RU2787201C2 - Cytokine conjugates for treatment of autoimmune diseases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, in particular to interleukin-2 conjugates; it can be used in medicine. The invention discloses a conjugate of an option of IL-2 peptide with water-soluble polymer. Such a conjugate has increased time of action compared to unmodified natural peptide. In addition, amino acid substitutions in an option of IL-2 polypeptide are selected in such a way that to reduce activity in a receptor transmission of a signal to a beta-gamma IL-2 receptor.

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

21 cl, 5 dwg, 5 tbl, 4 ex

Description

Link

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

Sequence listing

[2] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on August 3, 2018, has the name 46085-710_602_SL.txt and is 3703 bytes in size.

Background of the Invention

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

Brief summary of the present invention

[4] Disclosed herein are cytokine conjugates and their use in the treatment of one or more indications in certain embodiments. In some embodiments, interleukin 2 (IL-2) conjugates and use in the treatment of one or more indications are also described herein. In some cases, one or more indications include an autoimmune disease. In some instances, IL-2 conjugates are described herein to modulate the interaction between IL-2 and the IL-2 receptor to stimulate or expand populations of specifically regulatory T cells (Treg cells). In some instances, IL-2 conjugates with extended in vivo half-life, reduced toxicity, and/or extended therapeutic windows are described herein. In additional instances, pharmaceutical compositions and kits are described herein that contain one or more of the interleukin conjugates described herein (eg, IL-2 conjugates).

[5] In some embodiments, disclosed herein is an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid at a position that reduces activity in interleukin 2 receptor βγ (IL-2Rβγ) receptor signaling. or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, but retains significant activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), with reduced activity in receptor signaling compared to that in receptor signaling between the IL polypeptide -2 wild-type and IL-2Rβ, and moreover, recruitment is compared with recruitment of the IL-2Rγ subunit by the wild-type IL-2 polypeptide. In some embodiments, the position of at least one non-natural amino acid is selected from P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27 , N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81 , P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111 , A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, 192, L94, E95, E116, N119, R120, T123, A125 , Q126, S127, S130, T131, L132, and T133, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, at least one unnatural amino acid is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55 , Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108 , D109, E110, T111, A112, and T113, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, L12, E15, H16, L19, D20, Q22 , M23, N26, D84, N88, E95, and Q126, with the amino acid residue numbering according to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, and H16, with the amino acid residue numbering according to SEQ ID NO: NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from Q22, N26, N88, and Q126, with the amino acid numbering The number of residues corresponds to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from E15, D20, D84, and E95, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the position of at least one the unnatural amino acid is selected from L12, L19, and M23, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from Q22 and N26, with the amino acid residue numbering corresponding to SEQ ID NO: 1. According to in some embodiments, at least one non-natural amino acid: is a lysine analog; is a cysteine analog or a histidine analog; contains an aromatic side chain; contains an azido group; contains an alkyne group or contains an aldehyde or ketone group. In some embodiments, at least one non-natural amino acid does not contain an aromatic side chain. In some embodiments, at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine , allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m- acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylalanine-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3 -(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenyls elanil)propanoyl or selenocysteine. In some embodiments, at least one non-natural amino acid is incorporated into the modified IL-2 polypeptide via an orthogonal tRNA-synthetase/tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair contains at least one non-natural nucleotide base. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugating moiety through at least one non-natural amino acid. In some embodiments, the conjugating moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water-soluble polymer contains a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer contains a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid contains a fatty acid. In some embodiments, the fatty acid has about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms carbon, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbons. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein includes albumin, transferrin, or transthyretin. In some embodiments, the protein includes an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof comprise the Fc portion of the antibody. In some embodiments, the peptide includes an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the conjugating moiety is indirectly linked to at least one non-natural amino acid of modified IL-2 via a linker. In some embodiments, the linker includes a homobifunctional linker, a heterobifunctional linker, a zero length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the isolated and modified IL-2 polypeptide has a decrease in activity in receptor signaling relative to IL-2Rβγ, and the decrease in activity in receptor signaling is about 1-fold, 2-fold, 3-fold, 4-fold, 5x, 6x, 7x, 8x, 9x, 10x, 30x, 50x, 100x, 200x, 300x, 400x, 500x multiple, 1000-fold or more to IL-2Rβ relative to the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; recombinant IL-2 polypeptide; or recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide contains an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, an N-terminal deletion includes deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from N-terminus, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some embodiments, a C-terminal deletion includes deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from C- end, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises the IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130 , 10-125, 20-125, 30-125, 1-130, or 1-125, with residue positions referring to positions in SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide has a sequence identity of about 80 %, 85%, 90%, 95%, 96%, 97%, 98%, or 99% relative to SEQ ID NO: 1. In some embodiments, a modified IL-2 polypeptide with reduced activity in receptor signaling to IL- 2Rβγ is able to induce the expansion of CD4+ T regulatory (Treg) cells. In some embodiments, the conjugating moiety attenuates or blocks activity in IL-2 receptor signaling with IL-2Rβγ or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex. In some embodiments, the proliferation of CD4+ Treg cells by the modified IL-2/IL-2Rαβγ complex is equivalent to or greater than that of the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2/IL-2Rαβγ complex induces the proliferation of CD4+ Treg cells to a population sufficient to modulate the course of the disease in an animal model. In some embodiments, the modified IL-2 polypeptide exhibits a first IL-2Rβγ receptor signaling activity and a second IL-2Rαβγ receptor signaling activity, wherein the first receptor signaling activity is at least 1 fold, 2 fold, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 50 times, 100 times, 500 times, 1000 times or more lower than the second activity in receptor transmission signal. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rβγ. In some embodiments, the second activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some embodiments, the modified IL-2 polypeptide further provides an increase in the recruitment of the IL-2Rα subunit to the IL-2 polypeptide, resulting in the activation of the interleukin 2αβ receptor (IL-2Rαβγ), the increase in recruitment being compared to the recruitment of the IL-2Rα subunit by the IL-2 polypeptide wild-type. In some embodiments, the modified IL-2 polypeptide further provides a reduction in the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, wherein the reduction in recruitment is compared to the recruitment of the IL-2Rβ subunit and/or the IL-2Rγ subunit by the IL-2Rγ polypeptide. 2 wild type.

[6] In some embodiments, disclosed herein is an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid at a position that increases the recruitment of an IL-2Rα subunit to an IL-2 polypeptide leading to receptor activation. αβγ of interleukin 2 (IL-2Rαβγ), the increase in recruitment being compared to the recruitment of the IL-2Rα subunit by a wild-type IL-2 polypeptide. In some embodiments, the position of at least one non-natural amino acid is selected from P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27 , N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81 , P82, R83, D84, S87, N88, N89, V91, 192, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111 , A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125 , Q126, S127, S130, T131, L132, and T133, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, at least one unnatural amino acid is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55 , Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108 , D109, E110, T11, A112 and T113, and the numbering of amino acid residues corresponds to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, L12, E15, H16, L19, D20, Q22 , M23, N26, D84, N88, E95, and Q126, with the amino acid residue numbering according to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, and H16, with the amino acid residue numbering according to SEQ ID NO: NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from Q22, N26, N88, and Q126, with the amino acid numbering certain residues corresponds to SEQ ID NO: 1. In some embodiments, the position of at least one non-natural amino acid is selected from E15, D20, D84 and E95, and the numbering of amino acid residues corresponds to SEQ ID NO: 1. In some embodiments, the position of at least one the unnatural amino acid is selected from L12, L19, and M23, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from Q22 and N26, with the amino acid residue numbering corresponding to SEQ ID NO: 1. According to in some embodiments, at least one non-natural amino acid: is a lysine analog; is a cysteine analog or a histidine analog; contains an aromatic side chain; contains an azido group; contains an alkyne group or contains an aldehyde or ketone group. In some embodiments, at least one non-natural amino acid does not contain an aromatic side chain. In some embodiments, at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine , allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m- acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylalanine-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3 -(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenyl selanyl)propanoyl or selenocysteine. In some embodiments, at least one non-natural amino acid is incorporated into the modified IL-2 polypeptide via an orthogonal tRNA-synthetase/tRNA pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair contains at least one non-natural nucleotide base. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugating moiety through at least one non-natural amino acid. In some embodiments, the conjugating moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, the water-soluble polymer contains a PEG molecule. In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the water-soluble polymer contains a polysaccharide. In some embodiments, the polysaccharide includes dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid contains a fatty acid. In some embodiments, the fatty acid has about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms carbon, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 up to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein includes albumin, transferrin, or transthyretin. In some embodiments, the protein includes an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof comprise the Fc portion of the antibody. In some embodiments, the peptide includes an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the conjugating moiety is indirectly linked to at least one non-natural amino acid of modified IL-2 via a linker. In some embodiments, the linker includes a homobifunctional linker, a heterobifunctional linker, a zero length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the isolated and modified IL-2 polypeptide has a decrease in activity in receptor signaling relative to IL-2Rβγ, and the decrease in activity in receptor signaling is about 1-fold, 2-fold, 3-fold, 4-fold, 5x, 6x, 7x, 8x, 9x, 10x, 30x, 50x, 100x, 200x, 300x, 400x, 500x fold, 1000-fold or more to IL-2Rβ relative to wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is: a functionally active fragment of a full-length IL-2 polypeptide; recombinant IL-2 polypeptide; or recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide contains an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, an N-terminal deletion includes deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from N-terminus, with the positions of the residues referring to the positions in SEQ ID NO: 1. In some embodiments, the C-terminal deletion includes the deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, with the positions of the residues referring to those in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises the IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1- 130 or 1-125, with residue positions referring to positions in SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide has a sequence identity of about 80%, 85%, 90%, 95%, 96%, 97 %, 98%, or 99% of SEQ ID NO: 1. In some embodiments, a modified IL-2 polypeptide with decreased activity in IL-2Rβγ receptor signaling is capable of causing expansion of CD4+ T regulatory (Treg) cells. In some embodiments, the conjugating moiety attenuates or blocks activity in IL-2 receptor signaling with IL-2Rβγ or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex. In some embodiments, the proliferation of CD4+ Treg cells by the modified IL-2/IL-2Rαβγ complex is equivalent to or greater than that of the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2/IL-2Rαβγ complex induces the proliferation of CD4+ Treg cells to a population sufficient to modulate the course of the disease in an animal model. In some embodiments, the modified IL-2 polypeptide exhibits a first IL-2Rβγ receptor signaling activity and a second IL-2Rαβγ receptor signaling activity, wherein the first receptor signaling activity is at least 1 fold, 2 fold, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 50 times, 100 times, 500 times, 1000 times or more lower than the second activity in receptor transmission signal. In some embodiments, the first activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rβγ. In some embodiments, the second activity in receptor signaling of the modified IL-2 polypeptide is lower than the activity in receptor signaling of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some embodiments, the modified IL-2 polypeptide further provides for increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide resulting in activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), the increase in recruitment being compared to the recruitment of the IL-2Rα subunit by the IL-2 polypeptide. 2 wild type. In some embodiments, the modified IL-2 polypeptide further provides a reduction in the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, the reduced recruitment being compared to the recruitment of the IL-2Rβ subunit and/or the IL-2Rγ subunit by the IL polypeptide -2 wild type.

