Interleukin 10 conjugates and uses thereof

Abstract

Disclosed herein are interleukin-10 conjugates and their uses in treating one or more conditions, as well as pharmaceutical compositions and kits that include one or more IL-10 conjugates.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 757,690, filed November 8, 2018, and U.S. Provisional Patent Application No. 62 / 911,036, filed October 4, 2019, both of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 4, 2019, is named 46085-727_851_SL.txt and is 4,157 bytes in size. [Background technology]

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

[0004] Disclosed herein in certain embodiments are interleukin 10 (IL-10) conjugates and their use in treating one or more indications. In some embodiments, disclosed herein are IL-10 conjugates for treating cancer. In additional cases, disclosed herein are pharmaceutical compositions and kits comprising the IL-10 conjugates described herein.

[0005] Disclosed herein, in some embodiments, is a modified interleukin 10 (IL-10) polypeptide comprising an amino acid sequence comprising at least one non-natural amino acid linked to at least one conjugating moiety, the amino acid sequence being 99.0% identical to amino acids 33-158 of SEQ ID NO:1. In some embodiments, the amino acid sequence is at least 95.0% identical to amino acids 1-160 of SEQ ID NO:1. In some embodiments, the amino acid sequence is at least 97.0% identical to amino acids 1-160 of SEQ ID NO:1. In some embodiments, the amino acid sequence is at least 98.0% identical to amino acids 1-160 of SEQ ID NO:1. In some embodiments, the amino acid sequence comprises 13 lysine amino acid residues. In some embodiments, the non-natural amino acid is located at an amino acid position selected from E67, Q70, E74, E75, Q79, N82, K88, K125, N126, N129, K130, or Q132 in SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, or N82, where the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from Q70, E74, N82, K88, N126, K130, or Q132, where the residue positions correspond to positions 70, 74, 82, 88, 126, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid is located proximal to the N-terminus. In some embodiments, the at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the N-terminus.In some embodiments, at least one unnatural amino acid is located proximal to the C-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the C-terminus. In some embodiments, at least one unnatural amino acid: a) is a lysine analog; b) comprises an aromatic side chain; c) comprises an azide group; d) comprises an alkyne group; or e) comprises an aldehyde or ketone group. In some embodiments, at least one unnatural amino acid does not comprise an aromatic side chain. In some embodiments, at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid is selected from the group consisting of N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, phenyl fluoride, and the like. alanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, or selenocysteine.In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-10 polypeptide by an orthogonal tRNA synthetase / tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase / tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-10 polypeptide dimerizes with a second modified IL-10 polypeptide, thereby generating an IL-10 dimer comprising two unnatural amino acids. In some embodiments, the unnatural amino acid of the modified IL-10 polypeptide is located at a different residue position than the unnatural amino acid of the second modified IL-10 polypeptide. In some embodiments, both unnatural amino acids are located at the same residue position in the respective IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide dimerizes with an IL-10 polypeptide that does not comprise an unnatural amino acid. In some embodiments, the modified IL-10 polypeptide dimerizes with a wild-type IL-10 polypeptide. In some embodiments, at least one conjugating moiety comprises a water soluble polymer, a lipid, a protein, or a peptide. In some embodiments, the water-soluble polymer comprises a polyethylene glycol (PEG) molecule. In some embodiments, the water-soluble polymer comprises a polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), polysaccharide, poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer comprises a polysaccharide.In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin. In some embodiments, the protein comprises an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof comprises an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a homoamino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the conjugation moiety is indirectly linked to at least one unnatural amino acid of the modified IL-10 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the modified IL-10 polypeptide is: a) a functionally active fragment of a full-length IL-10 polypeptide; b) a recombinant IL-10 polypeptide; or c) a recombinant human IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, where residue positions are relative to those in SEQ ID NO:1.In some embodiments, the C-terminal deletion comprises deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, where the residue positions are relative to the positions in SEQ ID NO: 1. In some embodiments, the modified IL-10 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10 dimer binds to IL-10R to form an IL-10 / IL-10R complex. In some embodiments, the increased plasma half-life of the IL-10 dimer is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of the wild-type IL-10 dimer. In some embodiments, the increased plasma half-life of the IL-10 dimer is about 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, or 30 days. In some embodiments, at least one unnatural amino acid position does not substantially interfere with dimerization of the IL-10 polypeptide. In some embodiments, at least one unnatural amino acid position does not significantly interfere with binding of the IL-10 dimer to IL-10R. In some embodiments, the at least one unnatural amino acid position impairs IL-10R signaling by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%. In some embodiments, the at least one unnatural amino acid position does not significantly impair IL-10R signaling as defined by the ability of cells to phosphorylate Stat3 and / or activate IL-10R signaling. In some embodiments, the modified IL-10 polypeptide is isolated and purified. In some embodiments, the modified IL-10 polypeptide is formulated as a pharmaceutical formulation comprising a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical formulation is formulated for systemic delivery.

[0006] Disclosed herein, in some embodiments, is a modified IL-10 homodimer comprising two of the modified IL-10 polypeptides of claim 1.

[0007] Disclosed herein, in some embodiments, are compositions comprising a complex of a modified homodimer of the present disclosure bound to the IL-10 receptor (IL-10R).

[0008] Disclosed herein, in some embodiments, is a method for treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a modified IL-10 polypeptide of the present disclosure. In some embodiments, the proliferative disease or condition is cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.

[0009] Disclosed herein, in some embodiments, are nucleic acid molecules that encode the modified IL-10 polypeptides of the present disclosure.

[0010] Disclosed herein, in some embodiments, is a vector that comprises a nucleic acid molecule of the present disclosure.

[0011] Disclosed herein are isolated and modified interleukin 10 (IL-10) polypeptides that, in certain embodiments, include at least one unnatural amino acid at a position on the polypeptide that increases plasma half-life without significantly altering interaction with the IL-10 receptor, where the increased plasma half-life is compared to the plasma half-life of a wild-type IL-10 protein. In some embodiments, the at least one unnatural amino acid is located at a surface-exposed position of helices C, D or E. In some embodiments, the position of the at least one unnatural amino acid is selected from E67, Q70, E74, E75, Q79, N82, K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO:1. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, or N82, where the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from Q70, E74, N82, K88, N126, K130, or Q132, where the residue positions correspond to positions 70, 74, 82, 88, 126, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence is at least or about 99.0% identical to amino acids 33-158 of SEQ ID NO: 1. In some embodiments, the amino acid sequence includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 wild-type lysines.In some embodiments, the amino acid sequence comprises wild type lysines at one or more of K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 wild type lysines at K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, the amino acid sequence comprises wild type lysines at K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, at least one unnatural amino acid is located proximal to the N-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the N-terminus. In some embodiments, at least one unnatural amino acid is located proximal to the C-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the C-terminus. In some embodiments, at least one unnatural amino acid: a) is a lysine analog; b) comprises an aromatic side chain; c) comprises an azide group; d) comprises an alkyne group; or e) comprises an aldehyde or ketone group. In some embodiments, at least one unnatural amino acid does not comprise an aromatic side chain.In some embodiments, the at least one unnatural amino acid is selected from the group consisting of N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, phenyl fluoride, and the like. alanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, or selenocysteine. In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-10 polypeptide by an orthogonal tRNA synthetase / tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase / tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-10 polypeptide dimerizes with a second modified IL-10 polypeptide, thereby generating an IL-10 dimer comprising two unnatural amino acids. In some embodiments, the unnatural amino acid of the modified IL-10 polypeptide is located at a different residue position than the unnatural amino acid of the second modified IL-10 polypeptide.In some embodiments, both non-natural amino acids are located at the same residue position in each IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide dimerizes with an IL-10 polypeptide that does not include a non-natural amino acid. In some embodiments, the modified IL-10 polypeptide dimerizes with a wild-type IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide is covalently attached to a conjugation moiety via at least one non-natural amino acid. In some embodiments, the conjugation moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), polysaccharides, poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin.In some embodiments, the protein comprises an antibody or a binding fragment thereof. In some embodiments, the antibody or a binding fragment thereof comprises an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a homoamino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the conjugation moiety is indirectly linked to at least one unnatural amino acid of the modified IL-10 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the modified IL-10 polypeptide is: a) a functionally active fragment of a full-length IL-10 polypeptide; b) a recombinant IL-10 polypeptide; or c) a recombinant human IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, with residue positions relative to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion comprises 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 residue positions relative to the positions in SEQ ID NO: 1. In some embodiments, the modified IL-10 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10 dimer binds to IL-10R to form an IL-10 / IL-10R complex.In some embodiments, the increased plasma half-life of the IL-10 dimer is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of the wild-type IL-10 dimer. In some embodiments, the increased plasma half-life of the IL-10 dimer is about 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, or 30 days. In some embodiments, at least one unnatural amino acid position does not substantially interfere with dimerization of the IL-10 polypeptide. In some embodiments, at least one unnatural amino acid position does not significantly interfere with binding of the IL-10 dimer to IL-10R. In some embodiments, the at least one unnatural amino acid position impairs IL-10R signaling by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%. In some embodiments, the at least one unnatural amino acid position does not significantly impair IL-10R signaling as defined by the ability of cells to phosphorylate Stat3 and / or activate IL-10R signaling.

[0012] Provided herein, in certain embodiments, the at least one unnatural amino acid position is selected from N82, K88, N129, K130, or Q132, where the residue positions correspond to positions 82, 88, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from N82, N129, K130, or Q132, where the residue positions correspond to positions 82, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from N82, N129, K130, or Q132, where the residue positions correspond to positions 82, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from N82, N129, or Q132, where the residue positions correspond to positions 82, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from N82 or N129, where the residue positions correspond to positions 82 and 129 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from N82, where the residue position corresponds to position 82 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from N129, where the residue position corresponds to position 129 as set forth in SEQ ID NO: 1.

[0013] As disclosed herein, in certain embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid comprises N6-(propargylethoxy)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid comprises N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid comprises N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid comprises N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0014] As disclosed herein, in certain embodiments, the position of the at least one unnatural amino acid is selected from N82, K88, N129, K130, or Q132, where the residue positions correspond to positions 82, 88, 129, 130, and 132 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82, N129, K130, or Q132, where the residue positions correspond to positions 82, 129, 130, and 132 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82, N129, K130, or Q132, where the residue positions correspond to positions 82, 129, 130, and 132 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.In some embodiments, the position of the at least one unnatural amino acid is selected from N82, N129, or Q132, where the residue positions correspond to positions 82, 129, 132 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82 or N129, where the residue positions correspond to positions 82 and 129 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82, the residue position corresponding to position 82 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.In some embodiments, the at least one unnatural amino acid position is selected from N129, the residue position corresponding to position 129 shown in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0015] As disclosed herein, in certain embodiments, the at least one unnatural amino acid position is selected from N82, N129, or Q132, where the residue positions correspond to positions 82, 129, 132 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid position is selected from N82, N129, or Q132, where the residue positions correspond to positions 82, 129, 132 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(propargylethoxy)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid position is selected from N82, N129 or Q132, where the residue positions correspond to positions 82, 129, 132 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises an N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82, N129 or Q132, where the residue positions correspond to positions 82, 129, 132 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises an N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82, N129 or Q132, where the residue positions correspond to positions 82, 129, 132 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0016] As disclosed herein, in certain embodiments, the at least one unnatural amino acid position is selected from N82 or N129, the residue positions corresponding to positions 82 and 129 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid position is selected from N82 or N129, the residue positions corresponding to positions 82 and 129 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(propargylethoxy)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid position is selected from N82 or N129, the residue positions corresponding to positions 82 and 129 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82 or N129, where the residue positions correspond to positions 82 and 129 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises an N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82 or N129, where the residue positions correspond to positions 82 and 129 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises an N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0017] As disclosed herein, in certain embodiments, the at least one unnatural amino acid position is selected from N82, the residue position corresponds to position 82 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid position is selected from N82, the residue position corresponds to position 82 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(propargylethoxy)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid position is selected from N82, the residue position corresponds to position 82 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82, the residue position corresponding to position 82 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises an N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N82, the residue position corresponding to position 82 set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises an N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0018] As disclosed herein, in certain embodiments, the at least one unnatural amino acid position is selected from N129, the residue position corresponds to position 129 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid position is selected from N129, the residue position corresponds to position 129 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(propargylethoxy)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid position is selected from N129, the residue position corresponds to position 129 as set forth in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid position is selected from N129, a residue position corresponding to position 129 shown in SEQ ID NO:1, and the at least one unnatural amino acid comprises N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0019] As disclosed herein, in certain embodiments, the modified IL-10 polypeptide is covalently attached to the conjugation moiety via at least one unnatural amino acid. In some embodiments, the conjugation moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), polysaccharide, poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof.

[0020] As disclosed herein, in certain embodiments, the modified IL-10 polypeptide is covalently attached to a PEG molecule via at least one unnatural amino acid. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the weight average molecular weight of the PEG is about 100 Daltons to about 150,000 Daltons. Exemplary ranges include weight average molecular weights ranging from greater than 5,000 daltons to about 100,000 daltons, from about 6,000 daltons to about 90,000 daltons, from about 10,000 daltons to about 85,000 daltons, from about 10,000 daltons to about 85,000 daltons, from about 20,000 daltons to about 85,000 daltons, from about 53,000 daltons to about 85,000 daltons, from about 25,000 daltons to about 120,000 daltons, from about 29,000 daltons to about 120,000 daltons, from about 35,000 daltons to about 120,000 daltons, and from about 40,000 daltons to about 120,000 daltons.Exemplary weight average molecular weights of PEG include about 100 daltons, about 200 daltons, about 300 daltons, about 400 daltons, about 500 daltons, about 600 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 900 daltons, about 1,000 daltons, about 1,500 daltons, about 2,000 daltons, about 2,200 daltons, about 2,500 daltons, about 3,000 daltons, about 4,000 daltons, about 4,400 daltons, about 4,500 daltons, about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, and the like. about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. Branched versions of PEG having any of the foregoing total molecular weights (e.g., a branched 40,000 dalton PEG composed of two 20,000 dalton polymers) can also be used. In some embodiments, the molecular weight of PEG is about 5,000 daltons. In some embodiments, the molecular weight of PEG is about 7,500 daltons. In some embodiments, the molecular weight of PEG is about 8,000 daltons. In some embodiments, the molecular weight of PEG is about 9,000 daltons. In some embodiments, the molecular weight of PEG is about 10,000 daltons. In some embodiments, the molecular weight of PEG is about 11,000 daltons. In some embodiments, the molecular weight of PEG is about 12,000 daltons. In some embodiments, the molecular weight of PEG is about 13,000 daltons. In some embodiments, the molecular weight of PEG is about 14,000 daltons. In some embodiments, the molecular weight of PEG is about 15,000 daltons. In some embodiments, the molecular weight of PEG is about 16,000 daltons. In some embodiments, the molecular weight of PEG is about 17,000 daltons.In some embodiments, the molecular weight of PEG is about 18,000 daltons. In some embodiments, the molecular weight of PEG is about 19,000 daltons. In some embodiments, the molecular weight of PEG is about 20,000 daltons. In some embodiments, the molecular weight of PEG is about 21,000 daltons. In some embodiments, the molecular weight of PEG is about 22,000 daltons. In some embodiments, the molecular weight of PEG is about 23,000 daltons. In some embodiments, the molecular weight of PEG is about 25,000 daltons. In some embodiments, the molecular weight of PEG is about 30,000 daltons. In some embodiments, the molecular weight of PEG is about 40,000 daltons. In some embodiments, the molecular weight of PEG is about 50,000 daltons. In some embodiments, the molecular weight of PEG is about 60,000 daltons. In some embodiments, the molecular weight of PEG is about 70,000 daltons. In some embodiments, the molecular weight of PEG is about 80,000 daltons. In some embodiments, the molecular weight of PEG is about 90,000 daltons. In some embodiments, the molecular weight of PEG is about 100,000 daltons. In some embodiments, the molecular weight of PEG is about 120,000 daltons. In some embodiments, the molecular weight of PEG is about 130,000 daltons. In some embodiments, the molecular weight of PEG is about 140,000 daltons. In some embodiments, the molecular weight of PEG is about 150,000 daltons. In some embodiments, the molecular weight of PEG is about 175,000 daltons. In some embodiments, the molecular weight of PEG is about 200,000 daltons.

[0021] As disclosed herein, in certain embodiments, the modified IL-10 polypeptide is covalently linked to a monosaccharide or polysaccharide. In certain embodiments, the modified IL-10 polypeptide is covalently linked to a monosaccharide. In certain embodiments, the modified IL-10 polypeptide is covalently linked to a polysaccharide. In certain embodiments, the monosaccharide or polysaccharide is linked to the IL-10 polypeptide by a linker via a cysteine ​​or asparagine residue in the polypeptide. In certain embodiments, the monosaccharide or polysaccharide is linked to the IL-10 polypeptide by a linker via a cysteine ​​residue in the polypeptide. In certain embodiments, the monosaccharide or polysaccharide is linked to the IL-10 polypeptide by a linker via an asparagine residue in the polypeptide.

[0022] As disclosed herein, in certain embodiments, the modified IL-10 polypeptide is covalently linked to a PEG molecule through at least one unnatural amino acid and to a monosaccharide or polysaccharide through another amino acid in the polypeptide. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In certain embodiments, the modified IL-10 polypeptide is covalently linked to a PEG molecule through at least one unnatural amino acid and to a monosaccharide through another amino acid in the polypeptide. In certain embodiments, the modified IL-10 polypeptide is covalently linked to a PEG molecule through at least one unnatural amino acid and to a polysaccharide through another amino acid in the polypeptide. In certain embodiments, the monosaccharide or polysaccharide is linked to the IL-10 polypeptide by a linker through a cysteine ​​or asparagine residue in the polypeptide. In certain embodiments, the monosaccharide or polysaccharide is linked to the IL-10 polypeptide by a linker through a cysteine ​​residue in the polypeptide. In certain embodiments, the monosaccharide or polysaccharide is linked to the IL-10 polypeptide by a linker through an asparagine residue in the polypeptide.

[0023] Disclosed herein, in certain embodiments, are isolated and modified interleukin-10 (IL-10) polypeptides comprising at least one unnatural amino acid, wherein the isolated and modified IL-10 polypeptides are biologically active IL-10 polypeptides that bind to the IL-10 receptor (IL-10R) to form an IL-10 / IL-10R complex. In some embodiments, the biologically active IL-10 polypeptides have a substantially increased plasma half-life compared to the plasma half-life of a wild-type IL-10 protein. In some embodiments, the at least one unnatural amino acid is located in helices C, D, or E. In some embodiments, the at least one unnatural amino acid is located in a surface-exposed position of helices C, D, or E. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, N82, K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, or N82, where the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 88, 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from Q70, E74, N82, K88, N126, K130, or Q132, where the residue positions correspond to positions 70, 74, 82, 88, 126, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence is at least or about 99.0% identical to amino acids 33-158 of SEQ ID NO:1.In some embodiments, the amino acid sequence comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 wild type lysines. In some embodiments, the amino acid sequence comprises a wild type lysine at one or more of K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 wild type lysines at K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, the amino acid sequence includes wild type lysines at K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, at least one unnatural amino acid is located proximal to the N-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the N-terminus. In some embodiments, at least one unnatural amino acid is located proximal to the C-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the C-terminus. In some embodiments, at least one unnatural amino acid: a) is a lysine analog; b) comprises an aromatic side chain; c) comprises an azide group; d) comprises an alkyne group; or e) comprises an aldehyde or ketone group. In some embodiments, at least one unnatural amino acid does not comprise an aromatic side chain.In some embodiments, the at least one unnatural amino acid is selected from the group consisting of N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, phenyl fluoride, and the like. alanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, or selenocysteine. In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-10 polypeptide by an orthogonal tRNA synthetase / tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase / tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-10 polypeptide dimerizes with a second modified IL-10 polypeptide, thereby generating an IL-10 dimer comprising two unnatural amino acids. In some embodiments, the unnatural amino acid of the modified IL-10 polypeptide is located at a different residue position than the unnatural amino acid of the second modified IL-10 polypeptide.In some embodiments, both non-natural amino acids are located at the same residue position in each IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide dimerizes with an IL-10 polypeptide that does not include a non-natural amino acid. In some embodiments, the modified IL-10 polypeptide dimerizes with a wild-type IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide is covalently attached to a conjugation moiety via at least one non-natural amino acid. In some embodiments, the conjugation moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), polysaccharides, poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin.In some embodiments, the protein comprises an antibody or a binding fragment thereof. In some embodiments, the antibody or a binding fragment thereof comprises an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a homoamino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the conjugation moiety is indirectly linked to at least one unnatural amino acid of the modified IL-10 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the modified IL-10 polypeptide is: a) a functionally active fragment of a full-length IL-10 polypeptide; b) a recombinant IL-10 polypeptide; or c) a recombinant human IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, with residue positions relative to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion comprises 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 residue positions relative to the positions in SEQ ID NO: 1. In some embodiments, the modified IL-10 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10 dimer binds to IL-10R to form an IL-10 / IL-10R complex.In some embodiments, the increased plasma half-life of the IL-10 dimer is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of the wild-type IL-10 dimer. In some embodiments, the increased plasma half-life of the IL-10 dimer is about 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, or 30 days. In some embodiments, at least one unnatural amino acid position does not substantially interfere with dimerization of the IL-10 polypeptide. In some embodiments, at least one unnatural amino acid position does not significantly interfere with binding of the IL-10 dimer to IL-10R. In some embodiments, the at least one unnatural amino acid position impairs IL-10R signaling by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%. In some embodiments, the at least one unnatural amino acid position does not significantly impair IL-10R signaling.

