Pegylated il-2 for suppressing adaptive immune response to gene therapy

Abstract

The disclosure relates to methods of delivering a gene therapy agent to a subject, treating an individual in need of a gene therapy agent, increasing expression of a gene therapy agent, reducing an immune response to a gene therapy agent, and preventing immune-related adverse events in a subject by administering a gene therapy agent and an IL-2 conjugate to the subject. The IL-2 conjugate has at least one amino acid residue replaced by an unnatural amino acid linked to a conjugating moiety. The disclosure further relates to methods of selecting a subject for treatment with a gene therapy agent and an IL-2 conjugate.

Description

Attorney Docket No. 01183-0317-00PCT PEGYLATED IL-2 FOR SUPPRESSING ADAPTIVE IMMUNE RESPONSE TO GENE THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 625,113, filed January 25, 2024, and US Provisional Patent Application No. 63 / 641,189, filed May 1, 2024, which are each incorporated by reference herein in its entirety for all purposes. FIELD OF THE DISCLOSURE

[0002] This disclosure provides methods of delivering a gene therapy agent to a cell of a subject, methods of treating an individual in need thereof with a gene therapy agent, methods of increasing expression of a gene therapy agent, methods of reducing an immune response to a gene therapy agent, and methods of preventing immune-related adverse events in a subject in which an IL-2 conjugate is administered. In some embodiments, the IL-2 conjugate expands Treg cells, reduces an immune response to the gene therapy agent, facilitates increased expression of the gene therapy agent, and / or prevents immune-related adverse events in a subject receiving the gene therapy agent. SEQUENCE LISTING

[0003] The present application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “01183-0317-00PCT.xml” created on January 22, 2025, which is 40,250 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. INTRODUCTION AND SUMMARY

[0004] One of the current challenges with adeno-associated virus (AAV)-based and lipid nanoparticle (LNP)-based gene therapy is the host immune responses, resulting in immune- related adverse events (irAEs) and reduced therapeutic efficacy. Adaptive immune responses develop cytotoxic T-cell responses after exposure to AAV capsid and its transgene, and transduced cells get destroyed by the activated cytotoxic T cells (Ertl, 2022), resulting in the irAEs and loss of transgene expression. Similarly, expression of LNP-delivered transgene (ceDNA or RNA) can elicit cytotoxic T-cell responses to the therapeutic protein, leading to death of transduced cells and related toxicities. Regulatory T cells play crucial roles in regulating activation of immune cells including cytotoxic T cells. Therefore, it was hypothesized that byAttorney Docket No. 01183-0317-00PCT selectively and transiently expanding regulatory T cells, cytotoxic T-cell activation by the AAV and LNP can be hampered, leading to better safety and transgene expression. IL-2 is a crucial cytokine for regulatory T cells to proliferate. However, it is challenging to use recombinant human IL-2 (rhIL-2) to expand regulatory T cells in vivo as other lymphocytes, such as cytotoxic T cells, also express IL-2 receptors (IL-2Rs). Although regulatory T cells bind to IL-2 more strongly compared to other lymphocytes due to their constitutive expression of IL-2R alpha (Hernandez et al., 2022), its therapeutic window is very narrow to achieve selective expansion of regulatory T cells. In addition, rhIL-2 has shown a very poor half-life in vivo (<90 min.), which requires multiple intravenous (IV) infusions for a therapeutic purpose (see, e.g., www.accessdata.fda.gov / drugsatfda_docs / label / 2012 / 103293s5130lbl.pdf). Accordingly, there is a need for improved methods of delivering a gene therapy agent to a cell of a subject, methods of treating an individual in need thereof with a gene therapy agent, methods of increasing expression of a gene therapy agent, methods of reducing an immune response to a gene therapy agent, and methods of preventing immune-related adverse events in a subject. The present disclosure aims to meet one or more of these needs, provide other benefits, or at least provide the public with a useful choice.

[0005] The present inventors explored use of an IL-2 conjugate comprising a conjugating moiety in the above methods. IL-2 conjugates as described herein can prevent or reduce its binding to IL-2R beta, and can result in selective expansion of regulatory T cells in that the IL-2 conjugate can have reduced binding to other lymphocytes, such as cytotoxic T cells (i.e., cytotoxic T lymphocytes (CTL)). In addition, IL-2 conjugates as described herein can exhibit a longer serum half-life compared to IL-2, which allows routes of administration other than IV infusion, and can lead to better patient compliance. There has been a mouse study applying regulatory T cells to AAV gene therapy to modulate immune responses (Arjomandnejad M, Sylvia K, Blackwood M, Nixon T, Tang Q, Muhuri M, Gruntman AM, Gao G, Flotte TR, Keeler AM. Mol Ther Methods Clin Dev. 2021 Oct 28;23:490-506.), but its application of regulatory T cells is in a manner of adoptive cell transfer, which increases complexity and regulatory issues relative to methods described herein, which do not require adoptive cell transfer.