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

[8] In some embodiments, disclosed herein is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide and a conjugating fragment that binds to the isolated and purified IL-2 polypeptide at an amino acid residue selected from P2 , T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46 , K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, R83, D84, S87, N88, N89, V91 , 192, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125 , Q126, S127, S130, T131, L132, and T133, with the amino acid residue numbering corresponding to SEQ ID NO 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, 192, L94, E95, E116, N 119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, with the amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the position of at least one non-natural amino acid is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112 and T113, with the numbering of amino acid residues corresponds to SEQ ID NO: 1. In some embodiments, the position of at least one unnatural amino acid is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126, wherein the numbering amino acid residues corresponds to SEQ ID NO: 1. In some embodiments, the amino acid position is selected from K8, K9, and H16. In some embodiments, the amino acid position is selected from Q22, N26, N88, and Q126. In some embodiments, the amino acid position is selected from E15, D20, D84, and E95. In some embodiments, the amino acid position is selected from L12, L19, M23, and F78. In some embodiments, the amino acid position is selected from Q22 and N26. In some embodiments, the amino acid position is at Q22. In some embodiments, the amino acid position is at N26. In some embodiments, an amino acid residue selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, F78, D84, N88, E95, and Q126 further mutates to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, an amino acid residue selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, F78, D84, N88, E95, and Q126 further mutates to a non-natural amino acid. In some embodiments, the non-natural amino acid includes p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalamine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p- propargylphenylalanine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonothyrosine, p-bromophenylalanine, p-amino-L- phenylalanine or isopropyl-L-phenylalanine. In some embodiments, an additional mutated amino acid residue is associated with an additional conjugating fragment. In some embodiments, the IL-2 conjugate has reduced affinity for the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ), or a combination thereof compared to the wild-type IL-2 polypeptide. In some embodiments, reduced affinity is about a 10%, 20%, 30%, 40%, 50%, or 60% reduction in binding affinity for IL-2Rβ, IL-2Rγ, or a combination thereof relative to a wild-type IL-2 polypeptide. In some embodiments, the reduced affinity is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more relative to wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety reduces or blocks the binding of IL-2 to IL-2Rβ, IL-2Rγ, or a combination thereof. In some embodiments, the conjugating moiety down-regulates the modulation of IL-Rγ chain recruitment into the formed IL-2/IL-2Rβ chain complex. In some embodiments, the conjugating fragment prolongs the systemic half-life of the polypeptide without affecting its affinity for the α, β, and γ chains of the IL-2 receptor. In some embodiments, the conjugating moiety contains a water-soluble polymer. In some embodiments, the additional conjugating moiety contains a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), ethylene glycol-propylene glycol copolymers, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide ), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each of the water-soluble polymers independently contains PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently contains a polysaccharide. In some embodiments, the polysaccharide includes dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently contains a glycan. In some embodiments, each of the water-soluble polymers independently contains a polyamine. In some embodiments, the conjugating fragment contains a protein. In some embodiments, the additional conjugating portion comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently contains an Fc moiety. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugating fragment comprises a polypeptide. In some embodiments, the additional conjugating portion comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating fragment is directly linked to an isolated and purified IL-2 polypeptide. In some embodiments, the conjugating fragment is indirectly linked to an isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker includes a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobispropionimidate ( DTBP), 1,4-di-(3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), an aryl halide compound (DFDNB) such as, for example, 1,5-difluoro-2,4 -dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde , glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzy din, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker includes a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl-3-(2-pyridyldithio)propionate (sPDP), long chain N-succinimidyl-3-(2-pyridyldithio)propionate (LC-sPDP), water soluble long chain N-succinimidyl- 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), complex m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MB), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (Sulfo- sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinim ester yes (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl-6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl-6-[6-(((iodoacetyl)amino) hexanoyl)amino]hexanoate (sIAXX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl-6-(((((4-iodoacetyl)amino)methyl)cyclohexane- 1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N- maleimidomethyl)cyclohexane-1-carboxylhydrazide-8 (M ₂ C ₂ H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl 1,3'-dithiopropionate (sAsD), N- hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azide obenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH) , N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl )1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl-4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl- 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate (sulfo-CAMCA), p-nitrophenyldiazopyruvate ( pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p- azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG) . In some embodiments, the linker includes a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker includes a non-cleavable linker. In some embodiments, the linker contains a maleimide group optionally containing maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo -sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer includes p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative or analog thereof. In some embodiments, the conjugating fragment is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.

[9] In some embodiments, disclosed herein is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide and a conjugating fragment; wherein the IL-2 conjugate has reduced affinity for the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ), or a combination thereof compared to the wild-type IL-2 polypeptide. In some embodiments, the conjugating fragment is linked to an amino acid residue that interacts with IL-2Rβ or IL-2Rγ. In some embodiments, the conjugating fragment is linked to an amino acid residue selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94 , E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, with amino acid residue numbering corresponding to SEQ ID NO: 1. In some embodiments, the conjugating moiety contains a water-soluble polymer. In some embodiments, the additional conjugating moiety contains a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), ethylene glycol-propylene glycol copolymers, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide ), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or combinations thereof. In some embodiments, each of the water-soluble polymers independently contains PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently contains a polysaccharide. In some embodiments, the polysaccharide includes dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently contains a glycan. In some embodiments, each of the water-soluble polymers independently contains a polyamine. In some embodiments, the conjugating fragment contains a protein. In some embodiments, the additional conjugating portion comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently contains an Fc portion. In some embodiments, each of the proteins independently contains the Fc portion of an IgG. In some embodiments, the conjugating fragment comprises a polypeptide. In some embodiments, the additional conjugating portion comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating fragment is directly linked to an isolated and purified IL-2 polypeptide. In some embodiments, the conjugating fragment is indirectly linked to an isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker includes a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl suberate) (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST ), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyldimethylpimelimidate (DMP), dimethyl 3,3'-dithiobispropionimidate (DTBP), 1.4 -di-(3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), an aryl halide compound (DFDNB) such as, for example, 1,5-difluoro-2,4-dinitrobenzene or 1, 3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, diglycidyl ether 1,4-butanediol, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α '-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide). In some embodiments, the linker includes a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl-3-(2-pyridyldithio)propionate (sPDP), long chain N-succinimidyl-3-(2-pyridyldithio)propionate (LC-sPDP), water soluble long chain N-succinimidyl- 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio) toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), complex m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl(4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo- sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinim ester yes (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl-6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl-6-[6-(((iodoacetyl)amino) hexanoyl)amino]hexanoate (sIAXX), succinimidyl-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl-6-(((((4-iodoacetyl)amino)methyl)cyclohexane- 1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N- maleimidomethyl)cyclohexane-1-carboxylhydrazide-8 (M ₂ C ₂ H), 3-(2-pyridyldithio)propionylhydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N- hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azide obenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH) , N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl )1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl-4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl- 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyldiazopyruvate ( pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p- azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (ABH), 4-(p-azidosalicylamido)butylamine (AsBA), or p-azidophenylglyoxal (APG) . In some embodiments, the linker includes a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker includes a non-cleavable linker. In some embodiments, the linker contains a maleimide group optionally containing maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo -sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer includes p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative or analog thereof. In some embodiments, the conjugating fragment is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate.

[10] Disclosed herein, in some embodiments, is a pharmaceutical composition comprising: an IL-2 conjugate as described above and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

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

[12] In some embodiments, disclosed herein is a method for expanding a population of regulatory T cells (Treg) comprising: (a) contacting the cell with an IL-2 conjugate described above, and (b) interacting the IL-2 conjugate with IL-2Rα subunits , IL-2Rβ and IL-2Rγ to form an IL-2/IL-2Rαβγ complex; wherein the IL-2 conjugate has reduced affinity for IL-2Rβ and/or IL-2Rγ subunits, or down-modulates the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, or retains similar activity as IL-2 at its α receptor subunits , β and γ, but increases its half-life, and moreover, the IL-2/IL-2Rαβγ complex stimulates the expansion of Treg cells similarly or more effectively than native IL-2.

[13] Disclosed herein in some embodiments is a kit comprising an IL-2 conjugate as described above, or a pharmaceutical composition comprising an IL-2 conjugate as described above. In some embodiments, the present document also describes a kit containing a polynucleic acid molecule encoding an IL-2 polypeptide as described above.

Brief description of graphic materials

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

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

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

[17] FIG. 3A - fig. 3D shows illustrative non-natural amino acids. These unnatural amino acids (UAA) have been genetically encoded in proteins (Fig. 3A - UAA # 1-42; Fig. 3B - UAA # 43-89; Fig. 3C - UAA # 90-128; Fig. 3D - UAA # 129- 167). Fig. 3A-3D are taken from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69.

[18] FIG. 4A - fig. 4B shows dose-response curves of an exemplary IL-2 variant for pSTAT5 signaling in primary human LRS cell (FIG. 4A) and proliferation response in mouse CTLL-2 populations (FIG. 4B).

[19] FIG. 5 shows the improved PK profile of an exemplary IL-2 K35_30kD molecule at two different concentrations.

Detailed description of the present disclosure

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

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

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

[23] In some cases, IL-2 signaling through the "high affinity" IL-2Rαβγ complex modulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells (Treg) provide maintenance of immune homeostasis by suppressing effector cells such as CD8 ⁺ T cells, helper cells such as CD4 ⁺ Th1, Th2 and Th17, B- cells, NK cells and NK T cells. In some cases, Treg cells are derived from the thymus (tTreg cells) or induced from naive T cells in the periphery (pTreg cells). In some cases, Treg cells are considered as a mediator of peripheral tolerance. Indeed, in one study, the transfer of peripheral CD25-depleted CD4 ⁺ T cells caused various autoimmune diseases in nude mice, while the co-transfer of CD4 ⁺ CD25 ⁺ T cells suppressed the development of autoimmunity (Sakaguchi, et al., "Immunologic self- tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)," J. Immunol. 155(3): 1151-1164 (1995)). An increase in the population of Treg cells suppresses the proliferation of effector T cells and suppresses autoimmunity and antitumor T cell responses.

[24] Disclosed herein in some embodiments is a method for selectively activating a distinct population(s) of lymphocytes (eg, regulatory T cells) via cytokine/cytokine receptor signaling. In some cases, the cytokine includes an interleukin. In some cases, the cytokine is a cytokine conjugate, for example, an interleukin conjugate, an interferon conjugate, or a tumor necrosis factor conjugate. In additional cases described herein, pharmaceutical compositions and kits containing one or more of the cytokine conjugates described herein are included.