[0024] Disclosed herein, in certain embodiments, are isolated and modified interleukin 10 (IL-10) polypeptides that include at least one unnatural amino acid at a position on the polypeptide that increases exposure of a plurality of tumor cells to tumor infiltrating immune cells, where the increased exposure of a plurality of tumor cells is compared to exposure of a plurality of tumor cells in the presence of wild-type IL-10 protein. In some embodiments, also described herein are isolated and modified interleukin 10 (IL-10) polypeptides that include at least one unnatural amino acid at a position on the polypeptide that increases and / or maintains cytotoxicity and / or clonal expansion of tumor infiltrating immune cells, where the increased cytotoxicity and clonal expansion is compared to exposure of tumor infiltrating immune cells resulting from the same dose of wild-type IL-10 protein. In some embodiments, the at least one unnatural amino acid is located at a surface-exposed position of helix C, D, or E. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, N82, K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, or N82, where the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 88, 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1.In some embodiments, the at least one unnatural amino acid position is selected from Q70, E74, N82, K88, N126, K130, or Q132, where the residue positions correspond to positions 70, 74, 82, 88, 126, 130, and 132 as set forth in SEQ ID NO:1. In some embodiments, the amino acid sequence is at least or about 99.0% identical to amino acids 33-158 of SEQ ID NO:1. In some embodiments, the amino acid sequence comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 wild-type lysines. In some embodiments, the amino acid sequence comprises wild-type lysines at one or more of K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 wild type lysines of K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138 and K157. In some embodiments, the amino acid sequence comprises wild type lysines at K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138 and K157. In some embodiments, the position of at least one unnatural amino acid is located proximal to the N-terminus. In some embodiments, the position of at least one unnatural amino acid is located within the first 10, 20, 30, 40 or 50 residues from the N-terminus. In some embodiments, the position of at least one unnatural amino acid is located proximal to the C-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the C-terminus. In some embodiments, at least one unnatural amino acid: a) is a lysine analog; b) comprises an aromatic side chain; c) comprises an azide group; d) comprises an alkyne group; or e) comprises an aldehyde or ketone group. In some embodiments, at least one unnatural amino acid does not comprise an aromatic side chain.In some embodiments, the at least one unnatural amino acid is selected from the group consisting of N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, phenyl fluoride, and the like. alanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, or selenocysteine. In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-10 polypeptide by an orthogonal tRNA synthetase / tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase / tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-10 polypeptide dimerizes with a second modified IL-10 polypeptide, thereby generating an IL-10 dimer comprising two unnatural amino acids. In some embodiments, the unnatural amino acid of the modified IL-10 polypeptide is located at a different residue position than the unnatural amino acid of the second modified IL-10 polypeptide.In some embodiments, both non-natural amino acids are located at the same residue position in each IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide dimerizes with an IL-10 polypeptide that does not include a non-natural amino acid. In some embodiments, the modified IL-10 polypeptide dimerizes with a wild-type IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide is covalently attached to a conjugation moiety via at least one non-natural amino acid. In some embodiments, the conjugation moiety comprises a water-soluble polymer, a lipid, a protein, or a peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), polysaccharides, poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin.In some embodiments, the protein comprises an antibody or a binding fragment thereof. In some embodiments, the antibody or a binding fragment thereof comprises an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a homoamino acid polymer rich in glycine (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the conjugation moiety is indirectly linked to at least one unnatural amino acid of the modified IL-10 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the modified IL-10 polypeptide is: a) a functionally active fragment of a full-length IL-10 polypeptide; b) a recombinant IL-10 polypeptide; or c) a recombinant human IL-10 polypeptide. In some embodiments, the modified IL-10 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, with residue positions relative to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion comprises 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 residue positions relative to the positions in SEQ ID NO: 1. In some embodiments, the modified IL-10 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-10 dimer binds to IL-10R to form an IL-10 / IL-10R complex.In some embodiments, the increased plasma half-life of the IL-10 dimer is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of the wild-type IL-10 dimer. In some embodiments, the increased plasma half-life of the IL-10 dimer is about 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, or 30 days. In some embodiments, at least one unnatural amino acid position does not substantially interfere with dimerization of the IL-10 polypeptide. In some embodiments, at least one unnatural amino acid position does not significantly interfere with binding of the IL-10 dimer to IL-10R. In some embodiments, the at least one unnatural amino acid position impairs IL-10R signaling by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%. In some embodiments, the at least one unnatural amino acid position does not significantly impair IL-10R signaling. In some embodiments, the at least one unnatural amino acid position does not impair IL-10R signaling as measured by Stat3 phosphorylation and / or activation of IL-10R signaling.

[0025] Disclosed herein, in certain embodiments, are polynucleic acid polymers that encode the modified IL-10 polypeptides described herein.

[0026] Disclosed herein, in certain embodiments, is a vector comprising the polynucleic acid polymer described herein.

[0027] Disclosed herein, in certain aspects, is an interleukin 10 (IL-10) polypeptide comprising at least one unnatural amino acid, and an IL-10 conjugate comprising a conjugation moiety linked to the at least one unnatural amino acid. In some embodiments, the conjugation moiety is directly linked to the at least one unnatural amino acid. In some embodiments, the conjugation moiety is indirectly linked to the at least one unnatural amino acid via a linker. In some embodiments, the at least one unnatural amino acid is located in helices C, D or E. In some embodiments, the at least one unnatural amino acid is located at a surface-exposed position of helices C, D or E. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, N82, K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from E67, Q70, E74, E75, Q79, or N82, where the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 88, 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid position is selected from Q70, E74, N82, K88, N126, K130, or Q132, where the residue positions correspond to positions 70, 74, 82, 88, 126, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence is at least or about 99.0% identical to amino acids 33-158 of SEQ ID NO:1.In some embodiments, the amino acid sequence comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 wild type lysines. In some embodiments, the amino acid sequence comprises a wild type lysine at one or more of K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 wild type lysines at K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, the amino acid sequence includes wild type lysines at K34, K40, K49, K57, K88, K99, K117, K119, K125, K130, K134, K138, and K157. In some embodiments, at least one unnatural amino acid is located proximal to the N-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the N-terminus. In some embodiments, at least one unnatural amino acid is located proximal to the C-terminus. In some embodiments, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the C-terminus. In some embodiments, the IL-10 polypeptide includes two or more unnatural amino acids. In some embodiments, the IL-10 polypeptide dimerizes with a second IL-10 polypeptide that includes at least one unnatural amino acid. In some embodiments, the IL-10 polypeptide dimerizes with a second IL-10 polypeptide that does not include an unnatural amino acid. In some embodiments, the IL-10 polypeptide dimerizes with a wild-type IL-10 polypeptide. In some embodiments, the IL-10 dimer comprises two or more conjugation moieties, each of which is attached to an unnatural amino acid located at the same residue position in a respective IL-10 monomer.In some embodiments, the IL-10 dimer comprises two or more conjugation moieties, each of which is attached to a non-natural amino acid located at a different residue position within the IL-10 dimer. In some embodiments, the IL-10 polypeptide comprises an increased plasma half-life, the increased plasma half-life being compared to the plasma half-life of a wild-type IL-10 protein. In some embodiments, the IL-10 polypeptide is a biologically active IL-10 polypeptide that binds to the IL-10 receptor (IL-10R) to form an IL-10 / IL-10R complex and activates the IL-10R signaling pathway.

[0028] Disclosed herein, in certain embodiments, is an IL-10 / IL-10R complex comprising a modified IL-10 dimer comprising at least one unnatural amino acid and an IL-10R, wherein the modified IL-10 dimer has an increased plasma half-life compared to the plasma half-life of a wild-type IL-10 dimer. In some embodiments, the modified IL-10 dimer further comprises a conjugating moiety covalently attached to the at least one unnatural amino acid.

[0029] Disclosed herein in certain embodiments is a pharmaceutical composition comprising the isolated and modified IL-10 polypeptide described herein or the IL-10 conjugate described herein; and a pharma- ceutical acceptable excipient.In some embodiments, the pharmaceutical composition is formulated for systemic delivery.In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0030] Disclosed herein, in certain 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 an isolated and modified IL-10 polypeptide as described herein, an IL-10 conjugate as described herein; or a pharmaceutical composition as described herein. In some embodiments, the proliferative disease or condition is cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's lymphoma, and bladder cancer. Kit high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the isolated and modified IL-10 polypeptide described herein, the IL-10 conjugate described herein; or the pharmaceutical composition described herein and the additional therapeutic agent are administered simultaneously or formulated simultaneously. In some embodiments, the isolated and modified IL-10 polypeptide described herein, the IL-10 conjugate described herein; or the pharmaceutical composition described herein and the additional therapeutic agent are administered sequentially. In some embodiments, the isolated and modified IL-10 polypeptide described herein, the IL-10 conjugate described herein; or the pharmaceutical composition described herein is administered prior to the additional therapeutic agent.In some embodiments, the isolated and modified IL-10 polypeptide described herein, the IL-10 conjugate described herein; or the pharmaceutical composition described herein is administered following administration of an additional therapeutic agent.

[0031] Disclosed herein, in certain embodiments, is a kit comprising an isolated and modified IL-10 polypeptide described herein, an IL-10 conjugate described herein, or a pharmaceutical composition described herein.

[0032] Various aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0033] [Figure 1]

[0023] Figure 1 shows exemplary unnatural amino acids. This figure is taken from Figure 2 in Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry, 285(15):11039-11044 (2010). [Figure 2A]

[0023] Figure 2 shows exemplary unnatural amino acids: Figure 2A shows exemplary lysine derivatives; Figure 2B shows exemplary phenylalanine derivatives. [Figure 2B]

[0023] Figure 2A shows exemplary unnatural amino acids. Figure 2B shows exemplary phenylalanine derivatives. [Figure 3A] 3A-3D are taken from Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 3B] 3A-3D are taken from Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 3C] 3A-3D are taken from Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 3D] 3A-3D are taken from Dumas et al., Chemical Science 2015, 6, 50-69. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Cytokines include a family of cell signaling proteins, such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors that play a role in innate and adaptive immune cell homeostasis. Cytokines are produced by immune cells, such as macrophages, B lymphocytes, T lymphocytes, and mast cells, endothelial cells, fibroblasts, and various stromal cells. In some instances, cytokines regulate the balance between humoral and cellular immune responses.

[0035] Interleukins are signaling proteins that regulate the development and differentiation of T and B lymphocytes, monocytic cells, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4 T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue-resident cells. In some cases, there are about 15 interleukins: interleukin 1-13, interleukin 15, and interleukin 17.

[0036] IL-10 generates tumor immunity by activating tumor-infiltrating CD8+ T cells, inducing IFN-γ that increases MHC class I on tumor cells and MHC class II on macrophages, and inducing cytotoxic proteins that mediate target cell lysis. Increased T cell receptor stimulation on CD8+ T cells provides anti-apoptotic and proliferation signals. An unexpected role of IL-10 in the tumor microenvironment (TME) is the inhibition of pro-inflammatory Th17 cells and cytokines involved in tumor-associated inflammation resulting in suppression of anti-tumor effector cell responses. Preclinical studies have shown that IL-10 deficiency increases tumor incidence and reduces immune surveillance. Furthermore, treatment of Her2 transgenic mice with pegylated IL-10 resulted in tumor rejection, but required the expression of IFN-γ and granzyme-expressing CD8+ T cells, and was accompanied by a significant increase in CD8a / b+ T cells in the tumor.

[0037] IL-10 has a relatively short serum half-life in the body: in fact, its half-life in mice is approximately 2-6 hours, as measured by in vitro bioassays or by efficacy in a septic shock model system (see Smith et al., Cellular Immunology 173:207-214 (1996)).

[0038] Disclosed herein, in certain embodiments, are modified IL-10 polypeptides that have increased plasma half-life. In some embodiments, also described herein are modified IL-10 polypeptides that, by dimerization, increase the exposure of multiple tumor cells to tumor-infiltrating immune cells. In other embodiments, further described herein are modified IL-10 polypeptides that form biologically active IL-10 dimers.

[0039] Further described are IL-10 polypeptide conjugates, pharmaceutical compositions comprising one or more of the modified IL-10 polypeptides, and methods of treating a disease or indication.

[0040] Modified IL-10 Polypeptides In some embodiments, the present specification describes IL-10 polypeptides modified at an amino acid position. In some examples, the modification is a change to a natural amino acid. In some examples, the modification is a change to a non-natural amino acid. In some examples, the present specification describes isolated and modified IL-10 polypeptides that include at least one non-natural amino acid. In some examples, the IL-10 polypeptide is an isolated and purified mammalian IL-10, such as a rodent IL-10 protein, or a human IL-10 protein. In some cases, the IL-10 polypeptide is a human IL-10 protein. In some cases, the IL-10 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1. In some cases, the IL-10 polypeptide comprises the sequence of SEQ ID NO:1. In some cases, the IL-10 polypeptide consists of the sequence of SEQ ID NO:1. In additional cases, the IL-10 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2. In additional cases, the IL-10 polypeptide comprises the sequence of SEQ ID NO: 2. In additional cases, the IL-10 polypeptide consists of the sequence of SEQ ID NO: 2.

[0041] In some examples, the IL-10 polypeptide is a truncated variant. In some examples, the truncation is an N-terminal deletion. In other examples, the truncation is a C-terminal deletion. In additional examples, the truncation includes both N- and C-terminal deletions. For example, the truncation can be a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the N-terminus or C-terminus, or both termini. In some cases, the IL-10 polypeptide includes an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues. In some cases, the IL-10 polypeptide includes 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-10 polypeptide includes an N-terminal deletion of at least or about 2 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 3 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 4 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 5 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 6 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 7 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 8 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 9 residues. In some cases, the IL-10 polypeptide comprises an N-terminal deletion of at least or about 10 residues.

[0042] In some embodiments, the IL-10 polypeptide is a functionally active fragment. In some cases, the functionally active fragment comprises IL-10 regions 5-160, 10-160, 15-160, 20-160, 1-155, 5-155, 10-155, 15-155, 20-155, 1-150, 5-150, 10-150, 15-150, or 20-150, with residue positions relative to the positions in SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 5-160, with residue positions relative to the positions in SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 10-160, with residue positions relative to the positions in SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 15-160, with residue positions relative to the positions in SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 20-160, the residue positions being relative to the positions of SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 1-155, the residue positions being relative to the positions of SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 5-155, the residue positions being relative to the positions of SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 10-155, the residue positions being relative to the positions of SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 15-155, the residue positions being relative to the positions of SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 20-155, the residue positions being relative to the positions of SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 1-150, the residue positions being relative to the positions of SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 5-150, where the residue positions are relative to the positions in SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 10-150, where the residue positions are relative to the positions in SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 15-150, where the residue positions are relative to the positions in SEQ ID NO:1. In some examples, the functionally active fragment comprises IL-10 regions 20-150, where the residue positions are relative to the positions in SEQ ID NO:1.

[0043] In some embodiments, described herein are IL-10 polypeptides comprising at least one unnatural amino acid. In some examples, the at least one unnatural amino acid is located in helix C, D or E. In some instances, helix C comprises residues L60-N82, the positions of which are relative to the positions of SEQ ID NO:1. In some instances, helix D comprises residues I87-C108, the positions of which are relative to the positions of SEQ ID NO:1. In some instances, helix E comprises residues S118-L131, the positions of which are relative to the positions of SEQ ID NO:1. In some instances, the at least one unnatural amino acid is located in a surface-exposed position in helix C, D or E.

[0044] In some embodiments, described herein are IL-10 polypeptides comprising at least one unnatural amino acid at a position selected from E67, Q70, E74, E75, Q79, N82, K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some examples, the at least one unnatural amino acid is selected from E67, Q70, E74, E75, Q79, or N82, where the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some examples, the at least one unnatural amino acid position is selected from K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 88, 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some examples, the at least one unnatural amino acid position is selected from K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some examples, the at least one unnatural amino acid position is selected from Q70, E74, N82, K88, N126, K130, or Q132, where the residue positions correspond to positions 70, 74, 82, 88, 126, 130, and 132 set forth in SEQ ID NO: 1. In some examples, the at least one unnatural amino acid position is selected from E67. In some examples, the at least one unnatural amino acid position is selected from Q70. In some examples, the at least one unnatural amino acid position is selected from E74. In some examples, the at least one unnatural amino acid position is selected from E75. In some examples, the at least one unnatural amino acid position is selected from Q79. In some examples, the at least one unnatural amino acid position is selected from N82. In some examples, the at least one unnatural amino acid position is selected from K88. In some examples, the at least one unnatural amino acid position is selected from K125. In some examples, the at least one unnatural amino acid position is selected from N126.In some examples, the at least one unnatural amino acid position is selected from N129. In some examples, the at least one unnatural amino acid position is selected from K130. In some examples, the at least one unnatural amino acid position is selected from Q132.

[0045] In some instances, at least one unnatural amino acid is located proximal to the N-terminus. As used herein, proximal refers to a residue located at least one residue away from the N-terminus residue and up to about 50 residues away from the N-terminus residue. In some instances, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the N-terminus residue. In some instances, at least one unnatural amino acid is located within the first 10 residues from the N-terminus residue. In some instances, at least one unnatural amino acid is located within the first 20 residues from the N-terminus residue. In some instances, at least one unnatural amino acid is located within the first 30 residues from the N-terminus residue. In some instances, at least one unnatural amino acid is located within the first 40 residues from the N-terminus residue. In some instances, at least one unnatural amino acid is located within the first 50 residues from the N-terminus residue.

[0046] In some examples, at least one unnatural amino acid is the N-terminal residue.

[0047] In some instances, at least one unnatural amino acid is located proximal to the C-terminus. As used herein, proximal refers to a residue located at least one residue away from the C-terminus residue and up to about 50 residues away from the C-terminus residue. In some instances, at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the C-terminus residue. In some instances, at least one unnatural amino acid is located within the first 10 residues from the C-terminus residue. In some instances, at least one unnatural amino acid is located within the first 20 residues from the C-terminus residue. In some instances, at least one unnatural amino acid is located within the first 30 residues from the C-terminus residue. In some instances, at least one unnatural amino acid is located within the first 40 residues from the C-terminus residue. In some instances, at least one unnatural amino acid is located within the first 50 residues from the C-terminus residue.

[0048] In some examples, at least one unnatural amino acid is the C-terminal residue.

[0049] In some embodiments, the IL-10 polypeptide is a functionally active monomer that can bind to IL-10R and activate a signal transduction pathway. In some cases, the functionally active IL-10 monomer has an increased plasma half-life. In some cases, the increased plasma half-life is compared to the plasma half-life of a wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 polypeptide is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of a wild-type IL-10 protein. In some cases, the increased plasma half-life of the IL-10 polypeptide is about 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, or 30 days compared to the plasma half-life of a wild-type IL-10 protein.

