials, and any packaging material suitable for the composition chosen and the intended mode of administration and handling.

[350] For example, the container(s) contains one or more of the cytokine (e.g., IL-2) or cytokine (e.g., IL-2) conjugates described herein and optionally one or more of the pharmaceutical excipients described herein. herein, to facilitate the delivery of cytokine polypeptides (eg, IL-2) or cytokine conjugates (eg, IL-2). Such kits additionally optionally contain an identifying description or label or instructions relating to its use in the methods described herein.

[351] The kit typically contains labels with a list of contents and / or instructions for use, as well as inserts with instructions for use. The instruction set is also typically included.

[352] According to one embodiment, the label is on or associated with the container. According to one embodiment, the label is on the container when the letters, numbers, or other symbols forming the label are affixed, molded or etched into the container itself, the label is associated with the container when it is present inside the container or holder that also contains the container, such as package insert. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also provides directions for using the contents, such as in the methods described herein.

[353] In certain embodiments, pharmaceutical compositions are presented in a package or dispenser that contains one or more unit dosage forms containing a compound as provided herein. The package, for example, contains a metal or plastic foil, such as a blister pack. In one embodiment, the package or dispenser is accompanied by instructions for administration. In one embodiment, the package or dispenser is also accompanied by a notice associated with the container in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the form of the drug for use in humans or animals. For example, such a notice is a US Food and Drug Administration-approved label for drugs or an approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for the treatment of said condition.

Defined terms

[354] Unless otherwise defined, all technical and scientific terms used in this document are characterized by the same meaning, which is usually understood by a person skilled in the art to which the claimed subject matter belongs. It is to be understood that the detailed description is illustrative and explanatory only and is not intended to limit any claimed subject matter. In this application, the use of the singular includes the plural, unless specifically stated otherwise. It should be noted that, as used herein, singular forms include plurals unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or", unless otherwise indicated. In addition, the use of the term "including", as well as other forms such as "include", "includes" and "included", is not limiting.

[355] Although various features of the present invention may be described in the context of a single embodiment, the features may also be presented separately or in any suitable combination. Conversely, although the present invention may be described herein in the context of separate embodiments for the sake of clarity, the present invention may also be implemented according to one embodiment.

[356] Reference in the description to “certain embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included at least in some embodiments, but not necessarily all embodiments of the present invention.

[357] As used herein, ranges and amounts can be expressed as "approximately" a specific value or range. About also includes the exact amount. Therefore, "about 5 µl" means "about 5 µl", as well as "5 µl". Typically, the term "about" includes an amount that is expected to be within experimental error, such as, for example, within 15%, 10%, or 5%.

[358] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter of the description.

[359] As used herein, the terms "individual(s)", "subject(s)", and "patient(s)" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is not a human. Neither term requires or is limited to situations characterized by the supervision (eg, continuous or intermittent) of a healthcare professional (eg, physician, registered nurse, nurse practitioner, physician assistant, nurse, or hospice worker).

[360] As used herein, the term "significant" or "significantly" in relation to binding affinity means a change in the binding affinity of a cytokine (e.g., an IL-2 polypeptide) sufficient to affect the binding of a cytokine (e.g., an IL-2 polypeptide) to a target receptor. . In some cases, the term refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, 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 , 100x, 500x, 1000x or more.

[361] In some cases, the term "significant" or "significantly" in relation to the activation of one or more cell populations by a cytokine signaling complex means a change sufficient to activate the cell population. In some cases, the change in cell population activation is measured as receptor signaling activity. In such cases, the EC50 value may be indicated. In other cases, an ED50 value may be given. In additional cases, the concentration or dosage of the cytokine may be provided.