[0006] The present inventors have selectively expanded regulatory T cells with Compound A as a pre-treatment or a combination treatment while administering AAV-based or LNP-based gene therapies to mitigate irAEs and enhance transgene expression in various animal models, such as mice, rats, and non-human primates.

[0007] The following embodiments are encompassed. Embodiment 1 is a method of delivering a gene therapy agent to a cell of a subject, comprising administering an IL-2 conjugate to theAttorney Docket No. 01183-0317-00PCT subject, wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate, wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0008] Embodiment 2 is a method of treating an individual in need thereof with a gene therapy agent, comprising administering an IL-2 conjugate to the subject, wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate, and the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0009] Embodiment 3 is a method of increasing expression of a gene therapy agent, comprising: administering an IL-2 conjugate to a subject; wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate; and wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as toAttorney Docket No. 01183-0317-00PCT preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0010] Embodiment 4 is a method of reducing an immune response to a gene therapy agent, comprising: administering an IL-2 conjugate to a subject; wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate; and wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0011] Embodiment 5 is a method of preventing immune-related adverse events in a subject, comprising: administering an IL-2 conjugate to a subject; wherein a gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate; and wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25,Attorney Docket No. 01183-0317-00PCT G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0012] Embodiment 6 is the method of any one of the preceding embodiments, wherein the method further comprises, before the administration of the gene therapy agent and the IL-2 conjugate to the subject, a) incubating immune cells from the subject with the gene therapy agent and b) analyzing the immune cells for the expression of one or more activation biomarkers or increased expression of one or more activation biomarkers, wherein expression or increased expression of the one or more activation biomarkers following incubation with the gene therapy agent identifies the subject as being in need of the IL-2 conjugate.

[0013] Embodiment 7 is a method for selecting a subject for treatment with a gene therapy agent and an IL-2 conjugate, the method comprising a) incubating immune cells from the subject with the gene therapy agent, b) analyzing the immune cells for the expression of one or more activation biomarkers or increased expression of one or more activation biomarkers, wherein expression or increased expression of the one or more activation biomarkers following incubation with the gene therapy agent identifies the subject for treatment with the gene therapy agent and the IL-2 conjugate, and c) selecting the subject identified in step b) for treatment with the gene therapy agent and the IL-2 conjugate; wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0014] Embodiment 8 is method of the immediately preceding embodiment, further comprising steps of administering the IL-2 conjugate to the subject identified in step b), and administering the gene therapy agent to the subject identified in step b).Attorney Docket No. 01183-0317-00PCT

[0015] Embodiment 9 is the method of any one of embodiments 6-8, wherein the immune cell is a lymphocyte, a T cell, a CD8+ T cell, an effector T cell, a cytotoxic T cell, or an NK cell.

[0016] Embodiment 10 is the method of any one of the preceding embodiments, wherein the unnatural amino acid is linked to the conjugating moiety through a linker.

[0017] Embodiment 11 is the method of the immediately preceding embodiment, wherein the linker comprises a homobifunctional linker, a heterobifunctional linker, a cleavable or a non- cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.

[0018] Embodiment 12 is the method of any one of the preceding embodiments, wherein the unnatural amino acid is a substituted lysine, is a substituted phenylalanine, is a substituted histidine, is a substituted cysteine, comprises an azido group, comprises an alkyne group, comprises an aldehyde group, comprises an aromatic side chain, or comprises a ketone group.

[0019] Embodiment 13 is the method of any one of the preceding embodiments, wherein the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine, N6-((2-azidoethoxy)- carbonyl)-L-lysine, N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyllysine, p-acetyl-L-phenylalanine, p- azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, p- propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-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, phosphonotyrosine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid, or 2-amino-3-(phenylselanyl)propanoic acid.

[0020] Embodiment 14 is the method of any one of the preceding embodiments, wherein the unnatural amino acid is an azido-substituted lysine.

[0021] Embodiment 15 is the method of any one of the preceding embodiments, wherein the unnatural amino acid is N6-((2-azidoethoxy)-carbonyl)-L-lysine.

[0022] Embodiment 16 is the method of any one of the preceding embodiments, wherein the conjugating moiety comprises a water-soluble polymer.

[0023] Embodiment 17 is the method of the immediately preceding embodiment, wherein the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.Attorney Docket No. 01183-0317-00PCT

[0024] Embodiment 18 is the method of the immediately preceding embodiment, wherein the conjugating moiety comprises PEG.

[0025] Embodiment 19 is the method of the immediately preceding embodiment, wherein the conjugating moiety is PEG having a molecular weight of about 10-85 kDa or selected from about 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, 60kDa, 65kDa, 70kDa, 75 kDa, 80kDa, and 85 kDa.

[0026] Embodiment 20 is the method of any one of the preceding embodiments, wherein the conjugating moiety is PEG having a molecular weight of about 20-70 kDa or selected from about 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, 60kDa, 65kDa, and 70kDa.

[0027] Embodiment 21 is the method of any one of the preceding embodiments, wherein the conjugating moiety is PEG having a molecular weight of about 30-60 kDa or selected from about 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, and 60kDa.