[0050] In some embodiments, the IL-10 polypeptide forms an IL-10 dimer, e.g., a biologically active dimer. In some cases, the IL-10 dimer (e.g., a biologically active IL-10 dimer) comprises at least one unnatural amino acid. In other cases, the IL-10 dimer (e.g., a biologically active IL-10 dimer) comprises at least two or more unnatural amino acids. In additional cases, the IL-10 dimer (e.g., a biologically active IL-10 dimer) comprises at least three, four, five, six, seven, eight, or more unnatural amino acids.

[0051] In some embodiments, an IL-10 dimer (e.g., a biologically active IL-10 dimer) comprises two unnatural amino acids. In some cases, both unnatural amino acids are located on the same IL-10 polypeptide. In other cases, each of the IL-10 polypeptides comprises one unnatural amino acid. In such examples, each of the unnatural amino acids is located at a different residue position of the IL-10 polypeptide (i.e., one of the unnatural amino acids is located at a first residue position of one of the IL-10 polypeptides and a second unnatural amino acid is located at a second residue position of the other IL-10 polypeptide, the second residue position being different from the first residue position). In further examples, each of the unnatural amino acids is located at the same residue position of each of the IL-10 polypeptides.

[0052] In some embodiments, the IL-10 dimer (e.g., a biologically active IL-10 dimer) comprises one unnatural amino acid. In such cases, the modified IL-10 polypeptide dimerizes with an IL-10 polypeptide that does not comprise a unnatural amino acid. In some cases, the IL-10 polypeptide that does not comprise a unnatural amino acid comprises a mutation with a natural amino acid. In other cases, the IL-10 polypeptide that does not comprise a unnatural amino acid is an IL-10 variant, e.g., a truncated IL-10 protein. In some examples, the modified IL-10 polypeptide dimerizes with a wild-type IL-10 polypeptide.

[0053] In some examples, the at least one unnatural amino acid position does not substantially prevent dimerization of the IL-10 polypeptide.

[0054] In some cases, the at least one unnatural amino acid position further does not significantly interfere with binding of the IL-10 dimer to IL-10R.

[0055] In some embodiments, the at least one unnatural amino acid position impairs IL-10R signaling by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or less. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 80%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 70%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 60%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 50%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 40%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 30%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 20%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 10%. In some cases, the at least one unnatural amino acid position impairs IL-10R signaling by less than 5%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 2%. In some examples, the at least one unnatural amino acid position impairs IL-10R signaling by less than 1%. In some examples, the at least one unnatural amino acid position does not significantly impair IL-10R signaling. In additional cases, the at least one unnatural amino acid position does not impair IL-10R signaling.

[0056] In some examples, the biologically active IL-10 dimer has an increased plasma half-life. In some cases, the increased plasma half-life is compared to the plasma half-life of a wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of a wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 90 minutes, or longer than the plasma half-life of a wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 2 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 3 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 4 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 5 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 6 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 10 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 12 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 18 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 24 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 36 hours or longer than the plasma half-life of the wild-type IL-10 dimer.In some cases, the increased plasma half-life of the IL-10 dimer is at least 48 hours or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 3 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 4 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 5 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 6 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 7 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 10 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 12 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 14 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 21 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 28 days or longer than the plasma half-life of the wild-type IL-10 dimer. In some cases, the increased plasma half-life of the IL-10 dimer is at least 30 days, or longer than the plasma half-life of a wild-type IL-10 dimer.

[0057] In some examples, the biologically active IL-10 dimer has an increased plasma half-life of about 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, or 30 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 90 minutes. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 2 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 3 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 4 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 5 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 6 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 7 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 7 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 8 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 9 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 10 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 11 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 12 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 18 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 24 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 36 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 48 hours. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 3 days.In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 4 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 5 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 6 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 7 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 10 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 12 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 14 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 21 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 28 days. In some cases, the biologically active IL-10 dimer has an increased plasma half-life of about 30 days.

[0058] IL-10 conjugates In certain embodiments, the present specification describes IL-10 conjugates. In some embodiments, the IL-10 conjugates include an IL-10 polypeptide that includes at least one unnatural amino acid and a conjugation moiety bound to the at least one unnatural amino acid. In some examples, the conjugation moiety is directly bound to the at least one unnatural amino acid. In other examples, the conjugation moiety is indirectly bound to the at least one unnatural amino acid via a linker as described below.

[0059] As discussed above, the at least one unnatural amino acid is optionally located in helices C, D, or E, e.g., a surface-accessible residue. In some cases, the residue includes E67, Q70, E74, E75, Q79, N82, K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO:1. In some cases, the residue includes E67, Q70, E74, E75, Q79, or N82, where the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO:1. In some cases, the residues include K88, K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 88, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some cases, the residues include K125, N126, N129, K130, or Q132, where the residue positions correspond to positions 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some cases, the residues include Q70, E74, N82, K88, N126, K130, or Q132, where the residue positions correspond to positions 70, 74, 82, 88, 126, 130, and 132 as set forth in SEQ ID NO: 1.

[0060] In some embodiments, the at least one unnatural amino acid is located proximal to the N-terminus (e.g., proximal to the N-terminal residue). For example, the at least one unnatural amino acid is optionally located within the first 10, 20, 30, 40, or 50 residues from the N-terminus. In some cases, the at least one unnatural amino acid is located at the N-terminus (i.e., the at least one unnatural amino acid is the N-terminal residue of the IL-10 polypeptide).

[0061] In other embodiments, the at least one unnatural amino acid is located proximal to the C-terminus (e.g., proximal to the C-terminal residue). For example, the at least one unnatural amino acid is optionally located within the first 10, 20, 30, 40, or 50 residues from the C-terminus. In some cases, the at least one unnatural amino acid is located at the C-terminus (i.e., the at least one unnatural amino acid is the C-terminal residue of the IL-10 polypeptide).

[0062] In some examples, the IL-10 conjugate comprises one conjugating moiety attached to the unnatural amino acid.

[0063] In some examples, the IL-10 conjugate comprises an IL-10 monomer capable of activating the IL-10R signaling pathway. In other examples, the IL-10 conjugate comprises an IL-10 dimer that is functionally active.

[0064] In some examples, the IL-10 conjugate comprises two or more conjugation moieties, each of which is attached to a different non-natural amino acid. In some cases, the two or more conjugation moieties are conjugated to the same IL-10 polypeptide (e.g., a functionally active IL-10 monomer or in a functionally active IL-10 dimer). In other cases, the two or more conjugation moieties are each conjugated to a different IL-10 polypeptide within an IL-10 dimer. In additional cases, the IL-10 conjugate comprises three, four, five, six, or more conjugation moieties, each of which is attached to a different non-natural amino acid. In such an example, the two IL-10 polypeptides within the dimer have an uneven distribution of conjugation moieties, e.g., one IL-10 polypeptide has one conjugation moiety and the other IL-10 polypeptide has two or more conjugation moieties.

[0065] In some instances, the IL-10 conjugate comprises two or more conjugation moieties. In some instances, each of the two or more conjugation moieties is attached to a non-natural amino acid located at the same residue position within a respective IL-10 monomer. In other instances, each of the two or more conjugation moieties is attached to a non-natural amino acid located at a different residue position within the IL-10 dimer.

[0066] In some examples, the position of the conjugation moiety does not substantially prevent dimerization of the IL-10 polypeptide.

[0067] In some cases, the position of the conjugation moiety does not significantly interfere with binding of the IL-10 dimer to the IL-10R.

[0068] In some embodiments, the location of the conjugation moiety impairs IL-10R signaling by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or less. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 80%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 70%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 60%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 50%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 40%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 30%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 20%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 10%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 5%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 2%. In some examples, the location of the conjugation moiety impairs IL-10R signaling by less than 1%. In some cases, the location of the conjugation moiety does not significantly impair IL-10R signaling. In additional cases, the location of the conjugation moiety does not impair IL-10R signaling.

[0069] In some examples, the IL-10 conjugate has an increased plasma half-life. In some cases, the increased plasma half-life is compared to the plasma half-life of a wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of a wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 90 minutes, or longer than the plasma half-life of a wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 2 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 3 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 4 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 5 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 6 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 10 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 12 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 18 hours or longer than the plasma half-life of the wild-type IL-10 conjugate.In some cases, the increased plasma half-life of the IL-10 conjugate is at least 24 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 36 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 48 hours or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 3 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 4 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 5 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 6 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 7 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 10 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 12 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 14 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 21 days or longer than the plasma half-life of the wild-type IL-10 conjugate. In some cases, the increased plasma half-life of the IL-10 conjugate is at least 28 days or longer than the plasma half-life of the wild-type IL-10 conjugate.In some cases, the increased plasma half-life of the IL-10 conjugate is at least 30 days, or longer than the plasma half-life of the wild-type IL-10 conjugate.

[0070] In some embodiments, also described herein is an IL-10 / IL-10R complex comprising a modified IL-10 dimer comprising at least one unnatural amino acid and IL-10R, wherein the modified IL-10 dimer has an increased plasma half-life compared to the plasma half-life of a wild-type IL-10 dimer. In some examples, the modified IL-10 dimer further comprises a conjugating moiety covalently attached to the at least one unnatural amino acid.

[0071] IL-10 conjugate precursor Described herein are IL-10 conjugate precursors that include modified IL-10 polypeptides, in which one or more amino acids are mutated from wild-type amino acids. Such precursors are often used with the methods described herein for treating diseases or conditions. In some embodiments, the IL-10 precursors are not conjugated. Such mutations include various additions, deletions, or substitutions.

[0072] Natural and Unnatural Amino Acids In some embodiments, an amino acid residue described herein (e.g., in an IL-10 polypeptide) is mutated to lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine before binding to (or reacting with) a conjugation moiety. For example, the side chain of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine can be attached to a conjugation moiety described herein. In some examples, the amino acid residue is mutated to cysteine, lysine, or histidine. In some cases, the amino acid residue is mutated to cysteine. In some cases, the amino acid residue is mutated to lysine. In some cases, the amino acid residue is mutated to histidine. In some cases, the amino acid residue is mutated to tyrosine. In some cases, the amino acid residue is mutated to tryptophan. In some examples, the amino acid residue is located proximal to the N- or C-terminus, at the N- or C-terminus, or at an internal residue position. In some examples, the amino acid residue is an N- or C-terminal residue and the mutation is to a cysteine ​​or lysine. In some examples, the amino acid residue is located proximal to the N- or C-terminal residue (e.g., within 50, 40, 30, 20, or 10 residues of the N- or C-terminal residue) and the mutation is to a cysteine ​​or lysine.

[0073] In some instances, the amino acid residue is added to the N-terminus or C-terminus residue, i.e., the IL-10 polypeptide comprises an additional amino acid residue at either the N-terminus or C-terminus, and the additional amino acid residue is a cysteine ​​or lysine. In some instances, the additional amino acid residue is a cysteine. In some instances, the additional amino acid is conjugated to a conjugating moiety.

[0074] In some embodiments, an amino acid residue described herein (e.g., in an IL-10 polypeptide) is mutated to a non-natural amino acid. In some embodiments, the non-natural amino acid is not conjugated to a conjugating moiety. In some embodiments, an IL-10 polypeptide described herein comprises a non-natural amino acid, the IL-10 is conjugated to a protein, and the point of attachment is not the non-natural amino acid.

[0075] In some embodiments, the amino acid residues described herein (e.g., in IL-10 polypeptides) are mutated to non-natural amino acids before being attached to the conjugating moiety. In some cases, the mutation to a non-natural amino acid prevents or minimizes the autoantigen response of the immune system. As used herein, the term "non-natural amino acid" refers to an amino acid other than the 20 amino acids that naturally occur in proteins. Non-limiting examples of non-natural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronophenylalanine, O-propargyl tyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), unnatural analogues of tyrosine amino acid; unnatural analogues of glutamine amino acid; unnatural analogues of phenylalanine amino acid; unnatural analogues of serine amino acid; unnatural analogues of threonine amino acid; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, or combinations thereof; amino acids comprising photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; amino acids comprising biotin or biotin analogs; keto-containing amino acids;These include polyethylene glycol or polyether-containing amino acids; heavy atom substituted amino acids; chemically or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxy-containing acids; aminothioacids; α,α-disubstituted amino acids; β-amino acids; cyclic amino acids other than proline or histidine, and aromatic amino acids other than phenylalanine, tyrosine, or tryptophan;

[0076] Other examples of unnatural amino acids include N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0077] In some embodiments, the unnatural amino acid comprises a selectively reactive group, or a reactive group for site-selective labeling of a target polypeptide. In some instances, the chemical reaction is a bioorthogonal reaction (e.g., a biocompatible and selective reaction). In some cases, the chemistry is a metal-mediated process, such as Cu(I)-catalyzed or "copper-free" alkyne-azide triazole formation reaction, Staudinger ligation, inverse electron demand Diels-Alder (IEDDA) reaction, "photoclick" chemistry, or olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.

[0078] In some embodiments, the unnatural amino acid comprises a photoreactive group and crosslinks upon irradiation with, for example, UV light.

[0079] In some embodiments, the unnatural amino acid comprises a photocaged amino acid.

[0080] In some instances, the unnatural amino acids are para-, meta-, or ortho-substituted amino acid derivatives.

[0081] In some examples, the unnatural amino acid comprises p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, or isopropyl-L-phenylalanine.

[0082] In some cases, the unnatural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, or 3-iodotyrosine.

[0083] In some cases, the unnatural amino acid is phenylselenocysteine.

[0084] In some examples, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide containing phenylalanine derivative.

[0085] In some instances, the unnatural amino acid is Lysine derivatives containing benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide It is.

[0086] In some instances, the unnatural amino acid comprises an aromatic side chain.

[0087] In some instances, the unnatural amino acid does not contain an aromatic side chain.

[0088] In some examples, the unnatural amino acid comprises an azide group.

[0089] In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid comprises N6-(propargylethoxy)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid comprises N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid comprises N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid comprises N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0090] In some examples, the unnatural amino acid comprises a Michael-acceptor group. In some examples, the Michael-acceptor group comprises an unsaturated moiety capable of forming a covalent bond via a 1,2-addition reaction. In some examples, the Michael-acceptor group comprises an electron-deficient alkene or alkyne. In some examples, the Michael-acceptor group includes, but is not limited to, an α,β unsaturated: ketone, aldehyde, sulfoxide, sulfone, nitrile, imine, or aromatic.

[0091] In some examples, the unnatural amino acid is dehydroalanine.

[0092] In some instances, the unnatural amino acid comprises an aldehyde or ketone group.

[0093] In some examples, the unnatural amino acid is a lysine derivative that includes an aldehyde or ketone group.

[0094] In some examples, the unnatural amino acid is a lysine derivative that includes one or more O, N, Se, or S atoms at the beta, gamma, or delta position. In some examples, the unnatural amino acid is a lysine derivative that includes an O, N, Se, or S atom at the gamma position.

[0095] In some examples, the unnatural amino acid is a lysine derivative in which the epsilon N atom is replaced with an oxygen atom.

[0096] In some instances, the unnatural amino acid is a lysine derivative that is not a naturally occurring post-translationally modified lysine.

[0097] In some examples, the unnatural amino acid is an amino acid that comprises a side chain and the sixth atom from the alpha position comprises a carbonyl group. In some examples, the unnatural amino acid is an amino acid that comprises a side chain and the sixth atom from the alpha position comprises a carbonyl group and the fifth atom from the alpha position is nitrogen. In some examples, the unnatural amino acid is an amino acid that comprises a side chain and the seventh atom from the alpha position is an oxygen atom.

[0098] In some examples, the unnatural amino acid is a serine derivative that includes selenium. In some examples, the unnatural amino acid is selenoserine (2-amino-3-hydroselenopropanoic acid). In some examples, the unnatural amino acid is 2-amino-3-(((2-(3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid. In some examples, the unnatural amino acid is 2-amino-3-(phenylselanyl)propanoic acid. In some examples, the unnatural amino acid includes selenium, and oxidation of the selenium results in the formation of an alkene-containing unnatural amino acid.

[0099] In some examples, the unnatural amino acid comprises a cyclooctynyl group.

[0100] In some examples, the unnatural amino acid comprises a trans cyclotenyl group.

[0101] In some examples, the unnatural amino acid comprises a norbornenyl group.

[0102] In some examples, the unnatural amino acid comprises a cyclopropenyl group.

[0103] In some examples, the unnatural amino acid comprises a diazirine group.

[0104] In some examples, the unnatural amino acid comprises a tetrazine group.

[0105] In some examples, the unnatural amino acid is a lysine derivative and the side chain nitrogen is carbamylated. In some examples, the unnatural amino acid is a lysine derivative and the side chain nitrogen is acylated. In some examples, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-Boc-N6-methyllysine. In some examples, the unnatural amino acid is N6-acetyllysine. In some examples, the unnatural amino acid is pyrrolysine. In some examples, the unnatural amino acid is N6-trifluoroacetyllysine. In some examples, the unnatural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is 2-amino-6-{[(p-iodobenzyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-prolyl lysine. In some examples, the unnatural amino acid is 2-amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine. In some examples, the unnatural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some examples, the unnatural amino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some examples, the unnatural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some examples, the unnatural amino acid is 2-amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-((2-azidoethoxy)-carbonyl)-L-lysine. In some examples, the unnatural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid.In some examples, the unnatural amino acid is 2-amino-6-{[(2-cyclooctyloxy)carbonyl]amino}hexanoic acid. In some examples, the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. In some examples, the unnatural amino acid is 2-amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid. In some examples, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some examples, the unnatural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In some examples, the unnatural amino acid is N6-(pentenyl-5-oxycarbonyl)lysine. In some examples, the unnatural amino acid is N6-((but-3-yn-1-yloxy)carbonyl)-lysine. In some examples, the unnatural amino acid is N6-(((pent-4-yn-1-yloxy)carbonyl)-lysine. In some examples, the unnatural amino acid is N6-(thiazolidine-4-carbonyl)lysine. In some examples, the unnatural amino acid is 2-amino-8-oxononanoic acid. In some examples, the unnatural amino acid is 2-amino-8-oxooctanoic acid. In some examples, the unnatural amino acid is N6-(2-oxoacetyl)lysine.

[0106] In some examples, the unnatural amino acid is N6-propionyl lysine. In some examples, the unnatural amino acid is N6-butyryl lysine. In some examples, the unnatural amino acid is N6-(but-2-enoyl) lysine. In some examples, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl) lysine. In some examples, the unnatural amino acid is N6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl) lysine. In some examples, the unnatural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl) lysine. In some examples, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl) lysine. In some examples, the unnatural amino acid is cysteinyl lysine. In some examples, the unnatural amino acid is N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In some examples, the unnatural amino acid is N6-((metanitrobenyloxy)N6-methylcarbonyl)lysine. In some examples, the unnatural amino acid is N6-((bicyclo[6.1.0]non4-yn-9-ylmethoxy)carbonyl)-lysine. In some examples, the unnatural amino acid is N6-((cyclohept-3-en-1-yloxy)carbonyl)-L-lysine.

[0107] In some examples, the unnatural amino acid is 2-amino-3-(((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.

[0108] In some embodiments, the unnatural amino acid is incorporated into an IL-10 polypeptide via an alternative amber, opal, or ochre stop codon.

[0109] In some embodiments, the unnatural amino acid is incorporated into the IL-10 polypeptide via a four-base codon.

[0110] In some embodiments, the unnatural amino acid is incorporated into the IL-10 polypeptide by an alternative rare-sense codon.

[0111] In some embodiments, the unnatural amino acid is incorporated into the IL-10 polypeptide by a synthetic codon that comprises a unnatural nucleic acid.