[362] As used herein, the term "activity" refers to the amount of cytokine (eg, IL-2 polypeptide) required to obtain the desired effect. In some instances, the term "activity" refers to the amount of a cytokine (eg, IL-2 polypeptide) required to activate the target cytokine receptor (eg, IL-2 receptor). In other instances, the term "activity" refers to the amount of cytokine (eg, IL-2 polypeptide) required to activate the target cell population. In some cases, activity is measured as the ED50 (effective dose of 50), or the dose required to obtain 50% of the maximum effect. In other cases, activity is measured as EC50 (effective concentration of 50) or the dose required to achieve the target effect in 50% of the population.

EXAMPLES

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

EXAMPLE 1

[364] Kinase and cytokine receptor dimerization assays.

[365] Cell preparation

[366] PathHunter cell lines were expanded from freezer stock according to standard procedures. Cells were seeded in a total volume of 20 μl in 384-well white-walled microplates and incubated for the appropriate time before testing.

[367] Agonist Format

[368] To determine the agonist, cells were incubated with a sample to induce a response. An intermediate dilution of sample stocks was performed to obtain a 5X sample in assay buffer. About 5 μl of 5X sample was added to the cells and incubated at 37° C. for 6-16 hours depending on the assay. The filler concentration was 1%.

[369] Signal Detect

[370] The assay signal was generated by a single addition of 12.5 or 15 µl (50% by volume) PathHunter Detection Agonist and Antagonist Assay Reagent Kit, respectively, followed by one hour incubation at room temperature. In some assays, activity was detected using a highly sensitive detection reagent (PathHunter Flash Kit) to improve assay performance. In these assays, an equal volume of detection reagent (25 or 30 µl) was added to the wells, followed by one hour incubation at room temperature. The microplates were read after receiving the signal using a PerkinElmer Envision™ chemiluminescence signal detection instrument.

[371] Data Analysis

[372] Compound activity was analyzed using the CBIS data analysis kit (ChemInnovation, CA). For assays in agonist mode, percent potency was calculated using the following formula:

% activity = 100% × (average RLU of test sample - average RLU of vehicle control)/(average MAX RLU of ligand control - average RLU of vehicle control).

[373] For assays in antagonist mode, percent inhibition was calculated using the following formula:

% Inhibition = 100% × (1 - (mean RLU of test sample - mean RLU of vehicle control)/(mean RLU of EC80 control - mean RLU of vehicle control)).

EXAMPLE 2

[374] Cellular screening for identification of pegylated IL-2 compounds without involvement of IL-2Rα

[375] Structural data of the IL-2 signaling/heterotrimeric receptor complex (PDB:2ERJ) was used to target the design of nAA-pegylation sites to specifically abolish the interaction of IL-2 and the α-subunit of the IL-2 receptor (IL-2Rα). Exemplary IL-2 conjugates were subjected to functional analysis: K35, F42, K43, E62 and P65. IL-2 conjugates were expressed as inclusion bodies in E. coli, purified and refolded using standard procedures prior to specific PEGylation of the IL-2 product using DBCO mediated click chemistry without copper to attach stable mPEG covalent moieties to AzK.

[376] IL-2 conjugates were screened for functional activity in Discoverx (Fremont CA) using PathHunter's IL-2 cytokine receptor assay. This assay uses a recombinant human U2OS cell line that expresses the β (IL-2Rβ) and γ (IL-2Rγ) subunits of the IL-2 receptor, each fused to one half of the cleaved β-galactosidase reporter enzyme. A second cell line was further engineered to express the IL-2Rα subunit. A parallel study with these two cell lines allows evaluation of the activation variant of the αβγ IL-2 receptor as well as the basal βγ complex. IL-2 agonist activity on the βγ IL-2 receptor complex stimulates receptor dimerization and reduction of reporter β-galactosidase, resulting in a chemiluminescent signal. The assay was run in agonist mode to determine the EC-50 of each test product and comparison of the dose-response curve profiles between IL2Rα positive and negative cell types allows the contribution of IL2Rα to the observed activity to be determined.

[377] Table 1 shows EC50 data for IL-2 receptor agonism in cell screening for 30 kD PEGylated IL-2 conjugates.