[0028] Embodiment 22 is the method of any one of the preceding embodiments, wherein the amino acid linked to the conjugating moiety has the structure of Formula (I):W is a PEG group; and X has the structure:Attorney Docket No. 01183-0317-00PCTX-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.

[0029] Embodiment 23 is the method of the immediately preceding embodiment, wherein Z is.

[0030] Embodiment 24 is the method of the immediately preceding embodiment, wherein the structure of Formula (I) has the structure of Formula (IV) or Formula (V):Formula (V); wherein: W is a PEG group having a molecular weight of about 5-60 kDa or about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa.

[0031] Embodiment 25 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate further comprises an alanine or methionine N-terminal to the first amino acid of the sequence having at least 80% sequence identity to SEQ ID NO: 1.Attorney Docket No. 01183-0317-00PCT

[0032] Embodiment 26 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.

[0033] Embodiment 27 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate comprises the amino acid sequence of SEQ ID NO: 1 in which position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 is replaced with the unnatural amino acid.

[0034] Embodiment 28 is the method of any one of the preceding embodiments, wherein position K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108 in reference to the sequence of SEQ ID NO: 1 is replaced with the unnatural amino acid.

[0035] Embodiment 29 is the method of the immediately preceding embodiment, wherein position L18 in reference to the sequence of SEQ ID NO: 1 is replaced with the unnatural amino acid.

[0036] Embodiment 30 is the method of embodiment 28, wherein position H15 in reference to the sequence of SEQ ID NO: 1 is replaced with the unnatural amino acid.

[0037] Embodiment 31 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate is capable of expanding CD4+ T regulatory (Treg) cells.

[0038] Embodiment 32 is the method of any one of the preceding embodiments, wherein the unnatural amino acid and / or the conjugating moiety impairs or blocks the receptor signaling potency of the IL-2 conjugate to IL-2Rβγ, or reduces recruitment of IL-2Rγ subunit to an IL- 2 / IL-2Rβ complex.

[0039] Embodiment 33 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate has a receptor signaling potency to IL-2Rβγ that is lower than a receptor signaling potency of wild-type IL-2 to IL-2Rβγ.

[0040] Embodiment 34 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate has a receptor signaling potency to IL-2Rαβγ that is greater than or equal to a receptor signaling potency of wild-type IL-2 to IL-2Rαβγ.

[0041] Embodiment 35 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate expands a CD4+ Treg population in the subject.Attorney Docket No. 01183-0317-00PCT

[0042] Embodiment 36 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate suppresses CD8+ T cell proliferation in the subject.

[0043] Embodiment 37 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate suppresses effector memory CD8+ T cell proliferation in the subject.

[0044] Embodiment 38 is the method of any one of the preceding embodiments, wherein the gene therapy agent comprises a vector and the IL-2 conjugate suppresses vector-specific IFNγ- secreting CD8+ T cells in the subject.

[0045] Embodiment 39 is the method of any one of the preceding embodiments, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate suppresses transgene- product-specific IFNγ-secreting CD8+ T cells in the subject.

[0046] Embodiment 40 is the method of any one of the preceding embodiments, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate suppresses production of antibodies against the transgene product.

[0047] Embodiment 41 is the method of any one of the preceding embodiments, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate suppresses production of IgG1 antibodies against the transgene product.

[0048] Embodiment 42 is the method of any one of the preceding embodiments, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate prolongs the expression of the transgene product in the subject relative to a subject that is administered the gene therapy agent and without the IL-2 conjugate.

[0049] Embodiment 43 is the method of the immediately preceding embodiment, wherein the prolonged expression of the transgene product is at least about 5 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 14 weeks, or about 16 weeks.

[0050] Embodiment 44 is the method of any one of the preceding embodiments, wherein the gene therapy agent comprises a viral vector.

[0051] Embodiment 45 is the method of embodiment 44, wherein the IL-2 conjugate suppresses production of antibodies against the viral vector.

[0052] Embodiment 46 is the method of embodiment 44 or embodiment 45, wherein the IL-2 conjugate suppresses production of antibodies against a capsid protein of the viral vector.

[0053] Embodiment 47 is the method of any one of embodiments 44-46, wherein the viral vector is an adeno-associated viral (AAV) particle.

[0054] Embodiment 48 is the method of the immediately preceding embodiment, wherein the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, anAttorney Docket No. 01183-0317-00PCT AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a mouse AAV capsid rAAV2 / HBoV1 (chimeric AAV / human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid or an AAVPHP.eB capsid, or a functional variant thereof.

[0055] Embodiment 49 is the method of the immediately preceding embodiment, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.

[0056] Embodiment 50 is the method of any one of embodiments 47-49, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs is an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR.

[0057] Embodiment 51 is the method of the immediately preceding embodiment, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0058] Embodiment 52 is the method of the immediately preceding embodiment, wherein the one or more ITRs and the capsid of the AAV particles are derived from different AAV serotypes.