[0112] In some examples, unnatural amino acids are incorporated into IL-10 by orthogonal modified synthetase / tRNA pairs. Such orthogonal pairs include a) unnatural synthetases that can add unnatural amino acids to unnatural tRNAs while minimizing the addition of other endogenous amino acids onto the unnatural tRNAs, and b) the addition of unnatural amino acids onto other endogenous tRNAs. Such orthogonal pairs include tRNAs that can undergo addition by unnatural synthetases while avoiding the addition of other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, the orthogonal synthetase / tRNA pair includes components from a single organism. In some embodiments, the orthogonal synthetase / tRNA pair includes components from two different organisms. In some embodiments, the orthogonal synthetase / tRNA pair includes components that facilitate the translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthetase is a modified alanine synthetase. In some embodiments, the orthogonal synthetase is a modified arginine synthetase. In some embodiments, the orthogonal synthetase is a modified asparagine synthetase. In some embodiments, the orthogonal synthetase is a modified aspartate synthetase. In some embodiments, the orthogonal synthetase is a modified cysteine ​​synthetase. In some embodiments, the orthogonal synthetase is a modified glutamine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamate synthetase. In some embodiments, the orthogonal synthetase is a modified alanine lysine synthetase. In some embodiments, the orthogonal synthetase is a modified histidine synthetase. In some embodiments, the orthogonal synthetase is a modified leucine synthetase. In some embodiments, the orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, the orthogonal synthetase is a modified lysine synthetase. In some embodiments, the orthogonal synthetase is a modified methionine synthetase.In some embodiments, the orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, the orthogonal synthetase is a modified proline synthetase. In some embodiments, the orthogonal synthetase is a modified serine synthetase. In some embodiments, the orthogonal synthetase is a modified threonine synthetase. In some embodiments, the orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, the orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, the orthogonal synthetase is a modified valine synthetase. In some embodiments, the orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal RNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartate tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine ​​tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine lysine tRNA. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA.In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

[0113] In some embodiments, the unnatural amino acid is incorporated into an IL-10 polypeptide by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanococcus jannaschii (Mj-Tyr) aRS / tRNA pair, the E. coli TyrRS (Ec-Tyr) / Bacillus stearothermophilus tRNA pair, and the B. stearothermophilus tRNA pair. CUA pair, Escherichia coli LeuRS (Ec-Leu) / Bacillus stearothermophilus tRNA CUA In some examples, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) by Mj-TyrRS / tRNA pairs. Exemplary UAAs that can be incorporated by Mj-TyrRS / tRNA pairs include, but are not limited to, para-substituted phenylalanine derivatives, such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives, such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronophenylalanine; and o-nitrobenzyltyrosine.

[0114] In some instances, the unnatural amino acid is Ec-Tyr / tRNA CUA or Ec-Leu / tRNA CUA The Ec-Tyr / tRNA pair is incorporated into the IL-10 polypeptide. CUA or Ec-Leu / tRNA CUAExemplary UAAs that may be incorporated by pairs include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O-propargyl tyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3-(naphthalen-2-ylamino)-2-amino-propanoic acid.

[0115] In some instances, the unnatural amino acid is incorporated into the IL-10 polypeptide by a pyrrolysyl-tRNA pair. In some instances, the PylRS is obtained from an archaea, e.g., a methanogenic archaea. In some instances, the PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated by a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate substituted lysine, e.g., 2-amino-6-(((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N-ε- D -Prole- L -Lysine and N-ε-cyclopentyloxycarbonyl- L -Lysine;N-ε-Acryloyl- L -Lysine;N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]- L-lysine; and N-ε-(1-methylcyclopro-2-enecarboxamido)lysine. In some embodiments, the IL-10 conjugates disclosed herein are produced by using M. mazei tRNA to which an unnatural amino acid, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), has been selectively added by M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS). Other methods are known to those skilled in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682):644-647. Other examples of unnatural amino acids that may be used include, but are not limited to, N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0116] In some examples, unnatural amino acids are incorporated into IL-10 polypeptides by synthetases disclosed in U.S. Patent No. 9,988,619 and U.S. Patent No. 9,938,516. Exemplary UAAs incorporated by such synthetases include para-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 heterocycles. In some embodiments, such amino acids include azides, tetrazines, or other chemical groups that can be conjugated to coupling partners, such as water-soluble moieties. In some embodiments, such synthetases are expressed and used to incorporate UAAs into cytokines in vivo. In some embodiments, such synthetases are used to incorporate UAAs into cytokines using cell-free translation systems.

[0117] The host cell into which the construct or vector disclosed herein is introduced is cultured or maintained in an appropriate medium so that tRNA, tRNA synthetase and the protein of interest are produced. The medium also contains unnatural amino acid(s) so that the protein of interest incorporates the unnatural amino acid(s). In some embodiments, a nucleoside triphosphate transporter (NTT) from bacteria, plants or algae is also present in the host cell. In some embodiments, the IL-10 conjugate disclosed herein is produced by using a host cell that expresses NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter used in the host cell is selected from the group consisting of TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis), trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some embodiments, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, the NTT is PtNTT1. In some embodiments, the NTT is PtNTT2. In some embodiments, the NTT is PtNTT3. In some embodiments, the NTT is PtNTT4. In some embodiments, the NTT is PtNTT5. In some embodiments, the NTT is PtNTT6.Other NTTs that may be used are disclosed in Zhang et al., Nature 2017, 551(7682):644-647; Malyshev et al., Nature 2014, 509(7500):385-388; Zhang et al., Proc Natl Acad Sci USA 2017, 114:1317-1322.

[0118] In some embodiments, a method is provided for producing the IL-10 conjugate disclosed herein in a cell line expressing NTT and tRNA synthetase. In some embodiments described herein, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, and M. mazei. In some embodiments, the NTT is PtNTT1, and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT2 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT4 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT5 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT6 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei.

[0119] In some embodiments, the IL-10 conjugates disclosed herein comprise: (a) the nucleotide triphosphate transporter PtNTT2, including a truncated mutant in which the first 65 amino acid residues of the full-length protein are deleted; (b) a plasmid comprising a double-stranded oligonucleotide encoding an IL-10 mutant having a desired amino acid sequence and containing an unnatural base pair comprising a first unnatural nucleotide and a second unnatural nucleotide, the unnatural base pair being N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl) ... The IL-10 variant is produced in a cell such as E. coli that contains (c) a plasmid that provides a codon at the desired position where an unnatural amino acid, such as N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine, is incorporated; (d) a plasmid that encodes a tRNA derived from M. mazei and that contains unnatural nucleotides, where the plasmid provides a recognized anticodon (for the IL-10 variant codon) in place of the native sequence; and (e) a plasmid that encodes pyrrolysyl-tRNA synthetase from M. barkeri (Mb PylRS), which may be the same plasmid encoding the tRNA or a different plasmid. In some embodiments, the cells are further supplemented with deoxyribotriphosphates that include one or more unnatural bases. In some embodiments, the cells are further supplemented with ribotriphosphates that include one or more unnatural bases. In some embodiments, the cells are further supplemented with one or more unnatural amino acids, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.In some embodiments, the double-stranded oligonucleotide encoding the amino acid sequence of the desired IL-10 variant comprises, for example, the codon AXC at position 67, 70, 74, 75, 79, 82, 88, 125, 126, 129, 130, or 132 of the sequence encoding the protein having SEQ ID NO:1, where X is an unnatural nucleotide as disclosed herein, such as NaM. In some embodiments, the cell further comprises a plasmid, which may be a protein expression plasmid or another plasmid, encoding an orthogonal tRNA gene from M. mazei that comprises, in place of its native sequence, the AXC-compatible anticodon GYT, where Y is an unnatural nucleotide as disclosed herein, such as TPT3, which is complementary and may be the same or different as the unnatural nucleotide in the codon. In some embodiments, the unnatural nucleotide in the codon is different from and complementary to the unnatural nucleotide in the anticodon. In some embodiments, the unnatural nucleotide in the codon is the same as the unnatural nucleotide in the anticodon. In some embodiments, the first and second unnatural nucleotides comprising an unnatural base pair in the double-stranded oligonucleotide are.

[0120] [ka] In some embodiments, the first and second unnatural nucleotides comprising an unnatural base pair in the double stranded oligonucleotide can be derived from:

[0121] [ka] In some embodiments, the triphosphates of the first and second non-natural nucleotides can be derived from:

[0122] [ka] or a salt thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides are

[0123] [ka] or a salt thereof. In some embodiments, the mRNA derived from the double-stranded oligonucleotide comprising the first non-natural nucleotide and the second non-natural nucleotide comprises:

[0124] [ka] In some embodiments, the M. mazei tRNA may include an anticodon that includes a non-natural nucleotide that recognizes a codon in an mRNA that includes a non-natural nucleotide. The anticodon in the M. mazei tRNA may include an anticodon that includes a non-natural nucleotide that recognizes a codon in an mRNA that includes a non-natural nucleotide.

[0125] [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides derived from

[0126] [ka] In some embodiments, the mRNA comprises non-naturally occurring nucleotides derived from

[0127] [ka] In some embodiments, the mRNA comprises non-naturally occurring nucleotides derived from

[0128] [ka] In some embodiments, the tRNA comprises a non-naturally occurring nucleotide derived from

[0129] [ka] In some embodiments, the tRNA comprises a non-naturally occurring nucleotide derived from

[0130] [ka] In some embodiments, the tRNA comprises a non-naturally occurring nucleotide derived from

[0131] [ka] In some embodiments, the mRNA comprises non-naturally occurring nucleotides derived from

[0132] [ka] and the tRNA comprises a non-natural nucleotide derived from

[0133] [ka] In some embodiments, the mRNA comprises non-naturally occurring nucleotides derived from

[0134] [ka] and the tRNA comprises a non-natural nucleotide derived from

[0135] [ka] The host cells are cultured in a medium containing appropriate nutrients and supplemented with (a) triphosphates of deoxyribonucleosides containing one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene having the codons, (b) triphosphates of ribonucleosides containing one or more unnatural bases required for transcription of (i) an mRNA corresponding to the coding sequence of the cytokine and containing a codon containing one or more unnatural bases, and (ii) a tRNA containing an anticodon containing one or more unnatural bases, and (c) the unnatural amino acid(s) to be incorporated into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that allow for expression of the protein of interest.

[0136] The resulting protein containing one or more unnatural amino acids, AzK, N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine, for example, can be expressed and purified by methods known to those of skill in the art, and then reacted with an alkyne, such as DBCO, containing a PEG chain having the desired average molecular weight as disclosed herein under conditions known to those of skill in the art to provide the IL-10 conjugates disclosed herein. Other methods such as those disclosed in Zhang et al., Nature 2017, Vol. 551(7682):644-647; International Publication No. WO2015157555; International Publication No. WO2015021432; International Publication No. WO2016115168; International Publication No. WO2017106767; International Publication No. WO2017223528; International Publication No. WO2019014262; International Publication No. WO2019014267; International Publication No. WO2019028419; International Publication No. WO2019 / 028425 are known to those of skill in the art.

[0137] Alternatively, a cytokine (e.g., IL-10) polypeptide containing an unnatural amino acid(s) is produced by introducing into a host cell a nucleic acid construct as described herein, which contains a tRNA and an aminoacyl-tRNA synthetase and contains a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene with the new codon and anticodon, (b) ribonucleoside triphosphates required for transcription of the mRNA corresponding to (i) the cytokine sequence containing the codon and (ii) the orthogonal tRNA containing the anticodon, and (c) the unnatural amino acid(s). The host cell is then maintained under conditions that allow expression of the protein of interest. The unnatural amino acid(s) is incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more unnatural amino acids are incorporated into a cytokine (e.g., IL-10) polypeptide. Alternatively, two or more unnatural amino acids are incorporated into a cytokine (e.g., IL-10) polypeptide at two or more sites in the protein.

[0138] Once a cytokine (e.g., IL-10) polypeptide incorporating an unnatural amino acid(s) is produced within a host cell, it can be extracted from the host cell by a variety of techniques known in the art, including enzymatic, chemical and / or osmotic lysis and physical disruption. The cytokine (e.g., IL-10) polypeptide can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or any other suitable technique known to those of skill in the art.

[0139] In some examples, the unnatural amino acid is incorporated into the IL-10 polypeptide by a naturally occurring synthetase. In some embodiments, the unnatural amino acid is incorporated into the cytokine by an organism that is auxotrophic for one or more amino acids. In some embodiments, the synthetase corresponding to the auxotrophic amino acid can add the unnatural amino acid to the corresponding tRNA. In some embodiments, the unnatural amino acid is selenocysteine, or a derivative thereof. In some embodiments, the unnatural amino acid is selenomethionine, or a derivative thereof. In some embodiments, the unnatural amino acid is an aromatic amino acid, which comprises an aryl halide, such as an iodide. In embodiments, the unnatural amino acid is structurally similar to an auxotrophic amino acid.

[0140] In some examples, the unnatural amino acids include the unnatural amino acids shown in FIG.

[0141] In some examples, the unnatural amino acid comprises a lysine or phenylalanine derivative or analog. In some examples, the unnatural amino acid comprises a lysine derivative or lysine analog. In some examples, the unnatural amino acid comprises pyrrolysine (Pyl). In some examples, the unnatural amino acid comprises a phenylalanine derivative or phenylalanine analog. In some examples, the unnatural amino acid is an unnatural amino acid described in Wan et al., "Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool," Biochim Biophys Acta 1844(6):1059-4070 (2014). In some examples, the unnatural amino acid comprises an unnatural amino acid depicted in FIG. 2 (e.g., FIG. 2A and FIG. 2B).

[0142] In some embodiments, the unnatural amino acids include the unnatural amino acids depicted in Figures 3A-3D (adopted from Dumas et al., Chemical Science, 2015, 6, 50-69).

[0143] In some embodiments, the unnatural amino acids incorporated into the IL-10 polypeptide are disclosed in U.S. Patent No. 9,840,493; U.S. Patent No. 9,682,934; U.S. Patent Application Publication No. 2017 / 0260137; U.S. Patent No. 9,938,516; or U.S. Patent Application Publication No. 2018 / 0086734. Exemplary UAAs incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl, and lysine derivative unnatural amino acids. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycles. In some embodiments, such amino acids include azides, tetrazines, or other chemical groups that can be conjugated to coupling partners such as water-soluble moieties. In some embodiments, the UAAs include azides linked to aromatic moieties via alkyl linkers. In some embodiments, the alkyl linker is a C-C 10linker. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an alkyl linker. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an amino group. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an alkylamino group. In some embodiments, the UAA comprises an azide linked to the terminal nitrogen of an amino acid side chain via an alkyl chain (e.g., N6 of a lysine derivative, or N5, N4, or N3 of a derivative containing a shorter alkyl side chain). In some embodiments, the UAA comprises a tetrazine linked to the terminal nitrogen of an amino acid side chain via an alkyl chain. In some embodiments, the UAA comprises an azide or tetrazine linked to an amide via an alkyl linker. In some embodiments, the UAA is an azide or tetrazine-containing carbamate or amide of 3-aminoalanine, serine, lysine, or derivatives thereof. In some embodiments, such UAAs are incorporated into cytokines in vivo. In some embodiments, such UAAs are incorporated into cytokines in a cell-free system.

[0144] Conjugation Moiety In certain embodiments, disclosed herein are conjugated moieties attached to one or more modified IL-10 polypeptides described above. In some examples, the conjugated moiety is a molecule that disrupts the interaction of IL-10 with its receptor. In some examples, the conjugated moiety is any molecule that allows the IL-10 conjugate to modulate an immune response when bound to IL-10. In some examples, the conjugated moiety is attached to IL-10 via a covalent bond. In some examples, the IL-10 described herein is attached to a conjugated moiety having a triazole group. In some examples, the IL-10 described herein is attached to a conjugated moiety having a dihydropyridazine or pyridazine group. In some examples, the conjugated moiety comprises a water-soluble polymer. In other examples, the conjugated moiety comprises a protein or a binding fragment thereof. In additional examples, the conjugated moiety comprises a peptide. In additional examples, the conjugated moiety comprises a nucleic acid. In additional examples, the conjugated moiety comprises a small molecule. In additional examples, the conjugating moiety comprises a bioconjugate (e.g., a TLR agonist, such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand, such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or flagellin). In some cases, the conjugating moiety increases serum half-life and / or improves stability. In some cases, the conjugating moiety reduces cytokine interaction with one or more cytokine receptor domains or subunits. In additional cases, the conjugating moiety blocks interaction of IL-10 with one or more IL-10 domains or subunits or with its cognate receptor(s). In some embodiments, the IL-10 conjugates described herein comprise multiple conjugated moieties.In some embodiments, the conjugation moiety is attached to a non-natural or natural amino acid in the IL-10 polypeptide. In some embodiments, the IL-10 conjugate comprises a conjugation moiety attached to a natural amino acid. In some embodiments, the IL-10 conjugate is attached to a non-natural amino acid in the cytokine peptide. In some embodiments, the conjugation moiety is attached to the N- or C-terminal amino acid of the IL-10 polypeptide. Various combination sites are disclosed herein, for example, a first conjugation moiety is attached to a non-natural or natural amino acid in the IL-10 polypeptide and a second conjugation moiety is attached to the N- or C-terminal amino acid of the IL-10 polypeptide. In some embodiments, a single conjugation moiety is attached to multiple residues (e.g., staples) of the IL-10 polypeptide. In some embodiments, the conjugation moiety is attached to both the N- and C-terminal amino acids of the IL-10 polypeptide.

[0145] Water-soluble polymers In some embodiments, the conjugated moiety described herein is a water-soluble polymer. In some examples, the water-soluble polymer is non-peptidic, non-toxic, and biocompatible. As used herein, a substance is considered biocompatible if the beneficial effects associated with the use of the substance alone or with other substances (e.g., active agents such as IL-10 moieties) in relation to living tissue (e.g., administration to a patient) outweigh any adverse effects as assessed by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist. In some examples, the water-soluble polymer is also not immunogenic. In some examples, a substance is considered non-immunogenic if the intended use of the substance in vivo does not result in an undesirable immune response (e.g., formation of antibodies), or if an immune response does occur, such a response is not considered clinically significant or important as assessed by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.

[0146] In some examples, the water soluble polymer is characterized as having from about 2 to about 300 termini. Exemplary water soluble polymers include, but are not limited to, poly(alkylene glycols), such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers such as ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), polysaccharides, poly(α-hydroxy acids), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazenes, polyoxazolines ("POZ") (described in International Publication No. WO 2008 / 106186), poly(N-acryloylmorpholines), and combinations of any of the foregoing.

[0147] In some cases, the water-soluble polymer is not limited to a specific structure.In some cases, the water-soluble polymer is linear (e.g., end-capped, e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG, or PEG attached to a polyol core), dendritic (or star) structure, each of which may or may not have one or more degradable linkages.In addition, the internal structure of the water-soluble polymer can be organized in any number of different repeating patterns, and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.

[0148] In some embodiments, the weight-average molecular weight of the water-soluble polymer in the IL-10 conjugate is from about 100 Daltons to about 150,000 Daltons. Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5,000 daltons to about 100,000 daltons, the range of about 6,000 daltons to about 9,000 daltons, the range of about 10,000 daltons to about 85,000 daltons, the range of greater than about 10,000 daltons to about 85,000 daltons, the range of about 20,000 daltons to about 85,000 daltons, the range of about 53,000 daltons to about 85,000 daltons, the range of about 25,000 daltons to about 120,000 daltons, the range of about 29,000 daltons to about 120,000 daltons, the range of about 35,000 daltons to about 120,000 daltons, and the range of about 40,000 daltons to about 120,000 daltons.

[0149] Exemplary weight-average molecular weights for water soluble polymers include about 100 daltons, about 200 daltons, about 300 daltons, about 400 daltons, about 500 daltons, about 600 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 900 daltons, about 1,000 daltons, about 1,500 daltons, about 2,000 daltons, about 2,200 daltons, about 2,500 daltons, about 3,000 daltons, about 4,000 daltons, about 4,400 daltons, about 4,500 daltons, about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, and the like. 0 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. Branched versions of water-soluble polymers having any of the foregoing total molecular weights can also be used (e.g., a branched 40,000 dalton water-soluble polymer composed of two 20,000 dalton polymers). In one or more embodiments, the conjugate does not have any PEG moieties attached directly or indirectly to a PEG having a weight average molecular weight of less than about 6,000 daltons.