[378] Biochemical interactions of pegylated IL-2 with human IL-2 receptor subunits.

[379] The kinetics of interactions of the pegylated IL-2 compound with human IL-2 receptor subunits was measured using surface plasmon resonance (SPR) at Biosensor Tools LLC (Salt Lake City, UT). In these studies, human IgG1 Fc-fused Rα (Sino Biological #10165-H02H) and β (Sino Biological #10696-H02H) IL-2 extracellular domains were captured on the surface of a CM4 sensor chip coated with A Biacore protein. These surfaces were examined in duplicate, with 2-fold dilution series starting at 2 μM of either native IL-2 (wild-type IL-2; Thermo # PHC0021), P65_30kD, P65_5kD, E62_30kD, or E62_5kD using a Biacore 2000 SPR instrument. Test samples were injected for 60 s to allow association to be measured, followed by buffer only (wash) for 30 s to measure dissociation. Response units (RU, Y-axis) are plotted against time (s, X-axis).

[380] To evaluate the effect of the α subunit of the IL-2 receptor on IL-2 binding to β, α was captured in about a two-fold excess relative to β. Native IL-2 (wild-type IL-2), P65_30kD, P65_5kD, E62_30kD, or E62_5kD was applied to these surfaces in a three-fold dilution series starting at 2.5 μM. The binding data were consistent with a 1:1 interaction model that included array shifting, and the extracted kinetic parameters are summarized in Table 2A and Table 2B.

[381] On sensor surfaces containing immobilized IL-2Rα, native IL-2 showed fast association kinetics and slow dissociation kinetics, showing high affinity binding (FIG. 4A). In contrast, neither P65 30kD nor E62_30kD interacted with the surface of IL-2Rα even at the highest concentration tested, 2 μM (FIG. 4A). Surfaces containing immobilized IL-2Rβ showed comparable association and dissociation reactions with both native IL-2 (wild-type IL-2) and P65_30kD IL-2 (FIG. 4B). The small difference in K-D observed between compounds for the β subunit is due to a decrease in the rate of association of IL-2 P65_30kD compared to native IL-2, expected due to a change in the hydrodynamic radius of this pegylated compound (lower diffusion coefficient) as well as a non-specific screening effect large fragment of PEG to distant binding surfaces. These results suggest that P65_30kD and E62_30kD are defective in IL-2Rα interactions while retaining significant binding to IL-2Rβ.

[382] Ex-vivo immune response profiling of IL-2 P65_30kD and E62_kD in PBMC samples derived from the primary human leukocyte reduction system (LRS)

[383] To determine how the differential receptor specificity of IL-2 P65_30kD and E62_30kD influences the activation of primary immune cell subpopulations, concentration-response profiling of lymphocyte activation in LRS-derived human peripheral blood mononuclear cell (PBMC) samples was performed using multicolor flow chart. cytometry. These studies were performed at PrimityBio LLC (Fremont, CA). Fresh LRS-derived samples were treated with either native IL-2, P65_30kD or E62_30kD in a 5-fold dilution series starting at a maximum concentration of 30 μg/ml. After a 45-minute incubation, samples were fixed and stained with antibodies to detect the phosphorylated form of the STAT5 transcription factor (pSTAT5), a marker for the prior involvement and activation of IL-2 receptor signaling complexes, and a panel of surface markers (Table 3) to track the formation of pSTAT5 in specific T cell subpopulations. and natural killer (NK) cells.