[0059] Embodiment 53 is the method of any one of embodiments 44-46, wherein the viral vector is an adenoviral particle.

[0060] Embodiment 54 is the method of the immediately preceding embodiment, wherein the adenoviral particle comprises a capsid from Adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, , AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, or a functional variant thereof.

[0061] Embodiment 55 is the method of any one of embodiments 44-46, wherein the viral vector is a lentiviral particle.

[0062] Embodiment 56 is the method of the immediately preceding embodiment, wherein the lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross river virus (RRV), Ebola virus, Marburg virus, Mokala virus, Rabies virus, RD114, or a functional variant thereof.

[0063] Embodiment 57 is the method of any one of embodiments 44-46, wherein the viral vector is a Herpes simplex virus (HSV) particle.Attorney Docket No. 01183-0317-00PCT

[0064] Embodiment 58 is the method of the immediately preceding embodiment, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle, or a functional variant thereof.

[0065] Embodiment 59 is the method of any one of embodiments 1-41, wherein the gene therapy agent comprises a lipid nanoparticle.

[0066] Embodiment 60 is the method of any one of the preceding embodiments, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0067] Embodiment 61 is the method of the immediately preceding embodiment, wherein the heterologous transgene is operably linked to a promoter.

[0068] Embodiment 62 is the method of the immediately preceding embodiment, wherein the promoter is a constitutive promoter, a tissue- specific promoter, or an inducible promoter.

[0069] Embodiment 63 is the method of any one of embodiments 60-62, wherein the nucleic acid comprises closed-end DNA (ceDNA).

[0070] Embodiment 64 is the method of embodiment 60, wherein the nucleic acid comprises mRNA.

[0071] Embodiment 65 is the method of any one of the preceding embodiments, wherein the gene therapy agent is administered to the subject concurrently with the IL-2 conjugate.

[0072] Embodiment 66 is the method of any one of embodiments 1-64, wherein the gene therapy agent is administered to the subject before the IL-2 conjugate.

[0073] Embodiment 67 is the method of the immediately preceding embodiment, wherein the gene therapy agent is administered less than 14 days or less than 7 days before the IL-2 conjugate.

[0074] Embodiment 68 is the method of any one of embodiments 1-64, wherein the gene therapy agent is administered to the subject after the IL-2 conjugate.

[0075] Embodiment 69 is the method of the immediately preceding embodiment, wherein the gene therapy agent is administered less than 7 days, less than 3 days, or less than 1 day after the IL-2 conjugate.

[0076] Embodiment 70 is the method of any one of embodiments 1-64, wherein the IL-2 conjugate is administered before, at the same time, or after administration of the gene therapy agent.

[0077] Embodiment 71 is the method of any one of the preceding embodiments, wherein the individual has a disease or disorder suitable for treatment by gene therapy.

[0078] Embodiment 72 is the method of the immediately preceding embodiment, wherein the disease or disorder is a monogenic disease or disorder.Attorney Docket No. 01183-0317-00PCT

[0079] Embodiment 73 is the method of any one of the preceding embodiments, wherein the gene therapy agent is administered intravenously, intraperitoneally, intra-arterially, intramuscularly, subcutaneously, intracranially, intra-CSF, intra-DRG, intracerebroventricularly, intraocularly, intracisterna magna, or intrahepatically.

[0080] Embodiment 74 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate is administered parenterally and / or systemically.

[0081] Embodiment 75 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate is administered intravenously, intraperitoneally, intra-arterially, intramuscularly, subcutaneously, intracranially, intra-CSF, intra-DRG, intracerebroventricularly, intraocularly, intracisterna magna, or intrahepatically.

[0082] Embodiment 76 is the method of any one of the preceding embodiments, wherein the subject is a mammal.

[0083] Embodiment 77 is the method of any one of the preceding embodiments, wherein the subject is a primate.

[0084] Embodiment 78 is the method of any one of the preceding embodiments, wherein the subject is a human.

[0085] Embodiment 79 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate is administered about 1, 2, 3, 4, 5, 6, or 7 days before the gene therapy agent.

[0086] Embodiment 80 is the method of any one of embodiments 1-78, wherein the IL-2 conjugate is administered about 1, 2, 3, or 4 days after the gene therapy agent.

[0087] Embodiment 81 is the method of any one of embodiments 1-78, wherein the IL-2 conjugate is administered on the same day as the gene therapy agent.

[0088] Embodiment 82 is the method of any one of the preceding embodiments, wherein the IL- 2 conjugate is administered at a dose of about 0.02-0.5 mg / kg about 0.03-0.4 mg / kg, about 0.04- 0.1 mg / kg, or about 0.05-0.08 mg / kg.

[0089] Embodiment 83 is the method of the immediately preceding embodiment, wherein the IL-2 conjugate is administered at a dose of about 0.05 mg / kg.

[0090] Embodiment 84 is the method of embodiment 82, wherein the IL-2 conjugate is administered at a dose of about 0.08 mg / kg.