[0150] PEG typically contains multiple (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how PEG is defined]. As used herein, the number of repeat units is referred to as "(OCH2CH2) n" is identified by the subscript "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, about 450 to about 1930, about 1200 to about 1930, about 568 to about 2727, about 660 to about 2730, about 795 to about 2730, about 909 to about 2730, and about 1,200 to about 1,900. For any given polymer whose molecular weight is known, it is possible to determine the number of repeating units (i.e., "n") by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0151] In some instances, the water soluble polymer is an end-capped polymer, i.e., a polymer having a relatively inert group, e.g., a lower C 1-6 It is a polymer having at least one terminus capped with an alkoxy group, or a hydroxyl group. When the polymer is PEG, for example, methoxy-PEG (commonly referred to as mPEG) can be used, which is a linear form of PEG in which one terminus of the polymer is a methoxy (-OCH3) group, and the other terminus is a hydroxyl or other functional group that is optionally chemically modified.

[0152] In some embodiments, exemplary water-soluble polymers include, but are not limited to, linear or branched discrete PEG (dPEG) from Quanta Biodesign, Ltd.; linear, branched or forked PEG from Nektar Therapeutics; linear, branched or Y-shaped PEG derivatives from JenKem Technology.

[0153] In some embodiments, the IL-10 polypeptides described herein are conjugated to a water-soluble polymer selected from poly(alkylene glycols), e.g., polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers such as ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), polysaccharides, poly(α-hydroxy acids), poly(vinyl alcohol) (PVA), polyacrylamides (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazenes, polyoxazolines ("POZ"), poly(N-acryloylmorpholines), and combinations thereof. In some examples, the IL-10 polypeptides are conjugated to PEG (e.g., PEGylated). In some examples, the IL-10 polypeptides are conjugated to PPG. In some examples, the IL-10 polypeptide is conjugated to POZ. In some examples, the IL-10 polypeptide is conjugated to PVP.

[0154] In some examples, the water-soluble polymer comprises polyglycerol (PG). In some cases, the polyglycerol is highly branched PG (HPG) (e.g., 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 linear PG (LPG). In additional cases, the polyglycerol is a medium functionality PG, a linear-block-hyperbranched PG (e.g., 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 side chain functionality PG (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)).

[0155] In some examples, the IL-10 polypeptides described herein are conjugated to a PG, such as HPG, LPG, mid-functionality PG, linear-block-hyperbranched PG, or side chain functionality PG.

[0156] In some embodiments, the water-soluble polymer is a degradable synthetic PEG substitute.Representative degradable synthetic PEG substitutes include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone-modified PEG derivatives resulting from the polymerization of telechelic or di-terminally functionalized PEG-based macromonomers; PEG derivatives containing comonomers containing degradable linkages, such as poly[(ethylene oxide)-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; poly-(ε-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

[0157] In some examples, the IL-10 polypeptides described herein are conjugated to degradable synthetic PEG surrogates, such as POEGM; backbone-modified PEG derivatives resulting from polymerization of telechelic or di-terminally functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO and MPDL copolymerized with OEGMA; or PCL-g-PEO.

[0158] In some embodiments, the water-soluble polymer comprises a poly(zwitterion). Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). In some examples, the IL-10 polypeptide is conjugated to a poly(zwitterion) such as PSBMA, PCBMA, or PMPC.

[0159] In some embodiments, the water-soluble polymer comprises a polycarbonate. Exemplary polycarbonates include, but are not limited to, pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC6F5). In some examples, the IL-10 polypeptides described herein are conjugated to polycarbonates such as MTC-OC6F5.

[0160] In some embodiments, the water-soluble polymer includes a polymer hybrid, e.g., a polycarbonate / PEG polymer hybrid, a peptide / protein-polymer conjugate, or a hydroxyl-containing and / or zwitterion-derivatized polymer (e.g., a hydroxyl-containing and / or zwitterion-derivatized PEG polymer). In some examples, the IL-10 polypeptides described herein are conjugated to a polymer hybrid, e.g., a polycarbonate / PEG polymer hybrid, a peptide / protein-polymer conjugate, or a hydroxyl-containing and / or zwitterion-derivatized polymer (e.g., a hydroxyl-containing and / or zwitterion-derivatized PEG polymer).

[0161] In some examples, the water-soluble polymer comprises a polysaccharide. Exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some cases, the IL-10 polypeptide is conjugated to a polysaccharide. In some cases, the IL-10 polypeptide is conjugated to a dextran. In some cases, the IL-10 polypeptide is conjugated to a PSA. In some cases, the IL-10 polypeptide is conjugated to a HA. ​​In some cases, the IL-10 polypeptide is conjugated to an amylose. In some cases, the IL-10 polypeptide is conjugated to a heparin. In some cases, the IL-10 polypeptide is conjugated to a HS. In some cases, the IL-10 polypeptide is conjugated to a dextrin. In some cases, the IL-10 polypeptide is conjugated to a HES.

[0162] In some cases, the water-soluble polymer comprises a glycan. Exemplary types of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans. In some cases, the IL-10 polypeptide is conjugated to a glycan. In some cases, the IL-10 polypeptide is conjugated to an N-linked glycan. In some cases, the IL-10 polypeptide is conjugated to an O-linked glycan. In some cases, the IL-10 polypeptide is conjugated to a glycolipid. In some cases, the IL-10 polypeptide is conjugated to an O-GlcNAc. In some cases, the IL-10 polypeptide is conjugated to a glycosaminoglycan.

[0163] In some embodiments, the water-soluble polymer comprises a polyoxazoline polymer. The polyoxazoline polymer is a linear synthetic polymer, and like PEG, comprises low polydispersity. In some examples, the polyoxazoline polymer is a polydisperse polyoxazoline polymer characterized by average molecular weight. In some cases, the average molecular weight of the polyoxazoline polymer comprises, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. In some examples, the polyoxazoline polymer comprises poly(2-methyl 2-oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), or poly(2-propyl 2-oxazoline) (PPOZ). In some cases, the IL-10 polypeptide is conjugated to the polyoxazoline polymer. In some cases, the IL-10 polypeptide is conjugated to PMOZ. In some cases, the IL-10 polypeptide is conjugated to PEOZ. In some cases, the IL-10 polypeptide is conjugated to PPOZ.

[0164] In some examples, the water soluble polymer comprises a polyacrylic acid polymer. Optionally, the IL-10 polypeptide is conjugated to a polyacrylic acid polymer.

[0165] In some examples, the water-soluble polymer comprises a polyamine. A polyamine is an organic polymer that comprises two or more primary amino groups. In some embodiments, the polyamine comprises a branched polyamine, a linear polyamine, or a cyclic polyamine. In some cases, the polyamine is a low molecular weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine. In some cases, the IL-10 polypeptide is conjugated to a polyamine. In some cases, the IL-10 polypeptide is conjugated to putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, or piperazine.

[0166] In some examples, the water-soluble polymer is described in U.S. Patent Nos. 7,744,861, 8,273,833, and 7,803,777. In some examples, the IL-10 polypeptide is conjugated to a linker described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777.

[0167] Lipids In some embodiments, the conjugated moiety described herein is a lipid. In some examples, 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 containing 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.

[0168] In some embodiments, a lipid is conjugated to an IL-10 polypeptide described herein. In some examples, the lipid is a fatty acid, e.g., a saturated fatty acid or an unsaturated fatty acid. In some cases, the fatty acid is 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 fatty acid contains about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length. In some cases, the fatty acid includes caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylic acid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic acid), heneicosanoic acid (heneicosanoic acid), behenic acid (docosanoic acid), tricosanoic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosanoic acid (pentacosanoic acid), or cerotic acid (hexacosanoic acid).

[0169] In some embodiments, the IL-10 lipid conjugate increases serum stability and / or serum half-life.

[0170] protein In some embodiments, the conjugated moiety described herein is a protein or a binding fragment thereof. Exemplary proteins include albumin, transferrin, or transthyretin. In some examples, the protein or a binding fragment thereof comprises an antibody or a binding fragment thereof. In some cases, the IL-10 conjugate comprises a protein or a binding fragment thereof. In some cases, the IL-10 conjugate comprising the protein or a binding fragment thereof has an increased serum half-life and / or stability. In some cases, the IL-10 conjugate comprising the protein or a binding fragment thereof has a reduced IL-10 interaction with one or more IL-10R subunits. In additional cases, the protein or a binding fragment thereof blocks IL-10 interaction with one or more IL-10R subunits.

[0171] In some embodiments, the conjugated moiety is albumin. Albumins are a family of water-soluble globular proteins. They are commonly found in plasma, accounting for approximately 55-60% of the total plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide whose tertiary structure is divided into three domains, domain I (amino acid residues 1-195), domain II (amino acid residues 196-383), and domain III (amino acid residues 384-585). Each domain further contains a binding site that can reversibly or irreversibly interact with endogenous ligands, such as long- and medium-chain fatty acids, bilirubin, or hemin, or exogenous compounds, such as heterocyclic or aromatic compounds.

[0172] In some cases, the IL-10 polypeptide is conjugated to albumin. In some cases, the IL-10 polypeptide is conjugated to human serum albumin (HSA). In additional cases, the IL-10 polypeptide is conjugated to a functional fragment of albumin.

[0173] In some embodiments, the conjugated moiety is transferrin. Transferrin is a 679 amino acid polypeptide approximately 80 kDa in size that contains two Fe ligands, one in the N-terminal domain and one in the C-terminal domain. 3+ In some instances, human transferrin has a half-life of about 7 to 12 days.

[0174] In some examples, the IL-10 polypeptide is conjugated to transferrin. In some cases, the IL-10 polypeptide is conjugated to human transferrin. In additional cases, the IL-10 polypeptide is conjugated to a functional fragment of transferrin.

[0175] In some embodiments, the conjugated moiety is transthyretin (TTR), a transport protein localized in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine (T4) and retinol-binding protein bound to retinol.

[0176] In some examples, the IL-10 polypeptide is conjugated to transthyretin (through one of its termini or through the internal hinge region). In some cases, the IL-10 polypeptide is conjugated to a functional fragment of transthyretin.

[0177] In some embodiments, the conjugated moiety is an antibody, or a binding fragment thereof. In some examples, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide stabilized Fv protein (dsFv), a single domain antibody (sdAb), an IgNAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

[0178] In some examples, the conjugation moiety comprises an scFv, a bis-scFv, (scFv)2, a dsFv, or an sdAb. In some cases, the conjugation moiety comprises an scFv. In some cases, the conjugation moiety comprises a bis-scFv. In some cases, the conjugation moiety comprises an (scFv)2. In some cases, the conjugation moiety comprises a dsFv. In some cases, the conjugation moiety comprises an sdAb.

[0179] In some examples, the conjugating moiety comprises an Fc portion of an antibody, e.g., an Fc portion of an IgG, IgA, IgM, IgE, or IgD. In some examples, the moiety comprises an Fc portion of an IgG (e.g., an IgG1, IgG3, or IgG4).

[0180] In some cases, the IL-10 polypeptide is conjugated to an antibody or a binding fragment thereof. In some cases, the IL-10 polypeptide is conjugated to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an IgNAR, a camelid 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-10 polypeptide is conjugated to the Fc portion of an antibody. In additional cases, the IL-10 polypeptide is conjugated to the Fc portion of an IgG (e.g., IgG1, IgG3, or IgG4).

[0181] In some embodiments, the IL-10 polypeptide is conjugated to a water-soluble polymer (e.g., PEG) and an antibody or binding fragment thereof. In some cases, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a humabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an IgNAR, 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 binding fragment thereof comprises a scFv, a bis-scFv, (scFv)2, a dsFv, or a sdAb. In some cases, the antibody or binding fragment thereof comprises a scFv. In some cases, the antibody or binding fragment thereof directs the IL-10 conjugate to a target cell of interest, and the water soluble polymer increases stability and / or serum half-life.

[0182] In some examples, one or more IL-10 polypeptide-water-soluble polymer (e.g., PEG) conjugates are further attached to the antibody or binding fragment thereof. In some examples, the ratio of IL-10 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-10 conjugate to antibody is about 1:1. In other cases, the ratio of IL-10 conjugate to antibody is about 2:1, 3:1 or 4:1. In additional cases, the ratio of IL-10 conjugate to antibody is about 6:1 or more.

[0183] In some embodiments, one or more IL-10 polypeptide-water-soluble polymer (e.g., PEG) conjugates are directly attached to the antibody or binding fragment thereof. In other examples, the IL-10 conjugates are indirectly attached to the antibody or binding fragment thereof using a linker. Exemplary linkers include homobifunctional linkers, heterobifunctional linkers, maleimide-based linkers, zero trace linkers, self-impermeable linkers, spacers, and the like.

[0184] In some embodiments, the antibody or binding fragment thereof is directly or indirectly bound to the IL-10 polypeptide portion of the IL-10 polypeptide-water-soluble polymer (e.g., PEG) conjugate. In such cases, the conjugation site of the antibody to the IL-10 polypeptide is at a site that does not interfere with the binding of the IL-10 polypeptide to the IL-10R. In additional cases, the conjugation site of the antibody to the IL-10 polypeptide is at a site that partially blocks the binding of the IL-10 polypeptide to the IL-10R. In other embodiments, the antibody or binding fragment thereof is directly or indirectly bound to the water-soluble polymer portion of the IL-10 polypeptide-water-soluble polymer (e.g., PEG) conjugate.

[0185] peptide In some embodiments, the conjugated moiety described herein is a peptide. In some examples, the peptide is an unstructured peptide. In some cases, the IL-10 polypeptide is conjugated to a peptide. In some cases, the IL-10 conjugate comprising the peptide has an increased serum half-life and / or stability. In some cases, the IL-10 conjugate comprising the peptide has a reduced IL-10 interaction with one or more IL-10R subunits. In additional cases, the peptide blocks IL-10 interaction with one or more IL-10R subunits.

[0186] In some examples, the conjugated moiety is an XTEN™ peptide (Amunix Operating Inc.), and the modification is referred to as XTENylation. XTENylation is a genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding an XTEN™ peptide (Amunix Operating Inc.), which is a long, unstructured, hydrophilic peptide that contains different percentages of the six amino acids: Ala, Glu, Gly, Ser, and Thr. In some examples, the XTEN™ peptide is selected based on properties such as expression, genetic stability, solubility, aggregation resistance, increased half-life, increased potency, and / or increased in vitro activity in combination with the polypeptide of interest. In some cases, an IL-10 polypeptide is conjugated to an XTEN peptide.

[0187] In some examples, the conjugated moiety is a homo-amino acid polymer rich in glycine (HAP), and the modification is referred to as HAPylation. HAPylation is a genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a homo-amino acid polymer rich in glycine (HAP). In some examples, the HAP polymer is (Gly4Ser) nIt contains a repeat motif (SEQ ID NO:3) and is sometimes about 50, 100, 150, 200, 250, 300 or more residues in length. Optionally, the IL-10 polypeptide is conjugated to HAP.

[0188] In some embodiments, the conjugated moiety is a PAS polypeptide, and the modification is called PASylation. PASylation is a genetic fusion between the nucleic acid encoding the polypeptide of interest and the nucleic acid encoding the PAS polypeptide. PAS polypeptide is a hydrophilic, uncharged polypeptide consisting of Pro, Ala and Ser residues. In some examples, the length of the PAS polypeptide is at least about 100, 200, 300, 400, 500 or 600 amino acids. In some cases, IL-10 polypeptide is conjugated to PAS polypeptide.

[0189] In some embodiments, the conjugated moiety is an elastin-like polypeptide (ELP), and the modification is called ELPylation. ELPylation is a genetic fusion between a nucleic acid encoding a polypeptide of interest and a nucleic acid encoding an elastin-like polypeptide (ELP). ELP contains a VPGxG repeat motif (SEQ ID NO: 4), where x is any amino acid except proline. In some cases, the IL-10 polypeptide is conjugated to the ELP.

[0190] In some embodiments, the conjugating moiety is a CTP peptide. The CTP peptide is a 31 amino acid residue peptide FQSSSS * KAPPPS * LPSPS * RLPSGPS * DTPILPQ (SEQ ID NO: 5), * indicates an O-glycosylation site (OPKO). In some examples, the CTP peptide is genetically fused to the IL-10 polypeptide. In some cases, the IL-10 polypeptide is conjugated to the CTP peptide.

[0191] In some embodiments, the IL-10 polypeptide is modified by glutamylation, which is a reversible post-translational modification of glutamic acid in which the γ-carboxy group of glutamic acid forms a peptide-like bond with the amino group of free glutamic acid and the α-carboxy group elongates into a polyglutamic acid chain.

[0192] In some embodiments, the IL-10 polypeptide is modified by a gelatin-like protein (GLK) polymer. In some examples, the GLK polymer comprises multiple repeats of Gly-Xaa-Yaa, where Xaa and Yaa comprise predominantly proline and 4-hydroxyproline, respectively. In some cases, the GLK polymer further comprises amino acid residues Pro, Gly, Glu, Gln, 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.

[0193] Additional conjugation moieties In some examples, the conjugated moiety comprises an extracellular biomarker. In some examples, the extracellular biomarker is a tumor antigen. In some examples, exemplary extracellular biomarkers include CD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8, and B220. In some examples, the conjugated moiety is bound or conjugated to IL-10. In some cases, the conjugated moiety is genetically fused, for example, at the N-terminus or C-terminus of IL-10.

[0194] In some examples, the conjugated moiety comprises a molecule from a post-translational modification. In some examples, examples of post-translational modifications include myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypyration, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., addition of an alkyl group such as a methyl or ethyl group), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation. In some examples, IL-10 is modified by post-translational modifications such as myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypyration, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., addition of an alkyl group such as a methyl or ethyl group), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation.

[0195] Conjugation Linker In some embodiments, useful functional reactive groups for conjugating or attaching a conjugating moiety to an IL-10 polypeptide described herein include, for example, zero or higher order linkers. In some examples, a non-natural amino acid incorporated into an interleukin described herein includes a functional reactive group. In some examples, the linker includes a functional reactive group that reacts with a non-natural amino acid incorporated into an interleukin described herein. In some examples, the conjugating moiety includes a functional reactive group that reacts with a non-natural amino acid incorporated into an interleukin described herein. In some examples, the conjugating moiety includes a functional reactive group that reacts with a linker described herein (optionally previously attached to a cytokine peptide). In some embodiments, the linker includes a reactive group that reacts with a natural amino acid in an IL-10 polypeptide described herein. In some cases, the higher order linker includes a bifunctional linker, such as a homobifunctional linker or a heterobifunctional linker.Exemplary homobifunctional linkers include, but are not limited to, 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 carbonate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamide). butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB) such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido))ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0196] In some embodiments, the bifunctional linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-crosslinking linkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysuccinimide ester (Sulfo-MBs), 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)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMP ... succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-((((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-((((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA),Carbonyl-reactive and sulfhydryl-reactive cross-linkers, such as 4-(4-N-maleimidophenyl)butanoic acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers, such as N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), ), sulfosuccinimidyl-(4-azido-salicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl- 6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulf ... Succinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers, e.g.These include 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive cross-linkers such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as p-azidophenylglyoxal (APG).

[0197] In some examples, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on the binding moiety (e.g., on the conjugating moiety or IL-10). Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides. In some embodiments, the non-natural amino acids incorporated into the interleukins described herein comprise an electrophilic group.

[0198] 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.

[0199] In some embodiments, the linker is a peptide linker, e.g., comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some examples, the peptide linker comprises 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 comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0200] In some embodiments, the linker comprises an autoimmune linker moiety. In some embodiments, the autoimmune linker moiety comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker comprises a dipeptide linker moiety and an autoimmune linker moiety. In some embodiments, the autoimmune linker moiety is as described in U.S. Pat. No. 9,089,614 and WIPO Publication No. WO2015038426.

[0201] 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.

[0202] In some embodiments, the linker comprises a maleimide group. In some examples, the maleimide group is also referred to as a maleimide spacer. In some examples, the maleimide group further comprises caproic acid to form maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) as described above.