[25] In some embodiments, this document also describes a method for selectively activating a Treg population through IL-2/IL-2R signaling. In some cases, IL-2 is an IL-2 conjugate characterized by a reduced interaction of IL-2Rβ and IL-2Rγ in the IL-2Rαβγ complex relative to wild-type IL-2, or by down-modulating the recruitment of the IL-2Rγ subunit to the IL-2/IL complex -2Rβ, or retains activity similar to IL-2 in its α, β and γ receptor subunits, but prolongs its half-life. In some embodiments, this document further describes methods for treating an autoimmune disease using an IL-2 conjugate as described herein. In additional embodiments, pharmaceutical compositions and kits are described herein that contain one or more of the IL-2 conjugates described herein.

Cytokine conjugates

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

[27] In some embodiments, an interleukin conjugate is described herein. Illustrative interleukins include, without limitation, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 10 (IL-10), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18) and interleukin 21 (IL-21). In some cases, this document describes an interleukin conjugate, in which the interleukin is selected from IL-1, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18 and IL-21.

IL-2 conjugates

[28] In some embodiments, described herein are IL-2 conjugates modified at an amino acid position. In some instances, the IL-2 polypeptide is an isolated and purified IL-2 polypeptide. In some instances, the IL-2 polypeptide is mammalian IL-2, for example, a rodent IL-2 protein or a human IL-2 protein. In some instances, the IL-2 polypeptide is a human IL-2 protein. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some instances, the IL polypeptide -2 contains the sequence of SEQ ID NO: 1. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 1. In additional cases, the IL-2 polypeptide is characterized by a sequence identity of about 80%, 85%, 90%, 95% , 96%, 97%, 98% or 99% with respect to SEQ ID NO: 2. In additional cases, the IL-2 polypeptide contains the sequence of SEQ ID NO: 2. In additional cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 2.

[29] In some cases, the IL-2 polypeptide is a truncated version. In some cases, the truncation is an N-terminal deletion. In other cases, the truncation is a C-terminal deletion. In additional cases, truncation includes both N-terminal and C-terminal deletions. For example, the truncation may be a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues, either from N- end, either from the C-end, or from both ends. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more leftovers. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 2 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 3 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 4 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 5 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 6 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 7 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 8 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 9 residues. In some cases, the IL-2 polypeptide contains an N-terminal deletion of at least or about 10 residues.

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

[31] In some embodiments, an IL-2 conjugate described herein comprising an isolated and purified IL-2 polypeptide and a conjugating fragment has reduced affinity for the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ) or combinations thereof relative to a wild-type IL-2 polypeptide. In some embodiments, the IL-2 conjugate has reduced recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex compared to the wild-type IL-2 polypeptide. In some instances, the conjugating moiety is linked to an amino acid residue that interacts with IL-2Rβ (eg, at the IL-2/IL-2Rβ interface), with IL-2Rγ (eg, at the IL-2/IL-2Rβ interface), or their combination. In some instances, the conjugating moiety is linked to an amino acid residue that is proximal to the IL-2/IL-2Rβ interface, the IL-2/IL-2Rβ interface, or the IL-2Rβγ interface. In some cases, the amino acid residue is about 5Å, about 10Å, about 15Å, or about 20Å from the IL-2/IL-2Rβ interface, the IL-2/IL-2Rβ interface, or the IL-2Rβγ interface. As used herein, IL-2 residues involved in the IL-2/IL-2Rβ interface, IL-2/IL-2Rβ interface, or IL-2Rβγ interface include IL-2 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2Rβ subunit, IL-2Rγ subunit, or residues in the IL-2Rβγ contact region.

[32] In some cases, the conjugating fragment is linked to an amino acid residue selected from the amino acid position P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23 , N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78 , H79, R81, R82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109 , E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, with amino acid residue numbering according to SEQ ID NO: 1. In some cases, the amino acid position is selected from K8 , K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126 , S127, S130, T131, L132 and T133. In some cases, the amino acid position is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57 , E60, E67, N71, Q74, S75, K76, N77, F78, H79, R82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109 , E110, T111, A112 and T113. In some instances, the amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126. In some cases, the amino acid position is selected from K8, K9 and H16. In some cases, the amino acid position is selected from Q22, N26, N88 and Q126. In some cases, the amino acid position is selected from E15, D20, D84 and E95. In some cases, the amino acid position is selected from L12, L19 and M23. In some cases, the amino acid position is selected from Q22 and N26. In some cases, the amino acid position is at K8. In some cases, the position of the amino acid is in K9. In some cases, the amino acid position is at Q11. In some cases, the amino acid position is at L12. In some cases, the amino acid position is at E15. In some cases, the amino acid position is at H16. In some cases, the amino acid position is at L18. In some cases, the amino acid position is at L19. In some cases, the amino acid position is at D20. In some cases, the amino acid position is at Q22. In some cases, the amino acid position is at M23. In some cases, the amino acid position is at N26. In some cases, the position of the amino acid is in R81. In some cases, the amino acid position is at D84. In some cases, the amino acid position is in S87. In some cases, the amino acid position is in N88. In some cases, the amino acid position is in V91. In some cases, the amino acid position is at I92. In some cases, the amino acid position is at L94. In some cases, the position of the amino acid is at E95. In some cases, the amino acid position is at E116. In some cases, the amino acid position is at N119. In some cases, the amino acid position is in R120. In some cases, the amino acid position is at T123. In some cases, the amino acid position is at A125. In some cases, the amino acid position is at Q126. In some cases, the amino acid position is in S127. In some cases, the amino acid position is in S130. In some cases, the amino acid position is at T131. In some cases, the amino acid position is at L132. In some cases, the amino acid position is at T133.

[33] In some cases, the IL-2 conjugate additionally contains an additional mutation. In such cases, the amino acid is conjugated to an additional conjugating moiety to increase serum half-life, stability, or a combination of the two. Alternatively, the amino acid is first mutated to a natural amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine; or to a non-natural amino acid prior to binding to an additional conjugating moiety.

[34] In some embodiments, the IL-2 conjugate has reduced binding affinity for the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ), or a combination of the IL-2Rαβγ complex for the IL- 2 wild type. In some cases, the reduced affinity of an IL-2 conjugate for the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ), or a combination thereof compared to a wild-type IL-2 polypeptide is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or over 99%. In some cases, the decrease in affinity is about 10%. In some cases, the decrease in affinity is about 20%. In some cases, the decrease in affinity is about 40%. In some cases, the decrease in affinity is about 50%. In some cases, the reduction in affinity is about 60%. In some cases, the reduction in affinity is about 80%. In some cases, the reduction in affinity is about 90%. In some cases, the reduction in affinity is about 99%. In some cases, the reduction in affinity exceeds 99%. In some cases, the reduction in affinity is about 80%. In some cases, the reduction in affinity is about 100%.

[35] In some embodiments, the reduction in binding affinity of an IL-2 conjugate to an IL-2 receptor β subunit, an IL-2 receptor γ subunit, or a combination thereof relative to a wild-type IL-2 polypeptide is about 1-fold, 2-fold, 3- x, 4 x, 5 x, 6 x, 7 x, 8 x, 9 x, 10 x, 30 x, 50 x, 100 x, 200 x, 300 x, 400x, 500x, 1000x or more. In some cases, the decrease in affinity is about 1-fold. In some cases, the decrease in affinity is about 2-fold. In some cases, the decrease in affinity is about 4-fold. In some cases, the decrease in affinity is about 5-fold. In some cases, the decrease in affinity is about 6-fold. In some cases, the decrease in affinity is about 8-fold. In some cases, the decrease in affinity is about 10-fold. In some cases, the decrease in affinity is about 30-fold. In some cases, the reduction in affinity is about 50-fold. In some cases, the reduction in affinity is about 100-fold. In some cases, the decrease in affinity is about 300-fold. In some cases, the decrease in affinity is about 500-fold. In some cases, the reduction in affinity is about 1000-fold. In some cases, the reduction in affinity is more than 1000-fold.

[36] In some embodiments, the IL-2 conjugate has reduced recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex. In some instances, reduced recruitment is compared to recruitment of the IL-2Rγ subunit by an equivalent IL-2 polypeptide without the non-natural amino acid (eg, a wild-type IL-2 polypeptide). In some cases, the reduction in recruitment of IL-2Rγ subunits is characterized by a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99 % compared to the equivalent IL-2 polypeptide without unnatural amino acid modification. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 10%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 20%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 40%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 50%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 60%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 70%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 80%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 90%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 99%. In some cases, the reduction in recruitment of IL-2Rγ subunits exceeds 99%. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 100%. In some cases, the IL-2 conjugate further enhances the recruitment of IL-2Rα subunits.

[37] In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold , 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or more relative to the equivalent IL-2 polypeptide without non-natural amino acid modification (for example , wild-type IL-2 polypeptide). In some cases, the reduction in recruitment of IL-2Rγ subunits is about 1-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 2-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 4-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 5-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 6-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 8-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 10-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 30-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 50-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 100-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 300-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 500-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is about 1000-fold. In some cases, the reduction in recruitment of IL-2Rγ subunits is more than 1000-fold. In some cases, the IL-2 conjugate further enhances the recruitment of IL-2Rα subunits.

[38] In some embodiments, the IL-2 conjugate is characterized by increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide. In some instances, reduced recruitment has been compared to recruitment of the IL-2Rα subunit by an equivalent IL-2 polypeptide without the non-natural amino acid (eg, a wild-type IL-2 polypeptide). In some cases, the increase in recruitment of IL-2Rα subunits is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to the equivalent polypeptide IL-2 without unnatural modification of amino acids. In some cases, the increase in recruitment of IL-2Rα subunits is about 10%. In some cases, the increase in recruitment of IL-2Rα subunits is about 20%. In some cases, the increase in recruitment of IL-2Rα subunits is about 40%. In some cases, the increase in recruitment of IL-2Rα subunits is about 50%. In some cases, the increase in recruitment of IL-2Rα subunits is about 60%. In some cases, the increase in recruitment of IL-2Rα subunits is about 70%. In some cases, the increase in recruitment of IL-2Rα subunits is about 80%. In some cases, the increase in recruitment of IL-2Rα subunits is about 90%. In some cases, the increase in recruitment of IL-2Rα subunits is about 99%. In some cases, the increase in recruitment of IL-2Rα subunits exceeds 99%. In some cases, the increase in recruitment of IL-2Rα subunits is about 100%. In some instances, the IL-2 conjugate is further characterized by reduced recruitment of the IL-2Rβ subunit and/or the IL-2Rγ subunit.