[0091] Embodiment 85 is the method of embodiment 82, wherein the IL-2 conjugate is administered at a dose of about 0.3 mg / kg.

[0092] Embodiment 86 is a use of an IL-2 conjugate for the manufacture of a medicament for use in the method of any one of the preceding embodiments.Attorney Docket No. 01183-0317-00PCT

[0093] Embodiment 87 is an IL-2 conjugate for use in the method of any one of embodiments 1- 85. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] FIG. 1 provides data of the kinetics of CD4 Treg expansion by Compound A in mice as measured by percentage of CD4 Tregs. The graph shows the CD4 Treg expansion in the peripheral blood after mice received 0.3 mg / kg of Compound A subcutaneously.

[0095] FIGs. 2A and 2B provide data that Compound A effectively suppresses the expansion of effector memory CD8 T cells (CD8 TEM; FIG. 2A) and LacZ-specific CD8T cells stimulated by AAVrh32.33-LacZ administration in PBMC in mice (FIG. 2B).

[0096] FIGs. 3A and 3B provide data that Compound A effectively suppresses both AAV capsid-specific IFNγ secreting CD8 T (FIG. 3A) and LacZ-specific IFNγ secreting CD8 T cells (FIG. 3B) in the spleen in mice.

[0097] FIGs. 4A and 4B provide data that Compound A enhances the expression of the OVA gene delivered by AAVrh32.33 vector (FIG. 4A) and suppresses anti-OVA IgG1 production (FIG. 4B) in mice, indicating converse correlation between the level of OVA and anti-OVA IgG1 in serum. Results showed that Compound A enhanced the OVA expression while suppressing anti-OVA IgG1 production.

[0098] FIGs. 5A, 5B and 5C show graphs demonstrating that Compound A significantly increases the CD4 Treg population (FIG. 5A) and effectively suppresses CD8 T cell proliferation (FIG. 5B) and effector memory CD8 T-cell expansion (CD8 TEM; FIG. 5C) stimulated by AAVrh10-EGFP administration in PBMC in rats.

[0099] FIGs. 6A and 6B show data that Compound A administration significantly increases the CD4 Treg population (FIG. 6A) as measured by percentage of CD4 Treg cells out of singlet cells and enhances the expression of the OVA gene delivered by AAVrh32.33 vector measured in serum (FIG. 6B) in non-human primates.

[0100] FIGs. 7A and 7B show the dose response curves of an exemplary IL-2 variant for pSTAT5 signaling in human LRS primary cell (FIG. 7A) and proliferation response in mouse CTLL-2 populations (FIG. 7B).

[0101] FIG. 8 shows the plasma concentration profiles of IL-2 conjugates K9_30kD, L19_30kD, N88R / D109_30kD, H16_30kD, Q126_30kD, and N88_30kD (all dosed at 0.9 mg / kg) following dosing in C57 / BL6 mice from Example 5.

[0102] FIG. 9 shows the mean fold change of Treg (% in singlets) following the dosing of IL-2 conjugates in C57 / BL6 mice from Example 5.Attorney Docket No. 01183-0317-00PCT

[0103] FIG. 10 shows the proportion of the Treg (CD3+ CD4+ CD25+ FoxP3+) cell population within the total cell population (singlets) of IL-2 conjugates in C57 / BL6 mice from Example 5.

[0104] FIGs. 11A and 11B show the proportion of the CD8+ T cell (CD3+ CD4- CD8+) population within the total cell population (singlets) following a single dose of IL-2 conjugates. FIG. 11A shows the proportion of the CD8+ T cell population (CD3+ CD4- CD8+) within the total cell population (singlets) following a single dose of IL-2 conjugates K9_30kD, L19_30kD, Q126_30kD, and H16_30kD in C57 / BL6 mice from Example 5. FIG. 11B shows the proportion of the CD8+ T cell (CD3+ CD4- CD8+) population within the total cell population (singlets) following a single dose of IL-2 conjugates E100_30kD, N88R / D109_30kD, T123_30kD, N88_30kD, and V91_30kD in C57 / BL6 mice from Example 5.

[0105] FIG. 12 shows the plasma concentration profiles of IL-2 conjugates following dosing in Cynomolgus monkey from Example 5.

[0106] FIG. 13 shows the proportion of the Treg cell population within the total blood cell population (singlets) in Cynomolgus monkeys following dosing with IL-2 conjugates from Example 5.

[0107] FIG. 14 shows the proportion of the CD8+ T cell population within the total blood cell population (singlets) in Cynomolgus monkeys following dosing with IL-2 conjugates from Example 5.

[0108] FIG. 15 shows the plots of plasma concentration versus time for the H16_30kD variant in non-human primates at doses of 0.12 mg / kg and 0.67 mg / kg from Example 5, wherein the 0.12 mg / kg dose is shown as the lower trace, while the 0.67 mg / kg dose is shown as the upper trace.