[0203] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide and provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby removing the maleimide which undergoes elimination via a reverse Michael reaction. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol., 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0204] Conjugation Chemistry A variety of conjugation reactions are used to conjugate the linkers, conjugation moieties, and non-natural amino acids incorporated into the IL-10 polypeptides described herein. Such conjugation reactions are often compatible with aqueous conditions, such as "bio-orthogonal" reactions. In some embodiments, the conjugation reaction is mediated by chemical reagents, such as catalytic, light, or reactive chemical groups found on the linker, conjugation moiety, or non-natural amino acid. In some embodiments, the conjugation reaction is mediated by enzymes. In some embodiments, the conjugation reaction used herein is described in Gong, Y., Pan, L., Tett. Lett., 2015, 56, 2123. In some embodiments, the conjugation reaction used herein is described in Chen, X.; Wu. YW., Org. Biomol. Chem., 2016, 14, 5417.

[0205] 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 the reaction of a ketone with an aminoxy group to form an oxime. In some embodiments, the conjugation reaction comprises the reaction of a ketone with an aryl or heteroaryl amine group to form an imine. In some embodiments, the conjugation reaction comprises the reaction of an aldehyde with an aryl or heteroaryl amine group to form an imine. In some embodiments, the conjugation reaction described herein results in an IL-10 polypeptide comprising a linker or conjugation moiety linked via an oxime. In some embodiments, the conjugation reaction comprises the Pictet-Spengler reaction of an aldehyde or ketone with a tryptamine nucleophile. In some embodiments, the conjugation reaction comprises a hydrazino-Pictet-Spengler reaction. In some embodiments, the conjugation reaction comprises a Pictet-Spengler ligation.

[0206] 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 aryl phosphine. In some embodiments, the aryl phosphine comprises an orthoester group. In some embodiments, the phosphine comprises the structure of methyl 2-(diphenylphosphanyl)benzoate ester. In some embodiments, the conjugation reaction described herein results in an IL-10 polypeptide comprising a linker or conjugation moiety linked via an aryl amide. In some embodiments, the conjugation reaction described herein results in an IL-10 polypeptide comprising a linker or conjugation moiety linked via an amide.

[0207] In some embodiments described herein, the conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises a reaction of an azide with 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 an IL-10 polypeptide comprising a linker or conjugation moiety linked via a triazole. In some embodiments, the conjugation reaction described herein comprises a reaction of an azide with a strained olefin. In some embodiments, the conjugation reaction described herein comprises a reaction of an azide with a strained alkyne. In some embodiments, the conjugation reactions described herein include the reaction of an azide with a cycloalkyne, such as OCT, DIFO, DIFBO, DIBO, BARAC, TMTH, or other strained cycloalkyne, the structure of which is shown in Gong, Y., Pan, L., Tett. Lett., 2015, 56, 2123. In some embodiments, the 1,3-dipolar cycloaddition reaction is catalyzed by light ("photo-click"). In some embodiments, the conjugation reactions described herein include the reaction of a terminal allyl group with tetrazole and light. In some embodiments, the conjugation reactions described herein include the reaction of a terminal alkynyl group with tetrazole and light. In some embodiments, the conjugation reactions described herein include the reaction of an O-allyl amino acid with tetrazine and light. In some embodiments, the conjugation reactions described herein include the reaction of an O-allyl tyrosine with tetrazine and light.

[0208] In some embodiments, the conjugation reactions described herein include the reaction of an azide with a cycloalkyne, such as DBCO. In some embodiments, the conjugation reactions described herein include the reaction of an azide with a cycloalkyne, such as DBCO.

[0209] [ka] and X is a position in the IL-10 conjugate that comprises the unnatural amino acid. In some embodiments, the conjugating moiety comprises a water soluble polymer. In some embodiments, the reactive group comprises an alkyne or an azide. In some embodiments described herein, the conjugation reactions described herein comprise

[0210] [ka] and X is a position in the IL-10 conjugate that comprises the unnatural amino acid. In some embodiments described herein, the conjugation reaction described herein comprises

[0211] [ka] and X is a position in the IL-10 conjugate that comprises the unnatural amino acid.

[0212] Conjugation reactions such as the click reactions described herein can produce a single positional isomer or a mixture of positional isomers. In some examples, the ratio of positional isomers is about 1:1. In some examples, the ratio of positional isomers is about 2:1. In some examples, the ratio of positional isomers is about 1.5:1. In some examples, the ratio of positional isomers is about 1.2:1. In some examples, the ratio of positional isomers is about 1.1:1. In some examples, the ratio of positional isomers is greater than 1:1.

[0213] In some embodiments described herein, the conjugation reaction described herein comprises an inverse electron demand cycloaddition reaction involving a diene and a dienophile. In some embodiments, the diene comprises a tetrazine. In some embodiments, the dienophile comprises an alkene. In some embodiments, the dienophile comprises an alkyne. In some embodiments, the alkyne is a strained alkyne. In some embodiments, the alkene is a strained diene. In some embodiments, the alkyne is a trans-cyclooctyne. In some embodiments, the alkyne is a cyclooctene. In some embodiments, the alkene is a cyclopropene. In some embodiments, the alkene is a fluorocyclopropene. In some embodiments, the conjugation reaction described herein results in the formation of an IL-10 polypeptide linked to a linker or conjugation moiety via a six-membered heterocycle that includes two nitrogen atoms in the ring.

[0214] In some embodiments described herein, the conjugation reaction described herein comprises an olefin metathesis reaction. In some embodiments, the conjugation reaction described herein comprises the reaction of 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 the reaction of two alkynes with a ruthenium catalyst. In some embodiments, the conjugation reaction described herein comprises the reaction of an alkene or an alkyne with a ruthenium catalyst and an amino acid comprising an allyl group. In some embodiments, the conjugation reaction described herein comprises the reaction of an alkene or an alkyne with a ruthenium catalyst and an amino acid comprising an allyl sulfide or selenide. In some embodiments, the ruthenium catalyst is a Hoveda-Grubbs second generation catalyst. In some embodiments, the olefin metathesis reaction comprises the reaction of one or more strained alkenes or alkynes.

[0215] In some embodiments described herein, the conjugation reaction described herein comprises a cross-coupling reaction. In some embodiments, the cross-coupling reaction comprises a transition metal catalyst, such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or other transition metal catalyst, and one or more ligands. In some embodiments, the transition metal catalyst is water-soluble. In some embodiments described herein, the conjugation reaction described herein comprises a Suzuki-Miyaura cross-coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises a reaction of an aryl halide (or triflate, or tosylate), an aryl or alkenyl boronate, and a palladium catalyst. In some embodiments described herein, the conjugation reaction described herein comprises a Sonogashira cross-coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises a reaction of an aryl halide (or triflate, or tosylate), an alkyne, and a palladium catalyst. In some embodiments, the cross-linking reaction results in the attachment of a linker or conjugating moiety to the IL-10 polypeptide via a carbon-carbon bond.

[0216] In some embodiments described herein, the conjugation reactions described herein include a deprotection or "uncaging" reaction of the reactive group prior to conjugation. In some embodiments, the conjugation reactions described herein include uncaging of the reactive group by light, followed by a conjugation reaction. In some embodiments, the reactive group is protected with an aralkyl moiety that includes one or more nitro groups. In some embodiments, uncaging of the reactive group results in a free amine, sulfide, or other reactive group. In some embodiments, the conjugation reactions described herein include uncaging of the reactive group by a transition metal catalyst, followed by a conjugation reaction. In some embodiments, the transition metal catalyst includes palladium and one or more ligands. In some embodiments, the reactive group is protected with an allyl moiety. In some embodiments, the reactive group is protected with an allyl carbamate. In some embodiments, the reactive group is protected with a propargyl moiety. In some embodiments, the reactive group is protected with a propargyl carbamate. In some embodiments, the reactive group is protected with a dienophile, and exposure to a diene (such as a tetrazine) results in deprotection of the reactive group.

[0217] In some embodiments described herein, the conjugation reaction described herein comprises a ligand-directed reaction, where a ligand (optionally) bound to the reactive group facilitates the conjugation site between the reactive group and the IL-10 polypeptide. In some embodiments, the ligand is cleaved during or after the reaction of the IL-10 polypeptide with the reactive group. In some embodiments, the conjugation site of the IL-10 polypeptide is a natural amino acid. In some embodiments, the conjugation site of the IL-10 polypeptide is a lysine, cysteine, or serine. In some embodiments, the conjugation site of the IL-10 polypeptide is a non-natural amino acid as described herein. In some embodiments, the reactive group comprises a leaving group, such as an electron-deficient aryl or heteroaryl group. In some embodiments, the reactive group comprises a leaving group, such as an electron-deficient alkyl group, which is displaced by the IL-10 polypeptide. In some embodiments, the conjugation reaction described herein comprises a reaction of a radical scavenger with a radical species. In some embodiments, the conjugation reaction described herein comprises an oxidative radical addition reaction. In some embodiments, the radical scavenger is an arylamine. In some embodiments, the radical species is a tyrosyl radical. In some embodiments, the radical species is generated by a ruthenium catalyst (such as [Ru(bpy)3]) and light.

[0218] 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 reactions described herein include natural protein linkage (NPL) of terminal 1-amino-2-thio group with thioester to form amide bond.

[0219] Various conjugation reactions for reacting a linker or conjugating moiety with an IL-10 polypeptide are described herein, where the reaction occurs with a natural ("canonical") amino acid in the IL-10 polypeptide. In some embodiments, the natural amino acid is found at the conjugation position found in the wild-type sequence, or alternatively, the position is mutated. In some embodiments, the conjugation reaction involves the formation of a disulfide bond at an IL-10 residue. In some embodiments, the conjugation reaction involves a 1,4 Michael addition reaction of a cysteine ​​or lysine. In some embodiments, the conjugation reaction involves cyanobenzothiazole linkage of IL-10. In some embodiments, the conjugation reaction involves cross-linking with an acetone moiety, such as 1,3-dichloro-2-propion. In some embodiments, the conjugation reaction involves a 1,4 Michael addition to a dehydroalanine formed by reaction of a cysteine ​​with O-mesitylenesulfonylhydroxylamine. In some embodiments, the conjugation reaction comprises the reaction of tyrosine with a triazolinedione (TAD) or a TAD derivative, hi some embodiments, the conjugation reaction comprises the reaction of tryptophan with a rhodium carbenoid.

[0220] How to use Proliferative diseases or conditions In some embodiments, described herein 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 an IL-10 conjugate as described herein. In some embodiments, the proliferative disease or condition is cancer. In some cases, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the solid tumor is a metastatic cancer. In some cases, the solid tumor is a recurrent or refractory cancer from a previous treatment.

[0221] In some examples, the IL-10 conjugate described herein is administered to a subject in need thereof to treat a solid tumor. In such cases, the subject has bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer (or renal cell carcinoma), lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the IL-10 conjugate is administered to the subject to treat breast cancer. In some cases, the IL-10 conjugate is administered to the subject to treat colon cancer. In some cases, the IL-10 conjugate is administered to the subject to treat esophageal cancer. In some cases, the IL-10 conjugate is administered to the subject to treat head and neck cancer. In some cases, the IL-10 conjugate is administered to the subject to treat kidney cancer (or renal cell carcinoma, i.e., RCC). In some cases, the IL-10 conjugate is administered to the subject to treat lung cancer. In some cases, the IL-10 conjugate is administered to the subject for the treatment of melanoma. In some cases, the IL-10 conjugate is administered to the subject for the treatment of ovarian cancer. In some cases, the IL-10 conjugate is administered to the subject for the treatment of pancreatic cancer. In some cases, the IL-10 conjugate is administered to the subject for the treatment of prostate cancer. In some examples, the cancer is a metastatic cancer. In other examples, the cancer is a recurrent cancer. In additional cases, the cancer is a refractory cancer.

[0222] In some embodiments, the cancer is a treatment-naïve cancer. In such cases, the treatment-naïve cancer is a cancer that has never been treated. In some cases, the treatment-naïve cancer is a solid tumor, such as bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer (or RCC), lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some embodiments, the present specification describes a method for treating a treatment-naïve solid tumor in a subject in need thereof, comprising administering to the subject an IL-10 conjugate described herein.

[0223] In some embodiments, the cancer is a hematological malignancy. In some instances, the IL-10 conjugates described herein are administered to a subject in need thereof to treat a hematological malignancy. In some instances, the hematological malignancy includes leukemia, lymphoma, or myeloma. In some instances, the hematological malignancy is a T-cell malignancy. In other instances, the hematological malignancy is a B-cell malignancy. In some instances, the hematological malignancy is a metastatic hematological malignancy. In other instances, the hematological malignancy is a recurrent hematological malignancy. In additional instances, the hematological malignancy is a refractory hematological malignancy. In some instances, the subject has a T-cell malignancy. In some instances, the subject has a B-cell malignancy. In some cases, the subject is diagnosed with chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's high-grade lymphoma, The subject has high grade B cell lymphoma, primary mediastinal B cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some cases, the IL-10 conjugate is administered to the subject for the treatment of CLL. In some cases, the IL-10 conjugate is administered to the subject for the treatment of SLL. In some cases, the IL-10 conjugate is administered to the subject for the treatment of FL. In some cases, the IL-10 conjugate is administered to the subject for the treatment of DLBCL. In some cases, the IL-10 conjugate is administered to the subject for the treatment of MCL. In some cases, the IL-10 conjugate is administered to the subject for the treatment of Waldenstrom's macroglobulinemia. In some cases, the IL-10 conjugate is administered to the subject for the treatment of multiple myeloma.In some cases, the IL-10 conjugate is administered to the subject for the treatment of Burkitt's lymphoma.

[0224] Additional Therapeutic Agents In some embodiments, an additional therapeutic agent is further administered to the subject. In some cases, the additional therapeutic agent is administered simultaneously with the IL-10 conjugate and / or is co-formulated. In other cases, the additional therapeutic agent and the IL-10 conjugate are administered sequentially, for example, the IL-10 conjugate is administered prior to the additional therapeutic agent or the IL-10 conjugate is administered after the administration of the additional therapeutic agent.

[0225] In some cases, the additional therapeutic agent includes a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, a radiation therapy, or a combination thereof. Exemplary additional therapeutic agents include, but are not limited to, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as cyclosporine, ... topoisomerase I inhibitors, such as topotecan or irinotecan (CPT-11); topoisomerase II inhibitors, such as etoposide (VP-16), teniposide, or mitoxantrone; mitotic inhibitors, such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or corticosteroids, such as prednisone, methylprednisolone, or dexamethasone.

[0226] In some cases, the additional therapeutic agent comprises a first-line therapy. As used herein, "first-line therapy" includes a first-line therapy for a subject with cancer. In some cases, the cancer is a primary cancer or a local cancer. In other cases, the cancer is a metastatic or recurrent cancer. In some cases, chemotherapy is administered as the first-line treatment. In other cases, the first-line treatment is an immunotherapy, a targeted therapy, or a radiation therapy. Those skilled in the art will readily understand that different first-line treatments are applicable to different types of cancer.

[0227] In some cases, the IL-10 conjugate is administered in combination with an alkylating agent, such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; an antimetabolite, such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; an anthracycline, such as dacarbazine, is administered with an additional therapeutic agent selected from unorubicin, doxorubicin, epirubicin, 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 mitotic inhibitor, such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or a corticosteroid, such as prednisone, methylprednisolone, or dexamethasone.

[0228] In some examples, the IL-10 conjugates described herein are administered with an inhibitor of the enzyme poly ADP ribose polymerase (PARP). Exemplary PARP inhibitors include, but are not limited to, olaparib (AZD-2281, Lynparza®, from Astra Zeneca), rucaparib (PF-01367338, Rubraca®, from Clovis Oncology), niraparib (MK-4827, Zejula®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from AbbVie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene). In some cases, the IL-10 conjugate is administered in combination with a PARP inhibitor, such as olaparib, rucaparib, niraparib, talazoparib, veliparib, CK-102, E7016, iniparib, or pamiparib.

[0229] In some embodiments, the IL-10 conjugate described herein is administered together with a tyrosine kinase inhibitor (TKI).Exemplary TKIs include, but are not limited to, afatinib, alectinib, axitinib, bosutinib, cabozantinib, ceritinib, cobimetinib, crizotinib, dabrafenib, dasatinib, erlotinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, tofacitinib, and vandetanib.

[0230] In some examples, the IL-10 conjugates described herein are administered with an immune checkpoint inhibitor. Exemplary checkpoint inhibitors include:

[0231] PD-L1 inhibitors such as AstraZeneca's durvalumab (Imfinzi), Genentech's atezolizumab (MPDL3280A), EMD Serono / Pfizer's avelumab, CytomX Therapeutics' CX-072, Novartis Pharmaceuticals' FAZ053, 3D Medicine / Alphamab's KN035, Eli Lilly's LY3300054, or EMD Serono's M7824 (anti-PD-L1 / TGF-beta trap);

[0232] PD-L2 inhibitors such as GlaxoSmithKline's AMP-224 (Amplimmune) and rHIgM12B7;

[0233] PD-1 inhibitors such as nivolumab (Opdivo) from Bristol-Myers Squibb, pembrolizumab (Keytruda) from Merck, AGEN2034 from Agenus, BGB-A317 from BeiGene, Bl-754091 from Boehringer-Ingelheim Pharmaceuticals, CBT-501 (genolimuzumab) from CBT Pharmaceuticals, INCSHR1210 from Incyte, JNJ-63723283 from Janssen Research & Development, MEDI0680 from MedImmune, MGA012 from MacroGenics, PDR001 from Novartis Pharmaceuticals, PF-06801591 from Pfizer, REGN2810 (SAR439684) from Regeneron Pharmaceuticals / Sanofi, or TSR-042 from TESARO;

[0234] CTLA-4 inhibitors such as Bristol Meyers Squibb's ipilimumab (also known as Yervoy®, MDX-010, BMS-734016, and MDX-101), Pfizer's tremelimumab (CP-675,206, ticilimumab), or Agenus' AGEN 1884;

[0235] LAG3 inhibitors such as Bristol-Myers Squibb's BMS-986016, Novartis Pharmaceuticals' IMP701, Novartis Pharmaceuticals' LAG525, or Regeneron Pharmaceuticals' REGN3767;

[0236] B7-H3 inhibitors such as MacroGenics' enoblituzumab (MGA271);

[0237] KIR inhibitors such as Innate Pharma's lirilumab (IPH2101; BMS-986015);

[0238] CD137 inhibitors, such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor).

[0239] 13. PS inhibitors, for example, bavituximab.

[0240] and inhibitors, such as antibodies or fragments thereof (e.g., monoclonal antibodies, human, humanized, or chimeric antibodies), RNAi molecules, or small molecules against TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[0241] In some examples, the IL-10 conjugate is administered in combination with pembrolizumab, nivolumab, tremelimumab, or ipilimumab.

[0242] In some examples, the IL-10 conjugates described herein are administered with an antibody, such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumumab.

[0243] In some examples, the IL-10 conjugate is administered with an additional therapeutic agent selected from an additional cytokine. In some examples, the 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-2, IL-6, IL-7, IL-12, IL-15, IL-18, 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.

[0244] In some examples, the IL-10 conjugate is administered with an additional therapeutic agent selected from a receptor agonist. In some examples, the receptor agonist comprises a Toll-like receptor (TLR) ligand. In some cases, the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some cases, the TLR ligand comprises a synthetic ligand, such as 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 cases, the IL-10 conjugate is administered with one or more TLR agonists selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some cases, the IL-10 conjugate is administered with one or more TLRs 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 linker.

[0245] In some embodiments, the IL-10 conjugates are used with adoptive T cell transfer (ACT) therapy. In one embodiment, ACT involves the identification of autologous T lymphocytes in a subject, e.g., having anti-tumor activity, expanding the autologous T lymphocytes in vitro, and then reinfusing the expanded T lymphocytes to the subject. In another embodiment, ACT involves the use of allogeneic T lymphocytes, e.g., having anti-tumor activity, expanding the T lymphocytes in vitro, and then infusing the expanded allogeneic T lymphocytes to a subject in need thereof. In some examples, the IL-10 conjugates described herein are used with autologous T lymphocytes as part of an ACT therapy. In other examples, the IL-10 conjugates described herein are used with allogeneic T lymphocytes as part of an ACT therapy. In some cases, the IL-10 conjugates are administered to a subject in need thereof simultaneously with the ACT therapy. In other cases, the IL-10 conjugates are administered to a subject in need thereof sequentially with the ACT therapy.