[384] Flow cytometry data was analyzed to activate different subsets of T and NK cells in concentration-response mode, reading pSTAT5 accumulation after native IL-2 treatment (FIG. 5A), E62_30kD (FIG. 5B), and P65_30kD (FIG. 5C). ). In populations of NK- and effector T-cells (CD3+ CD8+), IL-2 P65_30kD and IL-2 E62_30kD retained activity relative to native IL-2, while EC-50 values for pSTAT5 production were 4-5 times higher than native IL-2 ( Fig. 5A). In contrast, the EC-50 values for pSTAT5 induction for IL-2 P65_30kD in the Treg subset (CD3+ CD4+ IL-2Rα+ CD127-) were reduced by 900-fold compared to native IL-2, but remained similar to the EC50 for the NK- and Teff cells (Fig. 5C). This significant increase in EC-50 for IL-2 P65_30kD specifically in the Treg population indicates that pegylation of IL-2 at these positions allows IL-2 receptor agonism but abolishes the bias of IL-2 to stimulate Tregs relative to effector T cells.

[385] EC50 values (pg/mL) were calculated from dose-response curves derived from MFI plots.

EXAMPLE 3

[386] PK/PD studies in intact (E3826-U1704) and tumor bearing B16-F10 (E3826-U1803) mice C57BL/6

[387] The study designs are summarized in Tables 5 and 6. Terminal blood samples were taken by cardiac puncture at the indicated time points. Study E3826-U1704, including 13 time points (0.13, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h), included 3 mice per each time point, and the E3826-U1803 study included 9 time points (2, 8, 12, 24, 48, 72, 120, 168, and 240 h) with 4-7 mice per time point. Plasma and blood cells (in both studies) and tumors in study E3826-U1803 were collected for PK and PD analysis.

[388] Bioanalysis of plasma samples was performed using a qualified human IL-2 ELISA assay (Abeam, Cambridge, UK). Aldesleukin, E62_30kD and P65_30kD and internal standard concentrations in plasma-derived samples were determined by ELISA analysis. PK data analysis was performed at NW Solutions (Seattle, WA). PK data was imported into Phoenix WinNonlin v6.4 (Certara/Pharsight, Princeton, NJ) for analysis. Mean plasma concentration versus time data were analyzed by non-commercial methods using an intravenous bolus model.

[389] Plasma concentration profiles of P65_30kD, E62_30kD, E62_5kD and 0.3 mg/kg aldesleukin are presented in FIG. 6.

[390] In study E3826-U1704, both P65_30kD and E62_30kD show superior PK profiles compared to aldesleukin as shown in Table 3. Following a single intravenous bolus dose of aldesleukin, Tmax was observed 0.03 h post-dose post-dose measurements) and mean plasma concentrations were measured up to 4 hours post-dose. Following a single intravenous bolus dose of P65_30 kDa and E62_30 kDa, Tmax was observed 0.03 h post-dose, and mean plasma concentrations were measured up to 120 h post-dose (last time point measured). In a separate study, following an intravenous dose of E625 kDa, Tmax was observed 0.133 hours post-dose, and mean plasma concentrations were measured up to 12 hours post-dose.

[391] The exposure based on C-max and AUC-0-t was as follows: P65_30kD>>E62_30kD>>E62_5kD>aldesleukin. E62_5kD with lower PEG had a PK profile closer to that of rIL-2. The exposure to P65_30kD was 5.5 and 200 times greater than that of aldesleukin based on Cmax and AUC0-t, respectively. In addition, P65_30kD showed a 23-fold increase in t1/2 (13.3 h vs. 0.57 h) and about a 198-fold decrease in CL (6.58 vs. 1300 ml/h/kg) compared with aldesleukin. For both P65_30kD and E62_30kD, the volume of distribution (82.4 and 92.3 ml/kg, respectively) decreased by about 4.2-4.7 times compared with aldesleukin and is similar to the blood volume in mice (85 ml/kg ; [Boersen 2013]). This suggests that P65_30kD and E62_30kD are mostly distributed within the systemic circulation.