[0109] FIG. 16 shows the plots of plasma concentration versus time for the H16_30kDa variant and the H16_50kDa variant in non-human primates at a dose of 0.12 mg / kg, and the H16_50kDa variant at a dose of 0.2 mg / kg from Example 5, wherein the trace for the 30 kDa variant is shown as the lower trace (squares) and the trace for the 50 kDa variant is shown as the upper trace (triangles).

[0110] FIG. 17 shows plots of Treg percent in singles versus time post-dose in non-human primates from Example 5 for the H16_30kDa variant at a dose of 0.12 mg / kg, and the H16_50kDa variant at a dose of 0.2 mg / kg, wherein the trace for the vehicle is the lower trace (squares), the trace for the 30 kDa variant is shown in the middle trace, and the trace for the 50 kDa variant is shown in the upper trace.

[0111] FIG. 18 shows the study design of Example 5 to assess the effects of H16_50kD on delayed-type hypersensitivity (DTH) in C57BL / 6 mice. DTH in mice was induced with keyholeAttorney Docket No. 01183-0317-00PCT limpet hemocyanin (KLH) (challenge at Day 7 following sensitization at Day 1 via subcutaneous injection) with dosing of H16_50kD (Day 0 and 3), at a dose of 0.03 mg / kg, 0.1 mg / kg, and 0.3 mg / kg from Example 5.

[0112] FIGs. 19A, 19B, and 19C show changes in ear thickness measurements and blood immunotypes of C57BL / 6 mice from Example 5. FIG. 19A shows Area Under Curve (AUC) of increased ear thickness as compared to the mice with KLH challenge on Day 7 only. FIG. 19B shows changes in ear thickness measurements in the C57BL / 6 mice prior to KLH challenge (on Day 7) and then subsequently on Days 8, 9 and 10. FIG. 19C shows changes over time in the relative percentage of CD4+ T cells within CD25+FoxP3+ cell population in whole blood samples from the mice. “KLH only” indicates KLH challenge on Day 7 only (without senitization on Day 1) with dosing of vehicle only. “Vehicle” indicates KLH sensitization (Day 1) and challenge (Day 7) with dosing of vehicle only. “0.03” indicates KLH sensitization and challenge with dosing of H16_50kD at a dose of 0.03 mg / kg. “0.1” indicates KLH sensitization and challenge with dosing of H16_50kD at a dose of 0.1 mg / kg. “0.3” indicates KLH sensitization and challenge with dosing of H16_50kD at a dose of 0. 3 mg / kg. “CsA” indicates KLH sensitization and challenge with dosing of Cyclosporine A. See also Table 10 in Example 5.

[0113] FIGs. 20A, 20B, and 20C show changes over time in the relative percentage of CD4+CD25+FoxP3+ cells within CD45+ cell population (FIG. 20A), within TCRβ+ cell population (FIG. 20B), and within CD4+ cell population (FIG. 20C) in whole blood samples from the mice from Example 5.

[0114] FIG. 21 shows the absolute counts of CD4+CD25+FoxP3+ cells on Day 10 in whole blood samples from the mice from Example 5.

[0115] FIGs. 22A and 22B provide data for Treg, CD8+ T cells, CD4+ T cells, NK cell, and B cell expansion by Compound A in mice. The Treg expansion as measured by percentage of CD4+ T cells in the peripheral blood was measured at the indicated times after mice received 0.3 mg / kg of Compound A subcutaneously (FIG. 22A). The CD4 Treg, CD8+ T cell, CD4+ T cell, NK cell, and B cell expansion as measured by fold change in the peripheral blood was measured at the indicated times after mice received 0.3 mg / kg of Compound A subcutaneously (FIG. 22B)

[0116] FIGs. 23A, 23B, and 23C provide data showing that Compound A effectively suppresses the expansion of effector memory CD8 T cells (CD8 TEM; FIG. 23A) that would otherwise occur following AAV administration. Compound A effectively suppresses both AAV capsid-specific IFNγ secreting CD8 T cells (FIG. 23B) and LacZ-specific IFNγ secreting CD8 T cells (FIG. 23C) following ex vivo spleen restimulation at day 21.Attorney Docket No. 01183-0317-00PCT

[0117] FIGs. 24A and 24B show graphs demonstrating that Compound A mitigates CD8+ T cell responses and enhances transgene levels following gene therapy. Compound A enhances the expression of the OVA gene delivered by AAVrh32.33 vector (FIG. 24A) in mice as measured by serum OVA concentration. Compound A significantly suppresses effector memory CD8 T- cell expansion (CD8 TEM; FIG. 24B) in mice after delivery of the OVA gene by AAVrh32.33 vector.

[0118] FIGs. 25A, 25B and 25C show graphs demonstrating that Compound A significantly increases the CD4 Treg population (FIG. 25A) and effectively suppresses effector memory CD8 T-cell expansion (CD8 TEM; FIG. 25B) and CD8 T cell proliferation (FIG. 25C) stimulated by AAVrh10-EGFP administration in PBMC in rats.