[0246] In some embodiments, the IL-10 conjugate is used for ex vivo activation and / or expansion of autologous and / or allogeneic T cell transplants. In such cases, the IL-10 conjugate is used to activate and / or expand a sample containing autologous and / or allogeneic T cells, and the IL-10 conjugate is optionally removed from the sample prior to administration of the sample to a subject in need thereof.

[0247] In some embodiments, the IL-10 conjugate is administered with a vaccine. In some examples, the IL-10 conjugate is utilized in combination with an oncolytic virus. In such cases, the IL-10 conjugate acts as a stimulant to regulate immune response. In some examples, the IL-10 conjugate is used with an oncolytic virus as part of an 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 cases, the IL-10 conjugate is used in combination with an oncolytic virus such as T-Vec, G47Δ, JX-594, CG0070, or Reolysin.

[0248] In some embodiments, the IL-10 conjugate is administered in combination with radiation therapy.

[0249] IL-10 Polypeptide Production In some examples, the IL-10 polypeptides described herein, including either natural or non-natural amino acid mutations, are recombinantly produced or chemically synthesized. In some examples, the IL-10 polypeptides described herein are recombinantly produced, e.g., by a host cell system or in a cell-free system.

[0250] In some examples, IL-10 polypeptide is recombinantly produced through a host cell system. In some cases, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell or a plant cell), a prokaryotic cell (e.g., a gram-positive or gram-negative bacterium), or an archaeal cell. In some cases, 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 the ability to integrate the desired genetic material into its genome and 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 integrated the desired genetic material into its genome and has no ability to express the product of the genetic material after many generations of cell division.

[0251] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293H cells, A549 cells, MDCK cells, CHODG44 cells, CHO-S cells, CHO-K1 cells, Expi293F( Trademark) cells, Flp-In(TM) T-REx(TM) 293 cell line, Flp-In(TM)-293 cell line, Flp-In(TM)-3T3 cell line, Flp-In(TM)-BHK cell line, Flp-In(TM)-CHO cell line, Flp -In(TM)-CV-1 cell line, Flp-In(TM)-Jurkat cell line, FreeStyle(TM) 293-F cells, FreeStyle(TM) CHO-S cells, GripTite(TM) 293MSR cell line, GS-CH O 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.

[0252] 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.

[0253] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0254] In some embodiments, the eukaryotic host cell is a plant host cell. In some examples, the plant cell comprises a cell derived from algae. Exemplary plant cell lines include Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0255] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0256] In some examples, suitable polynucleic acid molecules or vectors for producing the IL-10 polypeptides described herein include any suitable vector from either eukaryotic or prokaryotic origin. Exemplary polynucleic acid molecules or vectors include vectors from bacterial (e.g., Escherichia coli), insect, yeast (e.g., Pichia pastoris), algae, or mammalian origin. Examples of bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0257] Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.

[0258] Yeast vectors include, for example, a Gateway® pDEST™ 14 vector, a Gateway® pDEST™ 17 vector, a Gateway® pDEST™ 24 vector, a Gateway® pYES-DEST52 vector, a pBAD-DEST49 Gateway® destination vector, a pAO815 Pichia vector, a pFLD1 Pichia pastoris vector, a pGAPZA, B, and C Pichia pastoris vector, a pPIC3.5K Pichia vector, a pPIC6A, B, and C Pichia vector, a pPIC9K Pichia vector, a pTEF1 / Zeo, a pYES2 / yeast vector, a pYES2 / CT yeast vector, a pYES2 / NTA, B, and C yeast vector, or a pYES3 / CT yeast vector.

[0259] Algal vectors include, for example, pChlamy-4 vectors or MCS vectors.

[0260] Mammalian vectors include, for example, transient expression vectors or stable expression vectors.Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, b, c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0261] In some examples, cell-free systems are used to produce the IL-10 polypeptides described herein. In some cases, the cell-free system comprises a mixture of cytoplasmic and / or nuclear components from cells (e.g., composed of fully purified recombinant components or partially purified components) and is suitable for in vitro nucleic acid synthesis. In some examples, the cell-free system utilizes prokaryotic cell components. In other examples, the cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis is obtained in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, archaea, or HeLa cells. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.

[0262] The cell-free translation system includes various components such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and / or unnatural amino acids, and / or other components used in protein expression. Such components are optionally modified to improve yield, increase synthesis rate, increase fidelity of protein product, or incorporate unnatural amino acids. In some embodiments, the cytokines described herein are synthesized using the cell-free translation system described in U.S. Patent No. 8,778,631; U.S. Patent Application Publication No. 2017 / 0283469; U.S. Patent Application Publication No. 2018 / 0051065; U.S. Patent Application Publication No. 2014 / 0315245; or U.S. Patent No. 8,778,631. In some embodiments, the cell-free translation system includes modified release factors, or even removing one or more release factors from the system. In some embodiments, the cell-free translation system comprises a reduced protease concentration. In some embodiments, the cell-free translation system comprises a modified tRNA with a reassigned codon used to code for the unnatural amino acid. In some embodiments, the synthetase described herein for the incorporation of the unnatural amino acid is used in the cell-free translation system. In some embodiments, the tRNA is preloaded with the unnatural amino acid using enzymatic or chemical methods before being added to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organisms.

[0263] In some embodiments, the IL-10 polypeptide occurs in a circularly permuted form, either via an expression host system or via a cell-free system.

[0264] Production of IL-10 Polypeptides Containing Unnatural Amino Acids In the present disclosure, an orthogonal or extended genetic code can be used, where one or more specific codons present in the nucleic acid sequence of an IL-10 polypeptide are assigned to encode an unnatural amino acid such that it can be genetically incorporated into IL-10 by using an orthogonal tRNA synthetase / tRNA pair that can add the unnatural amino acid to a tRNA, which can incorporate the unnatural amino acid into a polypeptide chain in response to a codon.

[0265] In some instances, the codon is an amber, ochre, opal, or quartet codon. In some instances, the codon corresponds to an orthogonal tRNA used to deliver an unnatural amino acid. In some instances, the codon is an amber. In other instances, the codon is an orthogonal codon.

[0266] In some instances, the codon is a quadruplet codon and is decoded by the orthogonal ribosome ribo-Q1. In some instances, the quadruplet codon is as exemplified in Neumann et al., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature, 464(7287):441-444 (2010).

[0267] In some examples, the codons used in the present disclosure are recoded codons, e.g., synonymous codons, or rare codons replaced with alternative codons. In some cases, the recoded codons are as described in Napolitano et al., "Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli," PNAS, 113(38):E5588-5597 (2016). In some cases, the recoded codons are as described in Ostrov et al., "Design, synthesis, and testing toward a 57-codon genome," Science, 353(6301):819-822 (2016).

[0268] In some examples, unnatural nucleic acids are utilized that direct the incorporation of one or more unnatural amino acids into IL-10. Exemplary unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-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-propyluracil and cytosine. , 6-azo-uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, which increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, such as 2-aminopropyladenine , 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine,Other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7- Deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidines ([5,4-b][1,4]benzoxazin-2(3H)-ones), phenothiazine cytidines (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-ones), G-clamps, phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-ones), carbazole cytidines (2H-pyrimido[4 ,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those 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, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6 -dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine,and U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5,093,232; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,5 2,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653; and 6,005,096; International Publication No. WO 99 / 62923; Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, edited by Kroschwitz, JI, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613; and Sanghvi, Chapter 15, Antisense Research and Applications, edited by Crooke and Lebleu, CRC Press, 1993, pp. 273-288. Additional base modifications can be found, for example, in U.S. Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613; and Sanghvi, Chapter 15, Antisense Research and Applications, edited by Crooke and Lebleu, CRC Press, 1993, pp. 289-302.

[0269] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogues) are available in the art, and in some cases, the nucleic acid contains one or more heterocyclic bases other than the main five base components of naturally occurring nucleic acids. For example, the heterocyclic bases in some cases include uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2.3-d]pyrimidin-3-yl groups, and the purine is linked to the sugar moiety of the nucleic acid through the 9 position, to the pyrimidine through the 1 position, to the pyrrolopyrimidine through the 7 position, and to the pyrazolopyrimidine through the 1 position.

[0270] In some embodiments, nucleotide analogues are also modified at the phosphate moiety.Modified phosphate moieties include, but are not limited to, those that have modifications in the linkage between two nucleotides, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates.It is understood that these phosphates or modified phosphate linkages between two nucleotides are via 3'-5' linkages or 2'-5' linkages, and linkages include reverse polarity, such as 3'-5' to 5'-3', or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included. Numerous U.S. patents teach methods of making and using nucleotides containing modified phosphates, including, but not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,1 No. 31; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

[0271] In some embodiments, the non-naturally occurring nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleosides (PCT / US2002 / 006460), 5'-substituted DNA and RNA derivatives (PCT / US2002 / 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, vol. 87, pp. 3004-3020; PCT / JP2000 / 004720; PCT / JP2003 / 002342; PCT / JP2004 / 013216; PCT / JP2005 / 020435; PCT / JP2006 / 315479; PCT / JP2006 / 324484; PCT / JP2009 / 056718; PCT / JP2010 / 067560), or 5'-substituted monomers made as monophosphates with modified bases.

[0272] In some embodiments, the non-natural nucleic acid includes modifications at the 5' and 2' positions of the sugar ring (PCT / US94 / 02993), such as 5'-CH2-substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, vol. 83, pp. 1127-1143, and Wu et al., Bioconjugate Chem., 1999, vol. 10, pp. 921-924). In some cases, the non-natural nucleic acid includes amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, in which the 3'-linked nucleoside (5' to 3') in the dimer includes 2'-OCH3 and 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, pp. 1287-1290). Non-natural nucleic acids can include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Non-natural nucleic acids can include 5'-methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, vol. 34, pp. 2723-2726; Collingwood et al., Synlett, 1995, vol. 7, pp. 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, vol. 85, pp. 2777-2806). Non-natural nucleic acids can include 5'-phosphonate monomers with 2'-substitutions (US Patent Application Publication No. 2006 / 00074035) and other modified 5'-phosphonate monomers (International Publication No. WO1997 / 35869). Non-natural nucleic acids can include 5'-modified methylene phosphonate monomers (EP614907 and EP629633). Non-natural nucleic acids can include 5' or 6'-phosphonate ribonucleoside analogs that contain hydroxyl groups at the 5' and / or 6' positions (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033).Non-natural nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers with a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, vol. 16(1), pp. 68-82). Non-natural nucleic acids can include nucleosides with a 6'-phosphonate group, where the 5' and / or 6' positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and analogs); a methyleneamino group (CH2NH2) (and analogs) or a cyano group (CN) (and analogs) (Fairhurst et al., Synlett, 2001, vol. 4, pp. 467-472; Kappler et al., J. Med. Chem., 1986, vol. 29, pp. 1030-1033). 8; 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).

[0273] In some embodiments, the non-natural nucleic acids also include modifications of the sugar moiety. In some cases, the nucleic acid includes one or more nucleosides with modified sugar groups. Such sugar-modified nucleosides may confer increased nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents); bridging of two ring atoms to form bicyclic nucleic acids (BNAs); the modification of the ribosyl ring oxygen atom to S, N(R), or C(R1)(R2) (R=H, C1-C 12Examples of chemically modified sugars can be found in International Publication No. WO2008 / 101157, U.S. Patent Application Publication No. 2005 / 0130923, and International Publication No. WO2007 / 134181.

[0274] In some examples, modified nucleic acids include modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety is a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar is attached to the respective heterocyclic base in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogues, and the respective "nucleosides" in which such sugars or analogues are attached to a heterocyclic base (nucleobase), is known. Sugar modifications can also be made and combined with other modifications.

[0275] Modifications to the sugar moiety include natural modifications of ribose and deoxyribose as well as non-natural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl (alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C6 alkyl groups). 10 Alkyl, or C2-C 10alkenyl and alkynyl). 2' sugar modifications also include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2, and -O(CH2) n ON [(CH2) n CH3)]2 (n and m are from 1 to about 10).

[0276] Other modifications at the 2' position include, but are not limited to, C1-C 10Included are lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly at the 3' position of the sugar on the 3' terminal nucleotide, or at the 5' position of the sugar in 2'-5' linked oligonucleotides, and at the 5' position of the 5' terminal nucleotide. Modified sugars also include those that contain modifications of the bridging ring oxygens, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.There are numerous United States patents which teach the preparation of such modified sugar structures and detail a range of base modifications, e.g., U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,7 No. 86; No. 5,514,785; No. 5,519,134; No. 5,567,811; No. 5,576,427; No. 5,591,722; No. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658, 873; 5,670,633; 4,845,205; 5,130,302; 5,134,066; 5,175,273; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502 ,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,700,920, each of which is incorporated by reference in its entirety.

[0277] Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position also include allyl, amino, azido, thio, O-allyl, O-(C1-C 1O alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ) can be selected from each R m and R n are independently H or substituted or unsubstituted C-C 1O It is an alkyl.

[0278] In certain embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, and the bridge comprises a 4' to 2' bicyclic nucleic acid. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3 ...O-2' (S-2'); 4'-(CH2)-O-2' (S-2'); 4'-(CH2)-O-2' (S-2'); CH3)-O-2' and analogs thereof (see International Publication No. WO2009 / 006478, International Publication No. WO2008 / 150729, U.S. Patent Application Publication No. 2004 / 0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134, and International Publication No. WO2008 / 154401).See, for example, Singh et al., Chem. Commun., 1998, vol. 4, pp. 455-456; Koshkin et al., Tetrahedron, 1998, vol. 54, pp. 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, vol. 97, pp. 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, vol. 8, pp. 2219-2222; Singh et al., J. Org. Chem., 1998, vol. 63, pp. 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, vol. 129 (No. 26), pp. 8362-8379; Elayadi et al., Curr. Opinion. Invens.Drugs, 2001, Vol. 2, pp. 558-561; Braasch et al., Chem. Biol, 2001, Vol. 8, pp. 1-7; Oram et al., Curr. Opinion Mol.Ther., 2001, Volume 3, Pages 239-243; US Patent No. 4,849,513; US Patent No. 5,015,733; US Patent No. 5,118,80 No. 0; No. 5,118,802; No. 7,053,207; No. 6,268,490; No. 6,770,748; No. 6,794,499; Nos. 7,034,133; 6,525,191; 6,670,461; and 7,399,845; International Publication Nos. WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, WO2007 / 090071, and WO2007 / 134181; U.S. Patent Application Publication Nos. 2004 / 0171570, 2007 / 0287831, and 2008 / 0039618; U.S. Provisional Patent Application Nos. 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, PCT / US2008 / 068922, and PCT / DK98 / 00393.

[0279] In certain embodiments, the nucleic acid comprises a linked nucleic acid. The nucleic acids can be linked together using any internucleic acid linkage. Two major classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus containing nucleic acid linkage groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N * -dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, nucleic acid linkages with chiral atoms can be prepared as racemic mixtures, as separate enantiomers, for example, alkylphosphonates and phosphorothioates. Non-natural nucleic acids can contain a single modification. Non-natural nucleic acids can contain multiple modifications within one moiety or between different moieties.

[0280] Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which can be used in any combination. Other non-phosphate linkages can also be used.

[0281] In certain embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity to the modified nucleic acids and / or increase their stability in vivo.

[0282] In some examples, the phosphorus derivative (or modified phosphate group) is attached to a sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, and the like. Exemplary polynucleotides containing modified phosphate or non-phosphate linkages are described 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, eds.), John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates," in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS, 93:6657-6665; Jager et al., 1988, Biochem., 27:7247-7246; Nelson et al., 1997, JOC, 62:7278-7287; U.S. Patent No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem., 8:1157-1179.

[0283] In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties, such as anionic, neutral, or cationic groups. Examples of such modifications include anionic internucleoside linkages; N3' to P5' phosphoramidate modifications; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages, such as methylene phosphonates; amide-linked DNA; methylene (methylimino) linkages; formate acetal and thioform acetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic acid guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry, 8:1157-1179). Modified nucleic acids may include chimeric or mixed backbones that include one or more modifications, such as a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

[0284] Substituents of phosphates include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S and CH2 moieties. Numerous U.S. patents disclose methods of making and using these types of phosphate substitutes, including, but not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,96 7; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.It is also understood in nucleotide substitution that both the sugar and phosphate moieties of nucleotide are replaced, for example, by amide-type linkage (aminoethylglycine) (PNA). U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is incorporated herein by reference.See also Nielsen et al., Science, 1991, vol. 254, pp. 1497-1500.Other types of molecules (conjugates) can also be linked to the nucleotide or nucleotide analogue, for example to increase cellular uptake. The conjugate can be chemically linked to the nucleotide or nucleotide analogue.Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, vol. 86, pp. 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, vol. 4, pp. 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, vol. 660, pp. 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, vol. 3, pp. 2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids, 1996, vol. 1, pp. 1111-1115), and the like. Res., 1992, vol. 20, pp. 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5O J, 1991, vol. 10, pp. 1111-1118; Kabanov et al., FEBS Lett., 1990, vol. 259, pp. 327-330; Svinarchuk et al., Biochimie, 1993, vol. 75, pp. 49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, vol. 36, pp. 3651-3654; Shea et al., Nucl. Acids, 1996, vol. 20, pp. 533-538). Res., 1990, vol. 18, pp. 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, vol. 14, pp. 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. et al., 1995, vol. 36, pp. 3651-3654), palmityl moieties (Mishra et al., Biochem. Biophys. Acta, 1995, vol. 1264, pp. 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, vol. 277, pp. 923-937).Numerous United States patents teach the preparation of such conjugates, including, but not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; No. 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605, No. 735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4 ,958,013; 5,082,830; 5,112,963; 5,214,136; 5,112,963; 5,214,136; 5,245,022; 5,254,46 No. 9; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241, No. 5,391,723; No. 5,4 These include Nos. 16,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0285] In some cases, the non-natural nucleic acid further forms non-natural base pairs. Exemplary non-natural nucleotides that can form non-natural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, 5SICS, d5SICS, NAM, dNaM, and combinations thereof. In some embodiments, the non-natural nucleotides include:

[0286] [ka]

[0287] In some cases, unnatural nucleic acids further form unnatural base pairs. Exemplary unnatural nucleotides that can form unnatural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. Other examples of unnatural nucleotides capable of forming unnatural UBPs used to produce the IL-10 conjugates disclosed herein are described in Dien et al., J Am Chem Soc., 2018, 140:16115-16123; Feldman et al., J Am Chem Soc., 2017, 139:11427-11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res., 2014, 42:10235-10244; Malyshev et al., Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc., 2013, 135:18637-18643; Soc. 2013, 135:5408-5419; Malyshev et al., Proc Natl Acad Sci USA 2012, 109:12005-12010.

[0288] In some embodiments, the non-naturally occurring nucleotide used to make the IL-10 conjugates disclosed herein has the formula: [ka] wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide; and The wavy line indicates a bond to a ribosyl or 2'-deoxyribosyl, where the 5'-hydroxy group of the ribosyl or 2'-deoxyribosyl moiety is free or optionally attached to a monophosphate, diphosphate or triphosphate group. The compound may be derived from

[0289] In some embodiments, the non-naturally occurring nucleotides used to prepare the IL-10 conjugates disclosed herein include [ka] In some embodiments, the non-naturally occurring nucleotides used to prepare the IL-10 conjugates disclosed herein can be derived from:

[0290] [ka] or a salt thereof.

[0291] In some embodiments, the unnatural amino acid is incorporated into the IL-10 polypeptide by a synthetic codon / anticodon pair, including those listed below.

[0292] [Table 1]

[0293] wherein X and Y include unnatural bases as disclosed herein. For example, X can be [ka] and Y is a nucleotide derived from [ka] It may be a nucleotide derived from:

[0294] In some embodiments, the unnatural base pair results in an unnatural amino acid as described in Dumas et al., "Designing logical codon reassignment-Expanding the chemistry in biology," Chemical Science, 6:50-69 (2015).

[0295] Host cells into which the constructs or vectors disclosed herein are introduced are cultured or maintained in an appropriate medium so that the tRNA, tRNA synthetase and protein of interest are produced. The medium also contains an unnatural amino acid(s) so that the protein of interest incorporates the unnatural amino acid(s).