[39] In some embodiments, the increase in IL-2Rα subunit recruitment is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or more relative to the equivalent IL-2 polypeptide without non-natural amino acid modification (for example, polypeptide IL-2 wild type). In some cases, the increase in recruitment of IL-2Rα subunits is about 1-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 2-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 4-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 5-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 6-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 8-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 10-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 30-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 50-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 100-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 300-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 500-fold. In some cases, the increase in recruitment of IL-2Rα subunits is about 1000-fold. In some cases, the increase in recruitment of IL-2Rα subunits is more than 1000-fold. In some instances, the IL-2 conjugate is further characterized by reduced recruitment of the IL-2Rβ subunit and/or the IL-2Rγ subunit.

[40] In some embodiments, an IL-2 polypeptide described herein has a reduction in activity in IL-2Rβγ receptor signaling. In some cases, the decrease in activity in receptor signaling is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold , 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or more to IL-2Rβγ relative to wild-type IL-2 polypeptide. In some cases, the decrease in activity in receptor signaling is about 2-fold. In some cases, the decrease in activity in receptor signaling is about 5-fold. In some cases, the decrease in activity in receptor signaling is about 10-fold. In some cases, the decrease in activity in receptor signaling is about 20-fold. In some cases, the decrease in activity in receptor signaling is about 30-fold. In some cases, the decrease in activity in receptor signaling is about 40-fold. In some cases, the decrease in activity in receptor signaling is about 50-fold. In some cases, the decrease in activity in receptor signaling is about 100-fold. In some cases, the decrease in activity in receptor signaling is about 200-fold. In some cases, the decrease in activity in receptor signaling is about 300-fold. In some cases, the decrease in activity in receptor signaling is about 400-fold. In some cases, the decrease in activity in receptor signaling is about 500-fold. In some cases, the decrease in activity in receptor signaling is about 1000-fold.

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

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

[43] In some embodiments, an IL-2 polypeptide described herein has an extended therapeutic window compared to a wild-type IL-2 polypeptide. In some cases, the extended therapeutic window is due to decreased binding between the IL-2 polypeptide and interleukin 2 βγ receptor (IL-2Rβγ), decreased activity in IL-2Rβγ receptor signaling, decreased recruitment of IL-2Rγ subunits to the IL-2/IL- 2Rβ or increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide. In some cases, the IL-2 polypeptide does not have impaired apy interleukin 2 (IL-2Rαβγ) receptor activation.

[44] In some embodiments, the modified IL-2 polypeptide exhibits a first receptor signaling activity for the IL-2βγ signaling complex and a second receptor signaling activity for the IL-2αβγ signaling complex, wherein the difference between the first receptor signaling activity and the second activity in receptor signaling is at least 1-fold. In some cases, the difference is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30- multiple, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or more. In some cases, the first activity in receptor signaling is less than the second activity in receptor signaling. In some cases, the first activity in receptor signaling is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold times, 20 times, 30 times, 50 times, 100 times, 500 times, 1000 times lower than the second activity in receptor signaling. In some instances, the modified IL-2 polypeptide has lower activity in receptor signaling to the IL-2β signaling complex than the second activity in receptor signaling to the IL-2α signaling complex. In some instances, the first receptor signaling activity of the modified IL-2 polypeptide is at least 1-fold lower than the receptor signaling activity of the wild-type IL-2 polypeptide. In some cases, the first activity in receptor signaling of the modified IL-2 polypeptide is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200 times or 500 times lower than the receptor signaling activity of the wild-type IL-2 polypeptide. In some cases, both the first receptor signaling activity and the second receptor signaling activity are lower than the corresponding wild-type IL-2 polypeptide activities, but the first receptor signaling activity is lower than the second receptor signaling activity. In some instances, the difference between the first receptor signaling activity and the second receptor signaling activity increases the therapeutic window for the modified IL-2 polypeptide.

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

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

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

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

[49] In some embodiments, the IL-2 conjugate has a plasma half-life of at least about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, from about 2 hours to about 8 hours, about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to 10 o'clock.

[50] In some embodiments, the IL-2 conjugate has a plasma half-life in which it is capable of leading to proliferation and/or expansion of a Treg cell, but has no detrimental effects such as apoptosis.

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

[52] In some embodiments, the IL-2 conjugate has an increased plasma half-life, for example, from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours , from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours up to about 10 hours relative to wild-type IL-2.

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

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

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

[56] In some cases, the conjugating moiety attenuates or blocks activity in IL-2 receptor signaling with IL-2Rβγ or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex.

[57] In some cases, a modified IL-2 polypeptide with a decrease in activity in receptor signaling to IL-2Rβγ is able to cause the expansion of CD4+ T-regulatory (Treg) cells.

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

[59] In some embodiments, the IL-2/IL-2Rαβγ complex induces the proliferation of CD4+ Treg cells to a population sufficient to modulate the course of the disease in an animal model.

[60] In some embodiments, this document describes an interleukin 2 αβγ receptor (IL-2Rαβγ) binding protein, wherein the activity in receptor signaling for the interleukin 2 βγ receptor (IL-2Rβγ) of said binding protein is less than that of human IL- 2 wild type (hIL-2), and moreover, the specified binding protein contains at least one non-natural amino acid. In some instances, said binding protein is a modified IL-2 polypeptide or functionally active fragment thereof, wherein the modified IL-2 polypeptide contains at least one non-natural amino acid.

[61] In some embodiments, the present document describes a protein that binds the interleukin 2 apo receptor (IL-2Rαβγ), wherein the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex by said binding protein is less than that of wild-type human IL-2 type (hIL-2), and moreover, the specified binding protein contains at least one non-natural amino acid. In some instances, said binding protein is a modified IL-2 polypeptide or functionally active fragment thereof, wherein the modified IL-2 polypeptide contains at least one non-natural amino acid.

[62] In some embodiments, this document describes a protein that binds the interleukin 2 αβγ receptor (IL-2Rαβγ), wherein the binding affinity for the interleukin 2 βγ receptor (IL-2Rβγ) of said binding protein is less than that of human IL-2 (hIL -2) wild-type, and moreover, the specified binding protein contains at least one non-natural amino acid. In such cases, said binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide contains at least one unnatural amino acid.

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

[64] In some embodiments, an IL-2/IL-2Rαβγ complex is described herein comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex with said modified IL-2 polypeptide is less than the wild-type IL-2 polypeptide complex. In some instances, the modified IL-2 polypeptide further comprises a conjugating moiety covalently linked to a non-natural amino acid.

[65] In some embodiments, an IL-2/IL-2Rαβγ complex is described herein comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the modified IL-2 polypeptide has reduced binding affinity to IL-2Rβγ, and moreover, the reduced binding affinity is compared with the binding affinity between the wild-type IL-2 polypeptide and IL-2Rβγ. In some embodiments, an IL-2/IL-2Rαβγ complex is described herein comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the recruitment of an IL-2Rγ subunit into an IL-2/IL-2Rβ complex with said modified less IL-2 polypeptide than wild-type IL-2 polypeptide. In some instances, the modified IL-2 polypeptide further comprises a conjugating moiety covalently linked to a non-natural amino acid.

[66] In some embodiments, a CD4+ Treg cell activator is described herein that selectively causes expansion of CD4+ Treg cells in a cell population, said activator comprising a modified IL-2 polypeptide containing at least one unnatural amino acid. In some instances, said activator causes expansion of effector CD8+ T cells and/or natural killer cells by less than 20%, 15%, 10%, 5%, 1%, or 0.1% in a population of CD3+ cells when said activator is in contact with said population of CD3+ cells relative to the expansion of CD8+ effector T cells and/or natural killer cells in the population of CD3+ cells with which the wild-type IL-2 polypeptide comes into contact. In some instances, said cell population is an in vivo cell population. In some instances, said cell population is an in vitro cell population. In some instances, said cell population is an ex vivo cell population.

IL-10 cojugates

[67] In some embodiments, described herein are IL-10 conjugates modified at an amino acid position. IL-10, also known as human cytokine synthesis inhibitory factor or CSIF, is an immunomodulatory cytokine that limits the inflammatory response against pathogens, but it has also been identified as an antitumor cytokine due to its immunostimulatory activity on CD8 T cells. IL-10 suppresses the expression of Thl cytokines, MHC class II antigens, and costimulatory molecules on macrophages and further enhances B cell survival, proliferation, and antibody production. In addition, IL-10 can stimulate thymocyte proliferation and cytotoxicity of CD8 T cells. In some cases, the IL-10 conjugate contains an isolated and purified IL-10 polypeptide and a conjugating fragment. In some instances, the IL-10 conjugate has reduced affinity for the IL-10 receptor compared to the wild-type IL-10 polypeptide. In some instances, the conjugating moiety is linked to an amino acid residue that interacts with the IL-10 receptor (eg, at the IL-10/IL-10R interface). In some instances, the conjugating moiety is linked to an amino acid residue that is proximal to the IL-10/IL-10R interface (eg, about 5Å, about 10Å, about 15Å, or about 20Å from the IL-10/IL-10R interface). As used herein, residues involved in the IL-10/IL-10R interface include IL-10 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from IL-10R. In some instances, the conjugating moiety is linked to the N-terminus or C-terminus of an IL-10 polypeptide, either directly or indirectly via a linker peptide. In additional instances, the conjugating moiety modulates the interaction between IL-10 and IL-10R to enhance its immunosuppressive activity and decrease its immunostimulatory activity. In some cases, the IL-10 conjugate activates different populations of lymphocytes through IL-10/IL-10R signaling. In some cases, the IL-10 conjugate modulates immune activity.

Additional cytokine conjugates

[68] In some embodiments, one or more additional cytokine conjugates modified at an amino acid position are described herein. Illustrative cytokines include, without limitation, IL-1β, IL-7, IL-12, IL-15, IL-18, and IL-21. In some cases, the cytokine conjugate contains an isolated and purified cytokine polypeptide and a conjugating fragment. In some cases, the cytokine conjugate has reduced affinity for its respective receptor compared to the wild-type cytokine. In some instances, the conjugating moiety is linked to an amino acid residue that is proximal to the receptor's interface (eg, about 5Å, about 10Å, about 15Å, or about 20Å from the receptor's interface). In some instances, the conjugating moiety is linked to the N-terminus or C-terminus of the cytokine polypeptide, either directly or indirectly via a linker peptide. In additional instances, the conjugating moiety modulates the interaction between a cytokine and its receptor to enhance its immunosuppressive activity and decrease its immunostimulatory activity. In some cases, the cytokine conjugate activates different populations of lymphocytes through its receptor signaling. In some cases, the cytokine conjugate modulates immune activity.