EXAMPLE 4

[392] Pharmacodynamic observations in the peripheral blood compartment

[393] STAT5 phosphorylation and induction of cell proliferation (Ki-67 early molecular marker and cell number) were used as pharmacodynamic indications to assess the pharmacological profile of P65_30kD with respect to its pharmacokinetics. The pSTAT5 PD marker showed a good correlation with PK for both P65_30kD and aldesleukin in CD8+ effector T cells (Table 7). A persistent increase in pSTAT5 was observed in both NK- and CD8+ T cells up to 72 hours and up to 24 hours in Tregs. pSTAT5 induction only returned to baseline after 2 hours in mice treated with aldesleukin (FIG. 7). Phosphorylation of STAT5 was translated into delayed proliferative responses (72-120 hours) of CD8+ effector T cells and NK cells, but not from T regs (FIGS. 8A-8C). Phenotypic analysis of effector CD8+ T cells revealed a significant expansion of CD44+ memory cells in this population (FIGS. 9A-9B).

[394] Pharmacodynamic observations in the tumor compartment in B16-F10 (E3826-U1803) tumor-bearing C57BL/6 mice

[395] Table 8 shows plasma and tumor drug concentrations after a single dose of P65_30kD at 3 mg/kg in mice with B16-F10 tumors. The tumor half-life was twice the plasma half-life (24.4 vs. 12.6), indicating that P65_30kD enters the tumor and persists in the tumor. The tail end of the curves intersect, indicating that elimination from plasma is faster than from the tumor. The tumor:plasma AUC ratio was 9.7% and 8.4% for doses of 1 and 3 mg/kg, respectively.

[396] FIG. 10A - fig. 10B shows the expansion of NK- and CD+ T cells by P65_30kD in B16F10 tumors. In FIG. 10A shows the percentage of NK cells, CD8+ cells and Tregs in the CD3+ T cell tumor population after treatment with a single intravenous bolus dose of P65_30kD at 3 mg/kg. Tumor samples were analyzed for immune cell populations 5 days after exposure by flow cytometry. Each data point is the mean of three repetitions at each time point ± SEM. The cell population data shown are from day 5 tumor samples and the CD8/Treg ratio was calculated from day 7 samples. In FIG. 10B shows the ratio of CD8+ effector to CD4+ regulatory T cells 7 days after treatment with a single intravenous bolus dose of P65_30kD at 3 mg/kg. Each data point is the mean of three repetitions at each time point ± SEM.

[397] MTD study in Balb/c E3826-U1802 mice

[398] A P65_30kD dosing range study was performed on intact female Balb/c mice at Crown Biosciences, Inc. (San Diego, California). The study design is shown in Table 9. Blood samples were taken via the mandibular vein at 7 time points (0.25, 1, 4, 12, 24, 34, 48 and 72 hours). Both plasma and blood cells were collected for PK and PD analysis.

[399] All plasma samples were analyzed for human IL-2 as well as mouse cytokines IL-2, TNF-α, IFNγ, IL-5 and IL-6 using commercially available ELISA kits.

[400] Toxicological observations in the MTD study using Balb/c mice

[401] The main toxicity associated with high doses of IL-2 is vascular leakage syndrome and associated cytokine release syndrome (CRS). To evaluate this in mice, a single intravenous injection of P65_30kD was performed at doses ranging from 0.01 to 5.0 mg/kg (Table 10). The analysis performed was hematology, histopathology, body weight analysis and cytokine analysis. No abnormalities were observed in hematology, histopathology, or body weight compared to vehicle-treated mice with both P65_30kD and aldesleukin. With respect to cytokine analysis, aldesleukin was noted to increase plasma IL-5 levels from 1 mg/kg to 5 mg/kg (FIG. 11A). With P65_30kD, a modest increase in IL-5 was observed only at the 5 mg/kg dose, and this was less compared to aldesleukin (FIG. 11B). A temporary increase in the systemic content of IFNγ was observed for both aldesleukin and P65_30kD.

EXAMPLE 5

[402] PK/PD in cynomolgus monkeys - study no. 20157276

[403] The pharmacokinetic and pharmacodynamic profile of P65_30kD was evaluated in exposed cynomolgus monkeys following a single intravenous dose of 0.3 mg/kg. The study was conducted at Charles River Laboratories, Inc. (Reno, NV) and PK data analysis was performed at NW Solutions (Seattle, WA). 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 hours post-dose). Both plasma and blood cells were collected for PK and PD analysis. Selected time points were used for PK, PD, cell population and hematological analysis.