[0296] Orthogonal tRNA synthetase / tRNA pairs add unnatural amino acids to tRNAs and incorporate the unnatural amino acids into polypeptide chains in response to a codon. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair, the Escherichia coli TyrRS (Ec-Tyr) / Bacillus stearothermophilus tRNA pair, and the Bacillus subtilis tRNA pair. CUA vs. Escherichia coli LeuRS (Ec-Leu) / Bacillus stearothermophilus tRNA CUA pair, and pyrrolysyl-tRNA pair.

[0297] An IL-10 polypeptide comprising an unnatural amino acid(s) is produced by introducing into a host cell a nucleic acid construct as described herein, comprising a tRNA and a tRNA synthetase, and comprising a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons. The host cell is exposed to a physiological solution comprising the unnatural amino acid(s), and the host cell is then maintained under conditions that allow expression of the coding sequence of the protein of interest. The unnatural amino acid(s) is incorporated into the polypeptide chain in response to a codon. For example, one or more unnatural amino acids are incorporated into the IL-10 polypeptide. Alternatively, two or more unnatural amino acids are incorporated into the IL-10 polypeptide at two or more sites in the protein.

[0298] When multiple unnatural amino acids are incorporated into an IL-10 polypeptide, multiple codons must be incorporated into the encoding nucleic acid sequence at the desired positions such that a tRNA synthetase / tRNA pair can respond to the codon(s) to direct the incorporation of the unnatural amino acid. At least one, two, three, four, or more codons encoding a nucleic acid can be incorporated into the nucleic acid sequence of interest.

[0299] If it is desired to incorporate more than one type of unnatural amino acid into a single protein, a second or further orthogonal tRNA-tRNA synthetase pair can be used to incorporate the second or further unnatural amino acid; suitably, the second or further orthogonal tRNA-tRNA synthetase pair recognizes different codons in the nucleic acid encoding the protein of interest, such that two or more unnatural amino acids can be specifically incorporated into different defined sites in the protein in a single manufacturing step. In certain embodiments, therefore, two or more orthogonal tRNA-tRNA synthetase pairs can be used.

[0300] Once the IL-10 polypeptide incorporating the unnatural amino acid(s) is produced within the host cell, it can be extracted from the host cell by a variety of techniques known in the art, including enzymatic, chemical and / or osmotic lysis and physical disruption. The IL-10 polypeptide can be purified by standard techniques known in the art, such as preparative chromatography, affinity purification, or any other suitable technique.

[0301] Suitable host cells may include bacterial cells (e.g., E. coli), but most suitable host cells are eukaryotic cells, such as insect cells (e.g., Drosophila melanogaster, etc.), yeast cells, nematodes (e.g., Caenorhabditis elegans), mice (e.g., Mus musculus), or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH3T3 cells, and mouse erythroleukemia (MEL) cells), or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Suitably, the host cell is a mammalian cell, such as a human cell, or an insect cell.

[0302] Suitable host cells may include bacterial cells (e.g., E. coli, BL21(DE3)), but most suitably the host cell is a eukaryotic cell, for example an insect cell (e.g., a fruit fly such as Drosophila melanogaster), a yeast cell, a nematode (e.g., C. elegans), a mouse (e.g., Mus musculus), or a mammalian cell (e.g., Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH3T3 cells, and mouse erythroleukemia (MEL) cells), or a human cell or other eukaryotic cell. Other suitable host cells are known to those of skill in the art. Suitably, the host cell is a mammalian cell, such as a human cell or an insect cell. In some embodiments, a suitable host cell comprises E. coli.

[0303] Other suitable host cells commonly used in embodiments of the present invention are those mentioned in the Examples section. Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various well-known techniques for introducing foreign nucleic acid molecules (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

[0304] When making a cell line, it is generally preferred to produce a stable cell line. For example, for stable transfection of mammalian cells, it is known that depending on the expression vector and transfection technique used, only a few cells may integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selection marker (e.g., resistance to antibiotics) is generally introduced into the host cell together with the gene of interest. Preferred selection markers include markers that confer resistance to drugs such as G418, hygromycin, or methotrexate. The nucleic acid molecule encoding the selection marker can be introduced into the host cell on the same vector or can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have integrated the selection marker gene survive, while other cells die).

[0305] In one embodiment, the constructs described herein are integrated into the genome of the host cell. The advantage of stable integration is that uniformity between individual cells or clones is achieved. Another advantage is that the best producing cells may be selected. It is therefore desirable to create a stable cell line. In another embodiment, the constructs described herein are transfected into the host cell. The advantage of transfecting the constructs into the host cell is that protein yield is maximized. In one aspect, cells are described that contain the nucleic acid constructs or vectors described herein.

[0306] Pharmaceutical Compositions and Formulations In some embodiments, the pharmaceutical compositions and formulations described herein are administered to a subject by multiple administration routes, including but not limited to parenteral, oral, or transdermal administration routes.In some cases, parenteral administration includes intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration.In some examples, the pharmaceutical composition is formulated for local administration.In other examples, the pharmaceutical composition is formulated for systemic administration.

[0307] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, liposomal dispersions, aerosols, immediate release formulations, controlled release formulations, delayed release formulations, sustained release formulations, pulsatile release formulations, and mixed immediate and controlled release formulations.

[0308] In some embodiments, pharmaceutical preparations comprise carriers or carrier materials selected based on compatibility with the compositions disclosed herein and the release profile characteristics of desired dosage form.See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman, HA and Lachman, L. eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999).

[0309] In some cases, the pharmaceutical composition is formulated as an immunoliposome, which comprises a plurality of IL-10 conjugates directly or indirectly bound to the lipid bilayer of liposome.Exemplary lipids include, but are not limited to, fatty acids; phospholipids; sterols such as cholesterol; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, globosides and cerebrosides; surfactant amines such as stearyl, oleyl and linoleylamine.In some examples, the lipid comprises cationic lipid.In some examples, the lipid comprises phospholipid. Exemplary phospholipids include, but are not limited to, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerin ("PG"), phosphatidylethanolamine ("PE"), phosphatidylinositol ("PI"), and phosphatidylserine ("PS"), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-alpha-phosphatidylcholine ("DDPC"), dieridoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), linoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleoylphosphatidylcholine ("DLPC"). Phosphatidylcholine ("DOPC"), dipalmitoyl phosphatidylcholine ("DPPC"), distearoyl phosphatidylcholine ("DSPC"), 1-palmitoyl-2-oleoyl-phosphatidylcholine ("POPC"), diarachidoyl phosphatidylcholine ("DAPG"), didecanoyl-L-alpha-phosphatidylglycerol ("DDPG"), dielaidoyl phosphatidylglycerol ("DEPG"), dilauroyl phosphatidylglycerol ("DLPG"), dilinoleoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol ("DMPG"), dioleoyl phosphatidylglycerol ("DOPG"), dipalmitoyl phosphatidylglycerol ("DPPG"), distearoyl phosphatidylglycerol ("DSPG"),1-Palmitoyl-2-oleoyl-phosphatidylglycerol ("POPG"), Diarachidoyl phosphatidylethanolamine ("DAPE"), Didecanoyl-L-alpha-phosphatidylethanolamine ("DDPE"), Dielaidoyl phosphatidylethanolamine ("DEPE"), Dilauroyl phosphatidylethanolamine ("DLPE"), Dilinoleoyl phosphatidylethanolamine, Dimyristoyl phosphatidylethanolamine ("DMPE"), Dioleoyl phosphatidylethanolamine ("D OPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), distearoyl phosphatidylethanolamine ("DSPE"), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine ("POPE"), diarachidoyl phosphatidylinositol ("DAPI"), didecanoyl-L-alpha-phosphatidylinositol ("DDPI"), dieladoyl phosphatidylinositol ("DEPI"), dilauroyl phosphatidylinositol ("DLPI"), dilinoleo ... dilinositol, dimyristoyl phosphatidylinositol ("DMPI"), dioleoyl phosphatidylinositol ("DOPI"), dipalmitoyl phosphatidylinositol ("DPPI"), distearoyl phosphatidylinositol ("DSPI"), 1-palmitoyl-2-oleoyl-phosphatidoinositol ("POPI"), diarachidoyl phosphatidylserine ("DAPS"), didecanoyl-L-alpha-phosphatidylserine ("DDPS"), dieladyl phosphatidylserine ("DEPS"), Dilauroyl phosphatidylserine ("DLPS"), dilinoleoyl phosphatidylserine, dimyristoyl phosphatidylserine ("DMPS"), dioleoyl phosphatidylserine ("DOPS"), dipalmitoyl phosphatidylserine ("DPPS"), distearoyl phosphatidylserine ("DSPS"), 1-palmitoyl-2-oleoyl-phosphatidylserine ("POPS"), diarachidoyl sphingomyelin, didecanoyl sphingomyelin, dielaidoyl sphingomyelin, dilauroyl sphingomyelin,These include dilinoleoylsphingomyelin, dimyristoylsphingomyelin, sphingomyelin, dioleoylsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, and 1-palmitoyl-2-oleoylsphingomyelin.

[0310] In some examples, the pharmaceutical formulation further comprises a pH adjusting or buffering agent, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane, and buffers such as citric acid / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0311] In some examples, the pharmaceutical formulation contains one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0312] In some embodiments, the pharmaceutical formulation includes, but is not limited to, sugars such as trehalose, sucrose, mannitol, sorbitol, maltose, glucose, or salts such as potassium phosphate, sodium citrate, ammonium sulfate, and / or other agents such as heparin to increase the solubility and in vivo stability of the polypeptide.

[0313] In some instances, pharmaceutical formulations further comprise diluents that can provide a more stable environment and thus are used to stabilize the compound. Salts dissolved in buffer solutions (which can also provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to, phosphate buffered saline solutions.

[0314] Stabilizers include compounds such as any antioxidant, buffer, acid, preservative, etc. Examples of stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium diphosphate dihydrate, propylene glycol, metacresol or m-cresol, zinc acetate, polysorbate-20 or Tween® 20, or trometamol.

[0315] Surfactants include compounds such as sodium lauryl sulfate, docusate sodium, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic®. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkyl phenyl ethers, e.g., Octoxynol 10, Octoxynol 40. Sometimes surfactants are included to enhance physical stability or for other purposes.

[0316] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic use. In some embodiments, the pharmaceutical compositions are administered daily, every day, every other day, 5 days a week, once a week, every other week, 2 weeks a month, 3 weeks a month, once a month, twice a month, 3 times a month, or more. The pharmaceutical compositions are 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.

[0317] If the patient's condition improves, at the physician's discretion, administration of the composition may be continued or the dose of the composition administered may be temporarily reduced or stopped for a period of time (i.e., a "drug holiday"). In some examples, the length of the drug holiday may vary between 2 days and 1 year, including, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays can range from 10% to 100%, examples include 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0318] Once improvement of the patient's condition has occurred, a maintenance dose is administered if necessary, after which, as a function of symptoms, the dosage or frequency of administration, or both, can be reduced until the improved disease, disorder, or condition is maintained.

[0319] In some embodiments, the amount of a given agent that corresponds to such an amount will vary depending on factors such as the particular compound, the severity of the disease, the identity (e.g., body weight) of the subject or host requiring treatment, but is nevertheless routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, and the subject or host being treated. In some examples, the desired dose is conveniently presented as a single dose or divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example, as two, three, four or more sub-doses per day.

[0320] The above ranges are merely suggestive, since there are many variables associated with each individual treatment regimen, and substantial deviations from these recommendations are not uncommon. Such dosages will vary depending on a number of variables, including but not limited to, the activity of the compound being used, the disease or condition being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the physician.

[0321] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with minimal toxicity. Dosages vary within this range depending on the dosage form employed and the route of administration utilized.

[0322] Kits / Products In certain embodiments, the present specification discloses kits and products for use with one or more of the methods and compositions described herein.Such kits include carriers, packages, or containers that are compartmentalized to receive one or more containers, such as vials, tubes, and each of the containers (singular or plural) includes one of the separate elements used in the methods described herein.Suitable containers include, for example, bottles, vials, syringes, and test tubes.In one embodiment, the container is formed from various materials, such as glass or plastic.

[0323] The products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0324] For example, the container(s) contain one or more IL-10 polypeptides or conjugates disclosed herein, and optionally one or more pharmaceutical excipients described herein to facilitate delivery of the IL-10 polypeptides or conjugates. Such kits optionally further include an identifying description or label or instructions relating to their use in the methods described herein.

[0325] The kit typically includes a label listing the contents and / or instructions for use, and a package insert with the instructions. A set of instructions is also typically included.

[0326] In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letter, number or other character forming the label is attached, molded or etched on the container itself, and the label is associated with the container when it is present in a receptacle or carrier that also holds the container, for example, as a package insert. In one embodiment, the label is used to indicate that the contents should be used for a particular therapeutic application. The label also indicates instructions for the use of the contents, such as the methods described herein.

[0327] In certain embodiments, the pharmaceutical composition is provided in a pack or dispenser device that contains one or more unit dosage forms containing the compound provided herein. The pack includes, for example, metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is provided with instructions for administration. In one embodiment, the pack or dispenser also includes a notice associated with the container in a manner prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals. This notice reflects the approval by the agency of the drug form for human or veterinary administration. Such notice is, for example, a U.S. Food and Drug Administration approved drug label or an approved product insert. In one embodiment, compositions containing the compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in a suitable container, and labeled for the treatment of an indicated condition.

[0328] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the detailed description is merely exemplary and explanatory, and is not intended to limit the claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that, as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including", as well as other forms such as "include", "includes", and "included", is not limiting.

[0329] Although various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention is, for clarity, described herein in the context of separate embodiments, the invention may also be practiced in a single embodiment.

[0330] In this specification, when a reference is made to "some embodiments," "embodiments," "one embodiment," or "other embodiments," it is meant that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but not necessarily all embodiments.

[0331] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 5 μL" means "about 5 μL" and "5 μL". In general, the term "about" includes amounts that are expected to be within experimental error, such as within 15%, 10%, or 5%.

[0332] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0333] As used herein, the terms "individual(s)," "subject(s)," and "patient(s)" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of the terms require or are limited to situations characterized by the supervision (e.g., full-time or intermittent) of a medical professional (e.g., a physician, registered nurse, registered nurse, physician's assistant, janitor, or hospice worker).

[0334] As used herein, the terms "significant" and "significantly" with respect to receptor binding refer to a change sufficient to affect binding of an IL-10 polypeptide to a target receptor. In some examples, the terms refer to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some examples, the terms refer to a change of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more.

[0335] In some instances, the term "substantially" with respect to dimerization refers to an alteration sufficient to prevent the formation of IL-10 dimers.

[0336] As used herein, the term "tumor-infiltrating immune cell(s)" refers to immune cells that have infiltrated into an area (e.g., tumor microenvironment) that contains tumor cells. In some examples, tumor-infiltrating immune cells are associated with tumor cell destruction, reduced tumor cell proliferation, reduced tumor burden, or a combination thereof. In some examples, tumor-infiltrating immune cells include tumor-infiltrating lymphocytes (TILs). In some examples, tumor-infiltrating immune cells include T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, or basophils. In some examples, tumor-infiltrating immune cells include CD4+ or CD8+ T cells. EXAMPLES

[0337] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. EXAMPLES

[0338] Biochemical interaction of PEGylated IL-10 with human IL-10 receptor subunits The kinetic and equilibrium dissociation constants of the interaction of PEGylated IL-10 compounds with the human IL-10 receptor are measured using surface plasmon resonance (SPR) at Biosensor Tools LLC. For these studies, human IgG1 Fc-fused IL-10R extracellular domain is captured on the surface of a protein A-coated CM4 biosensor chip. Using a Biacore 2000 or similar SPR instrument, the surface is probed in duplicate with a two-fold dilution series starting with 2 μM of either native IL-10 or IL-10 mutant protein. Test samples are injected for 60 seconds or more and binding is measured until a plateau is reached, followed by injection of buffer only (wash) for 30 seconds or more and dissociation is measured. Response units (RU, Y-axis) are plotted against time (seconds, X-axis).

[0339] Ex vivo immune response profiling of IL-10 mutant proteins in PBMC samples from primary human leukopenic system (LRS) To determine how the differential receptor specificity of IL-10 muteins affects activation of primary immune cell subpopulations, concentration-response profiling of lymphocyte activation in human LRS-derived peripheral blood mononuclear cell (PBMC) samples is performed using multicolor flow cytometry. These studies are performed at PrimityBio LLC (Fremont, CA). Fresh LRS-derived samples are treated with either native IL-10 or IL-10 muteins in a 5-fold dilution series starting at a highest concentration of 30 μg / mL. After 45 min of incubation, samples are fixed and stained with antibodies to detect the phosphorylated form of the transcription factor STAT3 (pSTAT3), a marker of upstream binding and activation of the IL-10 receptor signaling complex, as well as a panel of surface markers (Table 1) that track pSTAT3 formation in specific T cell and natural killer (NK) cell subpopulations.

[0340] [Table 2] EXAMPLES

[0341] [Table 3]

[0342] Although preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the disclosed embodiments described herein can be used in implementing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. 1. A modified interleukin 10 (IL-10) polypeptide comprising at least one unnatural amino acid covalently attached to at least one conjugating moiety, the IL-10 polypeptide comprises an amino acid sequence that is 99% identical to amino acids 33-158 of SEQ ID NO:1; the at least one unnatural amino acid is an azide containing lysine derivative, the at least one unnatural amino acid being located at an amino acid position selected from N82, K99, K125, N129, and K130 of SEQ ID NO:1; The modified IL-10 polypeptide, wherein the conjugating moiety comprises a water soluble polymer.

2. The modified IL-10 polypeptide of claim 1, wherein the amino acid sequence is at least 95% identical to amino acids 1-160 of SEQ ID NO:

1.

3. The modified IL-10 polypeptide of claim 1, wherein the amino acid sequence is at least 97% identical to amino acids 1-160 of SEQ ID NO:

1.

4. The modified IL-10 polypeptide of claim 1, wherein the amino acid sequence is at least 98% identical to amino acids 1-160 of SEQ ID NO:

1.

5. The modified IL-10 polypeptide of claim 1, wherein the amino acid sequence comprises 13 lysine amino acid residues.

6. The IL-10 polypeptide is a modified IL-10 polypeptide described in any one of claims 1 to 5, wherein the IL-10 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 in which an amino acid position is modified with at least one non-natural amino acid.

7. The modified IL-1 according to any one of claims 1 to 6, wherein the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). 0 polypeptide.

8. The modified IL-10 polypeptide of claim 1, wherein the water-soluble polymer comprises polyethylene glycol (PEG).

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

10. The modified IL-10 polypeptide of claim 9, wherein the PEG has a molecular weight of about 20 kilodaltons.

11. The modified IL-10 polypeptide of any one of claims 1 to 10, wherein the modified IL-10 polypeptide is isolated and purified.

12. A pharmaceutical composition comprising a modified IL-10 polypeptide according to any one of claims 1 to 11, and a pharma- ceutically acceptable excipient.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is formulated for systemic delivery.

14. A modified IL-10 homodimer comprising two of the modified IL-10 polypeptides according to any one of claims 1 to 11.

15. 15. A composition comprising a complex of the modified IL-10 homodimer of claim 14 bound to the IL-10 receptor (IL-10R).

16. 14. A pharmaceutical composition according to claim 12 or 13 for use in treating a proliferative disease or condition in a subject in need thereof.

17. 17. The pharmaceutical composition of claim 16, wherein the proliferative disease or condition is cancer.

18. The pharmaceutical composition of claim 17 , wherein the cancer is a solid tumor cancer.

19. 19. The pharmaceutical composition of claim 18, wherein the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer.

20. 1. A nucleic acid molecule encoding an IL-10 polypeptide comprising an amino acid sequence comprising at least one unnatural amino acid, the amino acid sequence is 99% identical to amino acids 33-158 of SEQ ID NO:1, and the at least one unnatural amino acid is an azide containing lysine derivative; The nucleic acid molecule, wherein the at least one unnatural amino acid is located at an amino acid position selected from N82, K99, K125, N129, and K130 of SEQ ID NO:

1.

21. A vector comprising the nucleic acid molecule of claim 20.

Images