Precursors of cytokine conjugates

[69] Described herein are cytokine conjugate precursors containing a mutant cytokine (such as IL-2) in which one or more amino acids have been mutated from a wild-type amino acid. Such precursors are often used in the methods described herein for the treatment of diseases or conditions. In some embodiments, the cytokine precursor is not conjugated. Such mutations variously include additions, deletions or substitutions. In some embodiments, the mutation comprises a substitution of another naturally occurring amino acid. In some cases, the mutant cytokine contains a mutation at the amino acid position P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, R82, R83, D84, S87, N88, N89, V91, 192, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, with the amino acid residue numbering according to SEQ ID NO: 1. In some cases, the amino acid position is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132 and T133. In some cases, the amino acid position is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57 , E60, E67, N71, Q74, S75, K76, N77, F78, H79, R82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109 , E110, T111, A112 and T113. In some cases, the amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95 and Q126. In some cases, the amino acid position is selected from K8, K9 and H16. In some cases, the amino acid position is selected from Q22, N26, N88 and Q126. In some cases, the amino acid position is selected from E15, D20, D84 and E95. In some cases, the amino acid position is selected from L12, L19 and M23. In some cases, the amino acid position is selected from Q22 and N26. In some embodiments, the mutant cytokine contains a conjugating moiety, wherein the conjugating moiety is attached to a mutated site in the mutant cytokine.

Fusions of proteins or peptides

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

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

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

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

[74] In some cases, the IL-2 polypeptide is fused with the antibody or its binding fragments. In some cases, an IL-2 polypeptide is fused to a humanized antibody or binding fragment thereof, a mouse antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, monovalent Fab' fragments, divalent Fab ₂ , F(ab) ' ₃ , single chain variable fragment (scFv), bis-scFv, (scFv) ₂ , diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single domain antibody (sdAb), Ig NAR, camel antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the IL-2 polypeptide is fused to the Fc portion of the antibody. In additional instances, the IL-2 polypeptide is fused to the Fc portion of an IgG (eg, IgG1, IgG3, or IgG4). In some instances, an antibody or binding fragments thereof fused to an IL-2 polypeptide will not interfere with the binding of the IL-2 polypeptide to IL-2Rβγ. In some instances, an antibody or binding fragments thereof fused to an IL-2 polypeptide partially blocks the binding of the IL-2 polypeptide to IL-2Rβγ. In some instances, an IL-2 polypeptide fused to an antibody or binding fragments thereof is additionally conjugated to one or more of the conjugating fragments described below.

Natural and non-natural amino acids

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

[76] In some embodiments, the amino acid residue described herein (eg, in a cytokine such as IL-2) is mutated to a non-natural amino acid prior to binding to the conjugating moiety. In some cases, a mutation to a non-natural amino acid prevents or minimizes the response to the immune system's autoantigen. As used herein, the term "non-natural amino acid" refers to an amino acid other than the 20 amino acids that are naturally present in a protein. Non-limiting examples of non-natural amino acids include: p-acetyl-L-phenylalanine, p-iodine-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargylphenylalanine, L-3-(2 -naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-borophophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonothyrosine, p-bromophenylalanine, selenocysteine, p-amino- L-phenylalanine, isopropyl-L-phenylalanine, azido-lysine (AzK), a non-natural analogue of the amino acid tyrosine; non-natural analogue of the amino acid glutamine; non-natural analogue of the amino acid phenylalanine; non-natural analogue of the amino acid series; non-natural analogue of the amino acid threonine; alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine , aldehyde, hydroxylamine, keto or amino-substituted amino acid, or a combination thereof; amino acid with a photoactivated crosslinker; spin-labeled amino acid; fluorescent amino acid; metal-binding amino acid; a metal-containing amino acid; radioactive amino acid; photocell and/or photoisomerizable amino acid; biotin or a biotin analogue containing an amino acid; a ketogenic amino acid; amino acid containing polyethylene glycol or polyether; an amino acid substituted by a heavy atom; a chemically cleavable or photo-cleavable amino acid; an amino acid with an extended side chain; an amino acid containing a toxic group; sugar-substituted amino acid; a carbon-linked sugar-containing amino acid; redox amino acid; a-hydroxy acid; aminothioacid; α,α-disubstituted amino acid; β-amino acid; a cyclic amino acid other than proline or histidine; and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

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

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

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

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

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

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

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

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

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

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

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

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

[89] In some cases, the non-natural amino acid contains a Michael acceptor group. In some cases, Michael acceptor groups contain an unsaturated moiety capable of forming a covalent bond via a 1,2-addition reaction. In some cases, Michael acceptor groups include electron-deficient alkenes or alkynes. In some instances, Michael acceptor groups include, but are not limited to, alpha, beta unsaturated: ketones, aldehydes, sulfoxides, sulfones, nitriles, imines, or aromatics.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[116] In some cases, the non-natural amino acid includes the non-natural amino acid shown in FIG. 1.

[117] In some cases, the non-natural amino acid includes a derivative or analogue of lysine or phenylalanine. In some instances, the non-natural amino acid includes a lysine derivative or lysine analogue. In some cases, the non-natural amino acid includes pyrrolysine (Pyl). In some instances, the non-natural amino acid includes a phenylalanine derivative or phenylalanine analogue. In some cases, the non-natural amino acid is the non-natural amino acid described in Wan, et al., "Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool," Biocheim Biophys Aceta 1844(6): 1059-4070 (2014) . In some instances, the non-natural amino acid comprises the non-natural amino acid shown in FIG. 2 (for example, Fig. 2A and Fig. 2B).

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

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

Conjugating fragments

[120] In certain embodiments, disclosed herein are conjugating fragments that are associated with one or more of the cytokines (eg, interleukins, IFN, or TNF) described above. In some cases, the conjugating moiety is a molecule that interferes with the interaction of a cytokine with its receptor. In some cases, the conjugating moiety is any molecule that, by binding to a cytokine, allows the cytokine conjugate to modulate an immune response. In some cases, the conjugating fragment contains a water-soluble polymer. In other cases, the conjugating fragment contains a protein or a binding fragment. In additional cases, the conjugating fragment contains a peptide. In additional cases, the conjugating fragment contains a nucleic acid. In additional cases, the conjugating fragment contains a small molecule. In some cases, the conjugating fragment increases the serum half-life and/or improves stability. In some cases, the conjugating fragment reduces the interaction of cytokines with one or more receptor domains or cytokine subunits. In additional cases, the conjugating fragment blocks the cytokine interaction of one or more cytokine domains or subunits with its cognate receptor(s). In some embodiments, the cytokine conjugates described herein comprise a plurality of conjugating moieties. In some embodiments, the conjugating moiety is attached to a non-natural or naturally occurring amino acid in a cytokine peptide. In some embodiments, the cytokine conjugate comprises a conjugating moiety attached to a natural amino acid. In some embodiments, the cytokine conjugate is attached to a non-natural amino acid in the cytokine peptide. In some embodiments, the conjugating moiety is attached to the N- or C-terminal amino acid of the cytokine peptide. Various combination sites are disclosed herein, for example, a first conjugation moiety is attached to a non-natural or naturally occurring amino acid in a cytokine peptide, and a second conjugation moiety is attached to the N or C-terminal amino acid of the cytokine peptide. In some embodiments, one conjugating fragment is attached to a plurality of cytokine peptide residues (eg, brackets). In some embodiments, the conjugating moiety is attached to both the N- and C-terminal amino acids of the cytokine peptide.

Water soluble polymers

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

[122] In some cases, the water-soluble polymer is characterized by the presence of from about 2 to about 300 ends. Exemplary water-soluble polymers include, without limitation, poly(alkylene glycols) such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), ethylene glycol-propylene glycol copolymers, and the like, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), polysaccharides, poly(α-hydroxy acid), polyvinyl alcohol (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl )methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazolines ("POZ") (which are described in WO 2008/106186), poly(N-acryloylmorpholine), and combinations of any of the above.

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

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

[125] Exemplary weight average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 daltons, approx. 1000 daltons, approx. 1500 daltons, approx. 2000 daltons, approx. 2200 daltons, approx. 2500 daltons, approx. , about 7000 daltons, about 7500 daltons, about 8000 daltons, about 9000 daltons, about 10000 daltons, about 11000 daltons, about 12000 daltons, about 13000 daltons, about 14000 daltons, about 15000 daltons, about 20000 daltons, about 22500 daltons 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons and about 75,000 dalton. Branched versions of the water-soluble polymer (eg, a 40,000 dalton branched water-soluble polymer consisting of two 20,000 dalton molecular weight polymers) having a total molecular weight of any of the above factors can also be used. In one or more embodiments, the conjugate will not have any PEG moieties associated, directly or indirectly, with a PEG having a weight average molecular weight of less than about 6000 daltons.

[126] PEGs typically contain a range of (OCH ₂ CH ₂ ) monomers [or (CH ₂ CH ₂ O) monomers, depending on how PEG is defined]. As used herein, the number of repeating blocks is identified by the subscript "n" in "(OCH ₂ CH ₂ ) _n ". Thus, the value of (n) typically falls within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730; and from about 1200 to about 1900. For any given polymer whose molecular weight is known, the number of repeat units (i.e. "n") can be determined by dividing the total weight average molecular weight of the polymer by the molecular weight of the repeating monomer.

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

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

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

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

[131] In some cases, the water-soluble polymer contains polyglycerol (PG). In some cases, the polyglycerol is a hyperbranched PG (HPG) (eg, as described in knran, et al. "Influence of architecture of high molecular weight linear and branched polyglycerols on their biocompatibility and biodistribution," Biomaterials 33:9135 9147 (2012)) . In other cases, the polyglycerol is a linear PG (LPG). In additional instances, polyglycerol is a medium-functional 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 functional side chain 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).

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

[133] In some embodiments, the water-soluble polymer is a degradable synthetic alternative to PEG. Illustrative degradable synthetic alternatives to PEG include, without limitation, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone-modified PEG derivatives obtained by polymerization of telechelic or di-terminal PEG-based functionalized macromonomers; PEG derivatives containing comonomers containing a degradable bond, such as poly[(ethyleneoxy)-co-(methylene-ethylene oxide)] [P(EO-co-MEO)], cyclic ketene acetals, such as 5,6-benzo-2-methylene -1,3-dioxepane (BMDO), 2-methylene-1,3-dioxepane (MDO) and 2-methylene-4-phenyl-1,3-dioxolane (MPDL) copolymerized with OEGMA; or poly-(ε-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

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

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

[136] In some embodiments, the water-soluble polymer comprises a polycarbonate. Illustrative polycarbonates include, without limitation, pentafluorophenyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC ₆ F ₅ ). In some cases, a cytokine polypeptide (eg, interleukin, IFN, or TNF) described herein is conjugated to a polycarbonate, such as MTC-OC ₆ F ₅ . In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a polycarbonate such as MTC-OC ₆ F ₅ . [137] In some embodiments, the water-soluble polymer includes a polymer hybrid, such as, for example, a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., hydroxyl-containing and/or zwitter -ionic derivatized PEG polymer). In some instances, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a polymer fusion, such as a polycarbonate/PEG polymer fusion, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., hydroxyl-containing and /or zwitterionic derivatized PEG polymer). In some cases, the cytokine is an IL-2 polypeptide. In some instances, the IL-2 polypeptide is conjugated to a polymer hybrid, such as a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and/or zwitterionic derivatized PEG polymer).