[404] All plasma samples were analyzed for human IL-2 (PK reading) using commercially available ELISA kits.

[405] Table 10 shows PK parameters P65_30kD in cynomolgus monkeys.

[406] Following a single intravenous bolus, Tmax was observed 0.5 h post-dose (first measured time point post-dose), and mean plasma concentrations were measured up to 168 h post-dose (last measurement), t-1/2 and AUC for P65_30kD were 13.6 h and 121,000 h×ng/mL, respectively.

[407] Hematological parameters - cynomolgus macaques - Study No. 20157276

[408] For hematological parameters, the evaluation time points correspond to the pre-dose on day -1 and 1, 3, 6, 8, 10, 12, 14, 17, 19, 21 post-dose.

[409] In FIG. 12 shows the absolute content of leukocytes and differential indicators. Data are mean ± standard deviation (N=2 animals/dose group).

[410] Analysis of subpopulations of leukocytes (WBC) showed that a significant increase in the number of leukocytes was caused by an increase in the population of lymphocytes, which is consistent with the P65_30kD mechanism. There is no increase in eosinophils.

EXAMPLE 6

[411] Table 11 shows the IL-2 sequences described herein.

[412] While preferred embodiments of the present invention have been shown and described herein, those skilled in the art will appreciate that such embodiments are presented by way of example only. Those skilled in the art will appreciate that numerous variations, changes and substitutions will occur without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. It is intended that the following claims define the scope of the present invention and that methods and structures are thus covered within the scope of the claims and their equivalents.

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SEQUENCE LIST

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<120> CYTOKINE CONJUGATES FOR THE TREATMENT OF PROLIFERATIVE AND INFECTIOUS DISEASES

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<---

Claims (37)