[0119] FIGs. 26A, 26B, and 26C show data that Compound A administration significantly increases the CD4 Treg population (FIG. 26A) as measured by percentage of CD4 Treg cells out of singlet cells and enhances the expression of the OVA gene delivered by AAVrh32.33 vector measured in serum (FIG. 26C) in non-human primates relative to the expression of the OVA gene delivered by AAVrh32.33 vector without Compound A measured in serum (FIG. 26B) in non-human primates. Each line in FIGs. 26B and 26C represents serum OVA levels for an individual animal.

[0120] FIGs. 27A, 27B, 27C, and 27D show the longitudinal analysis of serum levels of anti AAV and anti-OVA IgG. FIGs. 27A and 27B: Wild-type C57BL / 6 mice (N>5 per group) were treated with rAAV1-OVA 2x1011ventrogluteal (VG) or AAVrh32.33-OVA 5x1010VG intramuscularly with or without a single subcutaneous dose of 0.3 mg / kg Compound A. Serum samples were collected via in-life serial bleeding at different time points over 16 weeks of the study. Levels of anti-AAV1 IgG (FIG. 27A), and anti-OVA IgG (FIG. 27B) in the serum were measured by ELISA. FIGs. 27C and 27D: Cynomolgus macaques (N>4 per group) were treated with rAAV1-OVA 3x1012VG intramuscularly with or without a single subcutaneous dose of 0.08 mg / kg Compound A. Serum samples were collected via in-life serial bleeding at different time points over 12 weeks of the study. Levels of anti-AAV1 IgG (FIG. 27C) and anti-OVA IgG (FIG. 27D) in the serum were measured by ELISA. Data is represented as mean ± SEM. Dotted line represents lower limit of detection. Statistical analysis performed by 2way ANOVA with test for repeated measures. *p < 0.05, **p < 0.01, ****p < 0.0001.

[0121] FIG. 28 shows data from Cynomolgus macaques treated with either AAVrh32.33 or rAAV1 expressing the OVA transgene intramuscularly with or without a single subcutaneous dose of Compound A with a dose up to 0.08 mg / kg. Immune cell analysis and / or serum transgene levels were measured serially via in-life bleeding. OVA protein concentrations in theAttorney Docket No. 01183-0317-00PCT serum of non-human primates (NHPs) were compared. Kaplan-Meier plot was generated for combined data from two studies comparing the duration for which the OVA expression lasts once expressed after AAV gene therapy. Statistical analysis performed by Cox proportional regression for combined data to determine the hazard ratio of the AAV treatment over the AAV + Compound A treatment group.

[0122] FIGs. 29A, 29B, 29C, and 29D show that wild-type C57BL / 6 mice were treated with AAVrh32.33-LacZ 2x1011ventrogluteal (VG) intramuscularly with or without a single subcutaneous dose of 0.3 mg / kg Compound A. The single dose of Compound A was administered either 4 days prior to AAV gene therapy (pre-treatment) or co-administered at the same time of AAV gene therapy (co-treatment). Mice from each group were sacrificed after at 21 days post AAV gene therapy treatment and CD8+ T cell immune responses were measured in the splenocytes by analyzing the proportion of proliferating CD8+ T cells (FIG. 29A) and the proportion of CD8+ T effector memory (CD8+TEM) cells (FIG. 29B). Proliferating CD8+ T cells were identified by CD8+ T cells expressing the proliferative marker Ki67. CD8+TEMcells were identified as CD62L+ and CD44+cells within the CD8 +T cell population (FIG. 29C and FIG. 29D). Splenocytes were restimulated ex-vivo for 6 hours with 1 µg / ml of either AAVrh32.33 peptide pool to determine AAV capsid specific T-cell responses (FIG. 29C) or 1 µg / ml of beta-galactosidase peptide pool to determine transgene specific T-cell responses (FIG. 29D). IFNγ expressing CD8+ T cell responses were measured in the splenocytes by Intracellular cytokine staining assay. Data is represented as mean ± SD. Statistical analysis performed by two- way ANOVA with test for multiple comparisons. *p < 0.05, **p < 0.01, ****p < 0.0001. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0123] This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. “About” indicates a degree of variation that does not substantially affect the properties of the described subject matter, e.g., within 10%, 5%, 2%, or 1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limitAttorney Docket No. 01183-0317-00PCT the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0124] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0125] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids, reference to “a cell” includes a plurality of cells, and the like.

[0126] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0127] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0128] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls. I. DefinitionsAttorney Docket No. 01183-0317-00PCT

[0129] As used herein, “peripheral blood mononuclear cells” or “PBMCs” refers to immune cells having a single, round nucleus that originate in bone marrow and are found in the peripheral circulation. Such cells include, e.g., lymphocytes (T cells, B cells, and NK cells) as well as monocytes, and are isolated from blood samples (such as from a whole blood sample collected from a subject) using density gradient centrifugation.