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

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

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

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

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

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

Squirrels

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

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

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

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

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

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

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

[151] In some embodiments, the conjugating moiety is transthyretin (TTR). Transthyretin is a transport protein located in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine (T4) and retinol-related retinol-binding protein.

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

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

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

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

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

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

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

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

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

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

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

Peptides

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

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

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

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

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

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

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

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

Additional conjugating fragments

[171] In some cases, the conjugating fragment contains an extracellular biomarker. In some cases, the extracellular biomarker is a tumor antigen. In some instances, an exemplary extracellular biomarker comprises CD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc 16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8 and B220. In some instances, the conjugating moiety is linked to or conjugated to a cytokine (eg, IL-2). In some cases, the conjugating fragment is genetically fused, for example, at the N-terminus or C-terminus of a cytokine (eg, IL-2).

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

Conjugation

Linkers

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

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

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

[176] 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.

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

[178] In some embodiments, the linker includes a self-cleaving linker moiety. In some embodiments, the self-cleaving linker moiety includes p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker includes a dipeptide linker moiety and a self-cleaving linker moiety. In some embodiments, the self-cleaving linker fragment is as described in US Pat. No. 9,089,614 and WIPO Application No. WO201503 8426.

[179] 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.

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

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

Chemistry of conjugation

[182] Various conjugation reactions are used to conjugate linkers, conjugating fragments, and non-natural amino acids included in the cytokine peptides described herein. Such conjugation reactions are often compatible with aqueous conditions such as "biorthogonal" reactions. In some embodiments, the conjugation reactions are mediated by chemicals such as catalysts, light, or reactive chemical groups found on linkers, conjugating moieties, or unnatural amino acids. In some embodiments, the conjugation reactions are mediated by enzymes. In some embodiments, the conjugation reaction described herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, the conjugation reaction described herein is described in Chen, X.; wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417. [183] According to some embodiments described herein, the conjugation reaction comprises the reaction of a ketone or aldehyde with a nucleophile. In some embodiments, the conjugation reaction comprises reacting a ketone with an amino group to form an oxime. In some embodiments, the conjugation reaction comprises reacting a ketone with an aryl or heteroarylamino group to form an imine. In some embodiments, the conjugation reaction comprises reacting an aldehyde with an aryl or heteroarylamino group to form an imine. In some embodiments, the conjugation reaction described herein results in a cytokine peptide containing a linker or conjugating moiety attached via an oxime. In some embodiments, the conjugation reaction comprises a Pictet-Spengler reaction of an aldehyde or ketone with a tryptamine nucleophile. In some embodiments, the conjugation reaction includes a hydrazino-Pictet-Spengler reaction. In some embodiments, the conjugation reaction includes a Pictet-Spengler ligation.

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

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

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

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

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

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

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

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

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

Methods of application

Autoimmune disease or disorder

[193] In some embodiments, this document also describes a method of treating an autoimmune disease or disorder in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) herein. In some instances, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment, wherein the IL-2 conjugate has reduced affinity for the IL-2 receptor β subunit (IL-2Rp), a subunit at the IL-2 receptor (IL-2Ry ) or combinations thereof relative to the wild-type IL-2 polypeptide. In some cases, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment that binds to the isolated and purified IL-2 polypeptide at an amino acid residue selected from P2, T3, S4, S5, S6, T7, K8, K9 , Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55 , Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, R82, R83, D84, S87, N88, N89, V91, 192, L94, E95, K97, G98, S99, E100 , T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and amino acid numbering residues corresponds to SEQ ID NO 1. In some cases, the amino acid residue is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, 192, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132 and T133. In some cases, the amino acid residue is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95 and Q126. In some cases, the IL-2 conjugate will interact with the IL-2Rαβγ complex but with reduced affinity for the IL-2Rβ and IL-2Rγ subunits, or will reduce the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex. In some instances, the modified IL-2 polypeptide retains binding affinity for IL-2Rα relative to the wild-type IL-2 polypeptide. In such cases, the IL-2/IL-2Rαβγ complex stimulates or enhances the expansion of CD4+ Treg cells. In additional cases, the modified IL-2 polypeptide increases the dose required to activate Teff and/or NK cells via the IL-2Rβγ complex, thereby expanding the dose ranges for activating Treg cells via the IL-2Rαβγ complex (or expanding the therapeutic window of IL-2 for activation of Treg cells through the IL-2Rαβγ complex).

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

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

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

[197] In some instances, a cytokine conjugate (eg, an IL-2 conjugate) is administered to a subject to treat graft-versus-host disease (GVHD).

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

[199] Exemplary additional therapeutic agents for the treatment of an autoimmune disease or disorder include, without limitation, corticosteroids such as prednisone, budesonide, or prednisolone; calcineurin inhibitors such as cyclosporine or tacrolimus; mTOR inhibitors such as sirolimus or everolimus; IMDH inhibitors such as azathioprine, leflunomide or mycophenolate; biologics such as abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ikekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, or vedolizumab; and monoclonal antibodies such as basiliximab, daclizumab, or muromonab.

[200] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is administered with an additional therapeutic agent selected from corticosteroids such as prednisone, budesonide, or prednisolone; calcineurin inhibitors such as cyclosporine or tacrolimus; mTOR inhibitors such as sirolimus or everolimus; IMDH inhibitors such as azathioprine, leflunomide or mycophenolate; biologics such as abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ikekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, or vedolizumab; and monoclonal antibodies such as basiliximab, daclizumab or muromonab.

Cell population expansion methods

[201] In some embodiments, methods for expanding a population of Treg cells are further described herein. In some instances, the method involves contacting a cell with a cytokine conjugate as described herein and interacting the cytokine with a cytokine receptor to form a complex, the complex stimulating the expansion of a distinct population of lymphocytes.

[202] In some embodiments, a method for expanding a population of CD4+ regulatory T cells (Treg) involves contacting the cell with the isolated and modified IL-2 polypeptide described above for a time period sufficient to induce complex formation with IL-2Rαβγ, thereby stimulating population expansion. Treg cells. In some embodiments, a method for expanding a population of CD4+ Treg cells comprises (a) contacting the cell with an IL-2 conjugate as described herein, and (b) reacting the IL-2 conjugate with IL-2Rα, IL-2Rβ, and IL-2Rγ subunits to form IL-2/IL-2Rαβγ complex; moreover, the IL-2 conjugate is characterized by reduced affinity for IL-2Rβ and IL-2Rγ subunits or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, and moreover, the IL-2/IL-2Rαβγ complex stimulates the expansion of Treg cells. In some cases, the IL-2 conjugate contains an isolated and purified IL-2 polypeptide and a conjugating fragment that binds to the isolated and purified IL-2 polypeptide at an amino acid residue selected from P2, T3, S4, S5, S6, T7, K8, K9 , Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55 , Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, R82, R83, D84, S87, N88, N89, V91, 192, L94, E95, K97, G98, S99, E100 , T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132 and T133, and the numbering of amino acid residues corresponds to SEQ ID NO: 1. In some cases, the amino acid residue is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91 , 192, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132 and T133. In some cases, the amino acid residue is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57 , E60, E67, N71, Q74, S75, K76, N77, F78, H79, R82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109 , E110, T111, A112 and T113. In some instances, the amino acid position is selected from K8, K9, L12, E15, HI6, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126. In some cases, the amino acid position is selected from K8, K9 and H16. In some cases, the amino acid position is selected from Q22, N26, N88 and Q126. In some cases, the amino acid position is selected from E15, D20, D84 and E95. In some cases, the amino acid position is selected from L12, L19, M23 and F78. In some cases, the amino acid position is selected from Q22 and N26.

[203] In some cases, the IL-2 conjugate leads to the expansion of CD4+ T regulatory cells (Treg) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95%, 99% or more. In some cases, the IL-2 conjugate leads to the expansion of CD4+ T regulatory cells (Treg) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99 % or more.

[204] In some cases, the IL-2 conjugate leads to the expansion of CD4+ T regulatory cells (Treg), constituting at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x , 100x, 200x, 300x, 400x, 500x, 1000x or more. In some cases, the IL-2 conjugate leads to the expansion of CD4+ T regulatory cells (Treg), which is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold. multiple, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 1000x or more.

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

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

[207] In other embodiments, the method is an in vitro method.

[208] According to additional embodiments, the method is an ex vivo method.

Production of cytokine polypeptides

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

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

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

[212] 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.

[213] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H and X-33 and Saccharomyces cerevisiae yeast strain such as FNVSc1.

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

[215] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Machl™, DH10B™, TORYU, DH5a, DHlOBac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™ or Stbl4™.

[216] In some instances, suitable polynucleic acid molecules or vectors for the production of an IL-2 polypeptide described herein include any suitable vectors derived from a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors from bacteria (eg, E. coli), insects, yeasts (eg, Pichia pastoris), algae, or mammals. Bacterial vectors include, for example, pACYC177 vector series, pASK75, pBAD, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A , pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-l, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC or pTAC-MAT-2.

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

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

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

[220] 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-MATl, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Exemplary mammalian stable expression vectors include includes pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, P3xFLAG-CMV 10, P3xFLAG-CMV 14, pFLAG-Myc- CMV 22, P3xFLAG-Myc-CMV 26, pBICEP-CMV 1 or pBICEP-CMV 2.

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

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

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

Obtaining a cytokine polypeptide containing a non-natural amino acid

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

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

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

[227] In some cases, the codon used in the present disclosure is a recoded codon, such as a synonymous codon or a rare codon that has been replaced by an alternative codon. In some cases, the transcoded codon is as described in Napolitano, et al, "Emergent rules for codon choice elucidated by editing rare argine codons in Escherichia coli," PNAS, 113(38): E5588-5597 (2016). In some cases, the recoded codon is as described in Ostrov et al., "Design, synthesis, and testing towards a 57-codon genome," Science 353(6301): 819-822 (2016).

[228] In some cases, non-natural nucleic acids are used, leading to the incorporation of one or more non-natural amino acids into the cytokine (eg, IL-2). Illustrative non-natural nucleic acids include, without limitation, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-mc-C), 5-hydroxymethylcytosine, xanthine , hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenin. Certain non-natural nucleic acids such as 5-substituted pyrimidines, 6-azapyrimidines and N-2-substituted purines, N-6-substituted purines, O-6-substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5 -methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, dissimilar nucleic acids, oversized nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl adenine and guanine derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C=C-CH ₃ ) uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleins oic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and others 8 -substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-P-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine(1Н -pyrimido[5,4]-b][1,4]benzothiazin-2(3H)-one), G-fixers, fenoxazinecytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b ][1,4]benzoxazin-2(3H)-one), carbazolcytidine(2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine(H-pyrido[3',2':4.5 ]pyrrolo[2,3-d]pyrimidin-2-one), in which the purine or pyrimidine base is replaced by other heterocycles, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclots itosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, ioduracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-ioduracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2 '-deoxyadenosine and those described in US Pat. No. 3,687,808; 4845205; 4910300; 4948882; 5093232; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121; 5596091; 5614617; 5645985; 5681941; 5750692; 5763588; 5830653 and 6005096; W099/62923; Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613 and Sanghvi, Chapter 15, Antisense Research and Applications, Crookeand Lebleu Eds., CRC Press, 1993, 273-288 USA No. 3687808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613 and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993.