1. An interleukin-2 (IL-2) conjugate for the treatment of a proliferative disease or infection, comprising: a) a modified interleukin-2 (IL-2) polypeptide containing at least one unnatural amino acid, wherein the modified interleukin-2 (IL-2) polypeptide comprises at least 80% sequence identity with SEQ ID NO: 1, where the position of 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, and wherein the residue positions correspond to positions 35, 37, 38, 41, 42, 43, 44, 45, 60, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107, as shown in SEQ ID NO: 1; b) a conjugating moiety covalently attached via a linker to at least one non-natural amino acid; moreover, the conjugating fragment is a water-soluble polymer, including polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(alpha-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA), or a combination thereof . 2. The interleukin-2 (IL-2) conjugate according to claim 1, wherein the modified interleukin-2 (IL-2) polypeptide conjugate comprises at least 90% sequence identity to SEQ ID NO: 1. 3. An interleukin-2 (IL-2) conjugate according to claim 1, wherein the position of at least one non-natural amino acid is selected from F42, K43, F44, E62 and P65, and the positions of the residues correspond to positions 42, 43, 44, 62 and 65 as shown in SEQ ID NO: 1. 4. An interleukin-2 (IL-2) conjugate according to claim 3, wherein the position of at least one non-natural amino acid is E62, and the positions of the residues correspond to position 62, as shown in SEQ ID NO: 1. 5. An interleukin-2 (IL-2) conjugate according to claim 3, wherein the position of at least one non-natural amino acid is P65, and the positions of the residues correspond to position 65, as shown in SEQ ID NO: 1. 6. The interleukin-2 (IL-2) conjugate of claim 1, wherein at least one non-natural amino acid is a lysine analog or contains an aromatic side chain. 7. An interleukin-2 (IL-2) conjugate according to claim 1, wherein the water-soluble polymer contains polyethylene glycol (PEG). 8. The interleukin-2 (IL-2) conjugate of claim 7, wherein the polyethylene glycol (PEG) has a weight average molecular weight of about 10 kDa to about 85 kDa. 9. The interleukin-2 (IL-2) conjugate of claim 8, wherein the polyethylene glycol (PEG) has a weight average molecular weight selected from about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa or about 50 kDa. 10. Interleukin-2 (IL-2) conjugate according to claim 1, wherein: a) the position of at least one non-natural amino acid is selected from F42, K43, F44, Y45, E62 and P65, with the positions of the residues corresponding to positions 42, 43, 44, 45, 62 and 65, as shown in SEQ ID NO: 1; b) the modified interleukin-2 (IL-2) polypeptide contains an N-terminal deletion, including the deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 residues from the N-terminus, moreover, the positions of the residues refer to the positions in SEQ ID NO: 1; c) the conjugating moiety is polyethylene glycol (PEG) having a weight average molecular weight of about 20 kDa to about 85 kDa; and d) the modified interleukin-2 (IL-2) polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1. 11. The interleukin-2 (IL-2) conjugate according to claim 10, characterized in that the modified interleukin-2 (IL-2) polypeptide contains an N-terminal deletion, including the deletion of the first 1, 2, 3, 4 or 5 residues with N-terminus, with the positions of the residues referring to the positions in SEQ ID NO: 1. 12. The interleukin-2 (IL-2) conjugate of claim 11, wherein the polyethylene glycol (PEG) has a weight average molecular weight selected from about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa or about 50 kDa. 13. The interleukin-2 (IL-2) conjugate according to any one of paragraphs. 1-12, in which at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine , methyltetrazine-lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAM), p-iodine-L- phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p- acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl- L-tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2 -((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid or sat enocysteine. 14. Pharmaceutical composition for the treatment of a proliferative disease or infection, containing an effective amount of an interleukin-2 (IL-2) conjugate according to any one of paragraphs. 1-13 and a pharmaceutically acceptable excipient. 15. Pharmaceutical composition according to claim 14, characterized in that the pharmaceutical composition is formulated for systemic or parenteral delivery. 16. A method of treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject an interleukin-2 (IL-2) conjugate according to any one of paragraphs. 1-13 or a pharmaceutical composition according to claim 14 or 15. 17. The method of claim 16 wherein the proliferative disease or condition is cancer. 18. The method of claim 17 wherein the cancer is a solid tumor. 19. The method of claim 18 wherein the solid tumor 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. 20. The method of claim 17, wherein the cancer is a hematological malignancy. 21. The method of claim 20 wherein the hematologic malignancy is chronic lymphocytic leukemia (CLL), small cell lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's large B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, Progenitor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis . 22. A method for expanding a population of CD4+ helper cells, CD8+ effector naive cells, CD8+ effector memory cells, natural killer (NK) cells, or natural killer T cells (NKT), comprising bringing the cell population into contact with an interleukin-2 (IL-2) conjugate ) according to any one of paragraphs. 1-13 or a pharmaceutical composition according to claim 14 or 15 for a time sufficient to induce the formation of a complex with IL-2

, thereby stimulating the expansion of the population of Teff and/or NK cells. 23. The method according to p. 22, in which the interleukin-2 (IL-2) conjugate causes the expansion of CD4+ T-regulatory (Treg) cells by less than 20%, 15% 10%, 5% or 1% in the population of CD3+ cells by compared with the expansion of CD4+ Treg cells in the population of CD3+ cells in contact with the wild-type IL-2 polypeptide. 24. The method of claim 22, wherein the interleukin-2 (IL-2) conjugate does not cause expansion of CD4+ Treg cells in the cell population. 25. The method of claim 22, wherein the ratio of Teff cells to Treg cells in the cell population after incubation with an interleukin-2 (IL-2) conjugate is approximately 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. 26. The method according to p. 22, characterized in that the method is an in vivo method. 27. The method according to p. 22, characterized in that the method is an in vitro method. 28. The method according to p. 22, characterized in that the method is an ex vivo method.

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