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

[230] In some embodiments, the nucleotide analogs are also modified in the phosphate moiety. Modified phosphate moieties include, without limitation, those that have a modification at the bond between two nucleotides and contain, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl, and other alkylphosphonates, including 3'-alkylenephosphonate and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates. It is understood that these phosphate or modified phosphate bonds between two nucleotides pass through a 3'-5' bond or a 2'-5' bond, and this bond contains a reversed polarity such as from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Numerous US patents describe how to make and use modified phosphate containing nucleotides and include, without limitation, 3,687,808; 4469863; 4476301; 5023243; 5177196; 5188897; 5264423; 5276019; 5278302; 5286717; 5321131; 5399676; 5405939; 5453496; 5455233; 5466677; 5476925; 5519126; 5536821; 5541306; 5550111; 5563253; 5571799; 5587361 and 5625050.

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

[232] In some embodiments, non-natural nucleic acids include modifications at the 5'-position and 2'-position of the sugar ring (PCT/US94/02993), such as 5'-CH ₂ -substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some cases, non-natural nucleic acids include amido-linked nucleoside dimers prepared for inclusion in oligonucleotides, where the 3' linked nucleoside in the dimer (from 5' to 3') contains 2'-OCH ₃ and 5'-(S)-CH ₃ (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids may include 2'-substituted 5'-CH ₂ (or O) modified nucleosides (PCT/US92/01020). Non-natural nucleic acids may include 5'-methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703- 705 and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids may include 5'-phosphonate monomers containing a 2'-substitution (US2006/0074035) and other modified 5'-phosphonate monomers (WO1997/35869). Non-natural nucleic acids may include 5'-modified methylene phosphonate monomers (EP 614907 and EP 629633). Non-natural nucleic acids may include analogs of 5' or 6'-phosphonate ribonucleosides containing a hydroxyl group at the 5' and/or 6' position (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933 and Hampton et al., J. Med. Chem. ., 1976, 19(8), 1029-1033). Non-natural nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers containing a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids may include nucleosides containing a b'-phosphonate group in which the 5' or/and 6' position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH ₃ ) ₃ ) (and its analogs); methyleneamino group (CH ₂ NH ₂ ) (and its analogs) or cyano group (CN) (and its analogs) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640 and Hampton et al., J. Am Chem Soc, 1973, 95(13), 4404-4414).

[233] In some embodiments, non-natural nucleic acids also include sugar moiety modifications. In some cases, nucleic acids contain one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer increased nuclease stability, increased binding affinity, or some other beneficial biological property. In some embodiments, the nucleic acids comprise a chemically modified ribofuranose ring fragment. Examples of chemically modified ribofuranose rings include, but are not limited to, addition of substituent groups (including 5' and/or 2' substituent groups, bridge of two ring atoms to form bicyclic nucleic acids (BNA), replacement of the oxygen atom of the ribosyl ring with S, N(R) or C(R ₁ )(R ₂ ) (R=H, C ₁ -C ₁₂ alkyl or protecting group) and combinations thereof Examples of chemically modified Sugars can be found in WO2008/101157, US2005/0130923 and WO2007/ 134181.

[234] In some cases, the modified nucleic acid contains modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar group may be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or an "analogue" of the cyclopentyl sugar group. The sugar may be in the pyranosyl or furanosyl form. The sugar moiety may be a ribose, deoxyribose, arabinose, or 2'-O-alkylribose furanoside, and the sugar may be attached to the appropriate heterocyclic bases in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, 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, the sugar modification may include 2'-O-methyluridine or 2'-O-methylcytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs and the corresponding "nucleosides" in which such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known. Sugar modifications can also be made and combined with other modifications.

[235] Sugar moiety modifications include natural modifications of ribose and deoxyribose, as well as non-natural modifications. Sugar modifications include, without limitation, the following modifications at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl and alkynyl. 2' sugar modifications also include, without limitation, -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n ONH ₂ and -O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ ] ₂ , where n and m are from 1 to about 10.

[236] Other modifications at the 2' position include, without limitation: C ₁ -C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH ₃ , OCN, Cl, Br , CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocyclo o alkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, an intercalator, a group for improving the pharmacokinetic properties of the oligonucleotide, or a group for improving the pharmacodynamic properties of the oligonucleotide and other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, in particular at the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5'-position of the 5'-terminal nucleotide. Modified sugars also include those containing modifications at the bridging ring oxygen, such as CH ₂ and S. Nucleotide sugar analogs can also include sugar mimetics, such as cyclobutyl moieties, instead of pentofuranosyl sugar. There are numerous United States patents that describe the preparation of such modified sugar structures and detail a number of base modifications, such as US Pat. Nos. 4,981,957; 5118800; 5319080; 5359044; 5393878; 5446137; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; 4845205; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121, 5596091; 5614617; 5,681,941 and 5,700,920, each of which is incorporated herein by reference in its entirety.

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

[238] In certain embodiments, the nucleic acids described herein include one or more bicyclic nucleic acids. In some such embodiments, the bicyclic nucleic acid contains a bridge between the 4' and 2' atoms of the ribosyl ring. In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, wherein the bridge contains a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, without limitation, one of the formulas: 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4'-(CH ₂ )2-O-2'(ENA);4'-CH(CH ₃ )-O-2' and 4'-CH(CH ₂ OCH ₃ )-O-2' and their analogs (see US patent No. 7399845); 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see WO2009/006478, WO2008/150729, US2004/0171570, US Patent No. 7427672, Chattopadhyaya et al., J. Org. Chem. , 209, 74, 118-134 and WO2008/154401). See also, for example: Singh et al., Chem. Commun, 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc, 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; US Pat. No. 4,849,513; 5015733; 5118800; 5118802; 7053207; 6268490; 6770748; 6794499; 7034133; 6525191; 6670461 and 7399845; international publications No. WO2004/106356, WO1994/14226, WO2005/021570, WO2007/090071 and WO2007/134181; US Patent Nos. US2004/0171570, US2007/0287831 and US2008/0039618; U.S. Provisional Applications Nos. 60/989574, 61/026995, 61/026998, 61/056564, 61/086231, 61/097787 and 61/099844 and International Applications Nos. PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922 and PCT/DK98/00393.

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

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

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

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

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

[244] Phosphate substituents include, for example, short chain alkyl or cycloalkyl internucleoside bonds, mixed heteroatomic and alkyl or cycloalkyl internucleoside bonds, or one or more short chain heteroatomic or heterocyclic internucleoside bonds. These include those containing morpholino bonds (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; foracetal and thioformacetal backbones; methyleneformacetyl and thioformacetal backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino main chains; sulfonate and sulfonamide backbones; amide backbones and others containing mixed N, O, S and CH ₂ constituents. Numerous US patents disclose how to make and use these types of phosphate substitutes and include, without limitation, US Pat. Nos. 5,034,506; 5166315; 5185444; 5214134; 5216141; 5235033; 5264562; 5264564; 5405938; 5434257; 5466677; 5470967; 5489677; 5541307; 5561225; 5596086; 5602240; 5610289; 5602240; 5608046; 5610289; 5618704; 5623070; 5663312; 5633360; 5,677,437 and 5,677,439. It is also understood that when a nucleotide is replaced, both the sugar and phosphate portions of the nucleotide can be replaced, for example by an amide type (aminoethylglycine) (PNA) bond. US Patents No. 5539082; 5714331 and 5719262 describe how to create and use PNA molecules, each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates may be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as cholesterol group (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), bile acid (Manoharan et al., Bioorg Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g. et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chain, e.g. dodecanediol or undecyl residues (Saison-Behmoaras et al., EM50J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49 -54), a phospholipid, e.g. dihexadecyl-rac-glycerol or triethylammonium-1-di-O-hexadecyl-rac-glycero-8H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237) or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous US patents describe the preparation of such conjugates and include, without limitation, US Pat. Nos. 4,828,979; 4948882; 5218105; 5525465; 5541313; 5545730; 5552538; 5578717, 5580731; 5580731; 5591584; 5109124; 5118802; 5138045; 5414077; 5486603; 5512439; 5578718; 5608046; 4587044; 4605735; 4667025; 4762779; 4789737; 4824941; 4835263; 4876335; 4904582; 4958013; 5082830; 5112963; 5214136; 5082830; 5112963; 5214136; 5245022; 5254469; 5258506; 5262536; 5272250; 5292873; 5317098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5510475; 5512667; 5514785; 5565552; 5567810; 5574142; 5585481; 5587371; 5595726; 5597696; 5599923; 5599928 and 5688941.

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

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

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

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

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

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

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

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

[253] Suitable host cells may include bacterial cells (eg, E. coli), but most suitable host cells are eukaryotic cells, for example, insect cells (eg, Drosophila, such as Drosophila melanogaster), yeast cells, nematodes (e.g., Celegans), mice (e.g., Mus musculus), or mammalian cells (such as Chinese Hamster Ovary (CHO) cells or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells) or human or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Accordingly, the host cell is a mammalian cell, such as a human cell or an insect cell.

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

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

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

Pharmaceutical compositions and formulations

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

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

[259] In some embodiments, the pharmaceutical formulations include a carrier or carrier materials selected based on compatibility with the composition disclosed herein and properties of the release profile of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carriers include, but are not limited to, gum, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid , phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sodium stearoyl lactylate sugars, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980 and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[260] In some cases, the pharmaceutical composition is formulated as an immunoliposome that contains a plurality of IL-2 conjugates associated directly or indirectly with the lipid bilayer of the liposomes. Illustrative lipids include, without limitation, fatty acids; phospholipids; sterols such as cholesterol; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, gloocides and cerebrosides; surfactant amines such as stearyl, oleyl and linoleyl amines. In some cases, the lipid contains a cationic lipid. In some cases, the lipid contains a phospholipid. Illustrative phospholipids include, without limitation, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phosphatidylethanolamine ("PE"), phosphatidylinositol ("P1"), and phosphatidylserine ("PS" ), sphingomyelin (including brain sphingomyelin), lecithin, l iso lecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-b-alpha-phosphatidylcholine ("DDPC"), dieladoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleoylphosphatidylcholine ("DOPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-palmitoyl-2-oleoylphosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol ("DAPG"), didecanoyl-b-alpha-phosphatidylglycerol ("DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DMPG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoilf osphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), 1-palmitoyl-2-oleoylphosphatidylglycerol ("POPG"), diarachidoylphosphatidylethanolamine ("DAPE"), didecanoyl-b-alpha-phosphatidylethanolamine ("DDPE"), dielaidoylphosphatidylethanolamine (" DEPE"), dilauroylphosphatidylethanolamine ("DLPE"), dilinoleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine ("DMPE"), dioleoylphosphatidylethanolamine ("DOPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), distearoylphosphatidylethanolamine ("DSPE"), 1-palmitoyl-2-oleoylphosphatidylethanolamine (" POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl-b-alpha-phosphatidylinositol ("DDPI"), dialaidoylphosphatidylinositol ("DEPI"), dilauroylphosphatidylinositol ("DLPI"), dilinoleoylphosphatidylinositol, dimyristoylphosphatidylinositol ("DMPI"), dioleoylphosphatidylinositol (" DOPI"), dipalmitoylphosphatidylinositol ("DPPI"), distearoylphosphatidylinositol ("DSPI"), 1-palmitoyl-2-oleoylphosphatidylinositol ("POP1"), diarachidoylphosphatidylserine ("DAPS"), didecanoyl-b-alpha- phosphatidylserine ("DDPS"), dielaidoylphosphatidylserine ("DEPS"), dilauroylphosphatidylserine ("DLPS"), dilinoleoylphosphatidylserine, dimyristoylphosphatidylserine ("DMPS"), dioleoylphosphatidylserine ("DOPS"), dipalmitoylphosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS") , 1-пальмитоил-2-олеоилфосфатидилсерин («POPS»), диарахидоилсфингомиелин, дидеканоилсфингомиелин, диэлаидоилсфингомиелин, дилауроилсфингомиелин, дилинолеоилсфингомиелин, димиристоилсфингомиелин, сфингомиелин, диолеоилсфингомиелин, дипальмитоилсфингомиелин, дистеароилсфингомиелин и 1-пальмитоил-2-олеоилсфингомиелин.

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

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

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

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

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

[266] In some cases, pharmaceutical formulations include excipients such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol , lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

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

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

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

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

[271] Suspension agents include compounds such as polyvinylpyrrolidone, for example, polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, for example, polyethylene glycol may have a molecular weight of from about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethyl cellulose, methyl cellulose a, hydroxypropyl methyl cellulose, hydroxymethyl cellulose acetate stearate, polysorbate-80, hydroxyethyl cellulose, sodium alginate, such gums such as gum tragacanth and gum arabic, guar gum, xanthans, including xanthan gum, sugars, celluloses such as carboxymethylcellulose sodium, methylcellulose, carboxymethylcellulose sodium, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, alginate sodium, polyethoxylated sorbitan monolaurate, povid he etc. .

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

[273] Viscosity enhancers include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose, phthalate, carbomer, polyvinyl alcohol, alginates, gums, chitosans, and combinations thereof.

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

Treatment regimens

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

[276] In the case where the patient's condition does improve, at the discretion of the physician, the composition is administered continuously, alternatively, the dose of the administered composition is temporarily reduced or temporarily suspended for a certain period of time (i.e., "drug rest"). In some cases, the duration of drug rest varies from 2 days to 1 year, including by way of example only: 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days or 365 days. Dose reduction during drug rest ranges from 10% to 100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

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

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

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

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

Kits/product

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

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

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

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

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

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

Defined terms

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

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

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

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

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

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

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

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

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

EXAMPLES

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

EXAMPLE 1

[297] Kinase and cytokine receptor dimerization assays.

[298] Cell preparation

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

[300] The agoist format

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

[302] Signal Detect

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

[304] Data Analysis

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

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

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

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

EXAMPLE 2

[307] Ex vivo immune response profiling of exemplary IL-2 compounds in PBMC samples derived from the primary human leukocyte recovery system (LRS)

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

[309] Flow cytometry data was analyzed to activate different subsets of T and NK cells in concentration-response mode, reading the accumulation of pSTATS after treatment with an exemplary variant of IL-2 K9 30kD.

[310] In FIG. 4A-fig. 4B shows dose-response curves for pSTATS signaling in primary human LRS cell (FIG. 4A) and proliferative response in mouse CTLL-2 populations (FIG. 4B).

[311] Table 2 shows the dose-response EC50 for pSTAT5 signaling (EC50) in human LRS or CTLL-2 proliferation samples treated with the indicated IL-2 variant.

[312] EC50 values (pg/mL) were calculated based on dose-response curves obtained from MFI plots.

[313] * Change in Treg activity compared to native IL-2 (wild-type IL-2) in each individual experiment.

EXAMPLE 3

[314] PK study in mice with C57BL/6 tumors

[315] Experimental details are given in Table 3.

[316] The pharmacokinetic properties of exemplary pegylated IL-2 compound K35_30kD were evaluated at two dose levels. The lyophilized study article was reconstituted in PBS and nine male C57BL/6 mice were administered 0.3 and 3 mg/kg via tail vein intravenous injection for each dose group (see collection details below). Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours post-dose. Abeam's hIL-2 ELISA kit (ab100566), which does not cross-react with native mouse IL-2, was used to detect and quantify test articles. To correct for ELISA-specific differences in sensitivity between native and pegylated compounds, native IL-2 and test article K35_30kD standard curves were generated using test article dilution buffer and the data analyzed against the respective standard curves. The data presented are the mean and standard deviation for three separate samples (biological repeats) as described above, and the PK parameters for the test articles K35_30kD were extracted and summarized in Table 4

[317] FIG. 5 shows the improved PK profile of K35_30kD at two different concentrations. Their PK profiles are compared with wild-type IL-2.

EXAMPLE 4

[318] Table 5 shows the IL-2 sequences described herein.

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

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SEQUENCE LIST

<110> SYNTORX INC.

<120> CYTOKINE CONJUGATES FOR THE TREATMENT OF AUTOIMMUNE DISEASES

<130> 46085-710.602

<140>

<141>

<150> 62/540.781

<151> 2017-08-03

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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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<---

Claims (30)

1. Interleukin-2 (IL-2) conjugate for the treatment of an autoimmune disease, containing: a. a modified intelukin-2 (IL-2) polypeptide comprising at least one unnatural amino acid, wherein the modified intelukin-2 (IL-2) polypeptide comprises at least 80% sequence identity with SEQ ID NO: 1, where the position of at least one unnatural amino acid is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95 and Q126, and moreover, the positions of the residues correspond to positions 8, 9, 12, 15, 16, 19, 20, 22, 23, 26, 84, 88, 95 and 126, as presented in SEQ ID NO: 1; and b. conjugating fragment, covalently attached through a linker to at least one non-natural amino acid; moreover, the conjugating fragment is a water-soluble polymer, including polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly (hydroxyalkyl methacrylate), poly(saccharides), poly(alpha-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA), or a combination thereof . 2. An interleukin-2 (IL-2) conjugate according to claim 1, wherein the modified interleukin-2 (IL-2) polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1. 3. An interleukin-2 (IL-2) conjugate according to claim 1, wherein the position of at least one unnatural amino acid is selected from K8, K9, and H16, with residue positions corresponding to positions 8, 9, and 16, as shown in SEQ ID NO : 1. 4. An interleukin-2 (IL-2) conjugate according to claim 3, wherein at least one non-natural amino acid position is K9, with residue positions corresponding to position 9, as shown in SEQ ID NO: 1. 5. An interleukin-2 (IL-2) conjugate according to claim 3, wherein at least one non-natural amino acid position is H16, with residue positions corresponding to position 16 as shown in SEQ ID NO: 1. 6. The interleukin-2 (IL-2) conjugate of claim 1, wherein at least one non-natural amino acid is a lysine analog or contains an aromatic side chain. 7. An interleukin-2 (IL-2) conjugate according to claim 1, wherein the water-soluble polymer contains polyethylene glycol (PEG). 8. The interleukin-2 (IL-2) conjugate of claim 7, wherein the polyethylene glycol (PEG) has a weight average molecular weight of about 10 kDa to about 85 kDa. 9. The interleukin-2 (IL-2) conjugate of claim 8, wherein the polyethylene glycol (PEG) has a weight average molecular weight selected from about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa or about 50 kDa. 10. Interleukin-2 (IL-2) conjugate according to claim 1, wherein: a. the position of at least one non-natural amino acid is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95 and Q126, with the positions of the residues corresponding to positions 8, 9, 12, 15, 16, 19, 20, 22, 23, 26, 84, 88, 95 and 126 as shown in SEQ ID NO: 1; b. modified interleukin-2 (IL-2) polypeptide contains an N-terminal deletion containing a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 residues from the N-terminus, with positions residues refer to positions in SEQ ID NO: 1; c. the conjugating moiety is polyethylene glycol (PEG) having a weight average molecular weight of about 20 kDa to about 85 kDa; and d. the modified interleukin-2 (IL-2) polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1. 11. The interleukin-2 (IL-2) conjugate according to claim 10, characterized in that the modified interleukin-2 (IL-2) polypeptide contains an N-terminal deletion containing the deletion of the first 1, 2, 3, 4 or 5 residues with N-terminus, with the positions of the residues referring to those in SEQ ID NO: 1. 12. The interleukin-2 (IL-2) conjugate of claim 11, wherein the polyethylene glycol (PEG) has a weight average molecular weight selected from about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa or about 50 kDa. 13. The interleukin-2 (IL-2) conjugate according to any one of paragraphs. 1-12, in which at least one non-natural amino acid includes N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene-lysine, TCO-lysine, methyltetrazine lysine , allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodine-L-phenylalanine, m- acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acylalanine-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonothyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3 -(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid or 2-amino-3-(phenylselanyl)propanoyl. 14. Pharmaceutical composition for the treatment of an autoimmune disease, containing an effective amount of an interleukin-2 (IL-2) conjugate according to any one of paragraphs. 1-13 and a pharmaceutically acceptable excipient. 15. Pharmaceutical composition according to claim 14, characterized in that the pharmaceutical composition is formulated for systemic or parenteral delivery. 16. A method of treating an autoimmune disease or disorder in a subject in need thereof, comprising administering to the subject an interleukin-2 (IL-2) conjugate according to any one of paragraphs. 1-13 or a pharmaceutical composition according to claim 14 or 15. 17. The method of claim. 16, characterized in that the autoimmune disease or disorder includes alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes mellitus, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Vegenesis granulomatosis. 18. A method of increasing the population of CD4+ regulatory T (Treg) cells, including bringing the cell into contact with an interleukin-2 (IL-2) conjugate according to any one of paragraphs. 1-13 or a pharmaceutical composition according to claim 14 or 15 for a time sufficient to induce the formation of a complex with IL-2R

, thereby stimulating an increase in the population of Treg cells. 19. The method according to p. 18, characterized in that the method is an in vivo method. 20. The method according to p. 18, characterized in that the method is an in vitro method. 21. The method according to p. 18, characterized in that the method is an ex vivo method.

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