[0130] As used herein, “isolated” refers to a biological component (such as a nucleic acid molecule, protein, or cell) that has been substantially separated, produced apart from, or purified away from other components (for example, other components in a sample, cell, or organism in which the component naturally occurs). Nucleic acid molecules, proteins, or cells that have been “isolated” include those purified using standard purification methods. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched in a preparation than the biological component is in its natural environment within a cell, organism, sample, or production vessel (for example, a cell culture system). For example, an isolated biological component can represent at least 50%, such as at least 70%, at least 80%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.

[0131] As used herein, “subject” refers to an animal, such as a member of a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject”. For example, the subject can be an individual who is in need of gene therapy, e.g., due to having a disease such as an autoimmune disease or a developmental, neurological, or other genetic disorder. As another example, the subject can be a female individual who is pregnant or who is planning on becoming pregnant, who may have been diagnosed of or suspected of having a disease, e.g., a cancer, an auto-immune disease.

[0132] As used herein, the term “potency” refers to the amount of a cytokine (e.g., IL-2 polypeptide) required to produce a target effect. In some embodiments, the term “potency” refers to the amount of cytokine (e.g., IL-2 polypeptide) required to activate a target cytokine receptor (e.g., IL-2 receptor). In other instances, the term “potency” refers to the amount of cytokine (e.g.,Attorney Docket No. 01183-0317-00PCT IL-2 polypeptide) required to activate a target cell population. In some embodiments, potency is measured as ED50 (Effective Dose 50), or the dose required to produce 50% of a maximal effect. In other cases, potency is measured as EC50 (Effective Concentration 50), or the dose required to produce the target effect in 50% of the population.

[0133] As used herein, an “IL-2 conjugate” is an IL-2 polypeptide attached (such as through a linker) to a conjugating moiety, e.g., comprising a PEG group; the IL-2 conjugate may be but is not necessarily in the form of a pharmaceutically acceptable salt, solvate, or hydrate. As described in detail elsewhere herein, the IL-2 polypeptide may comprise an unnatural amino acid, which can serve as the site of attachment to the conjugating moiety.

[0134] As used herein, the terms “operably linked” and “in functional connection with” with respect to promoters, refer to a relationship between a coding sequence and a promoter element. The promoter is operably linked or in functional connection with the coding sequence when expression from the coding sequence via transcription is regulated, or controlled by, the promoter element. The terms “operably linked” and “in functional connection with” are utilized interchangeably herein with respect to promoter elements.

[0135] As used herein, the term “gene therapy agent” refers to a nucleic acid (e.g., expression construct, miRNA, antisense, shRNA, siRNA) or a nucleic acid in combination with an agent used to deliver the nucleic acid to an individual or a cell to modify or manipulate the expression of one or more nucleic acids (e.g., gene, mRNA) in an individual or a cell to alter the biological propertied of living cells. Examples of gene therapy agents include, but are not limited to, viral vectors (e.g., adeno-associated virus, adenovirus, lentivirus, Herpes simples virus, baculovirus), bacterial vectors, and non-viral vectors (e.g., lipid nanoparticles encapsulating a therapeutic nucleic acid or plasmid DNAs (e.g., close ended DNA) comprising a therapeutic nucleic acid and / or encoding a therapeutic polypeptide).

[0136] As used herein, a “vector” refers to a recombinant plasmid or virus that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo.

[0137] The term “polynucleotide” or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, comprising ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the nucleic acid can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the nucleic acid can comprise a polymer of synthetic subunits such asAttorney Docket No. 01183-0317-00PCT phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate- phosphodiester oligomer. In addition, a double-stranded nucleic acid can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0138] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full- length proteins and fragments thereof are encompassed by the definition. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (which may be conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0139] A “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (a nucleic acid sequence that does not naturally occur in the virus from which the vector is derived, e.g., a sequence that is not of viral origin or from a different virus). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one, e.g., two, inverted terminal repeat sequences (ITRs).

[0140] A “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one, e.g., two, AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. A rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linearAttorney Docket No. 01183-0317-00PCT artificial chromosomes, complexed with lipids, encapsulated within liposomes, and, in embodiments, encapsidated in a viral particle, particularly an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle)”.

[0141] An “rAAV virus” or “rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0142] A “recombinant adenoviral vector” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of adenovirus origin) that are flanked by at least one adenovirus inverted terminal repeat sequence (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeat sequences (ITRs). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell that is expressing essential adenovirus genes deleted from the recombinant viral genome (e.g., E1 genes, E2 genes, E4 genes, etc.). When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the recombinant viral vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of adenovirus packaging functions. A recombinant viral vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, for example, an adenovirus particle. A recombinant viral vector can be packaged into an adenovirus virus capsid to generate a “recombinant adenoviral particle.”

[0143] A “recombinant lentivirus vector” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of lentivirus origin) that are flanked by at least one lentivirus terminal repeat sequences (LTRs). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral terminal repeat sequences (LTRs). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper function. A recombinant lentiviral vector can be packaged into a lentivirus capsid to generate a “recombinant lentiviral particle.”

[0144] A “recombinant herpes simplex vector (recombinant HSV vector)” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of HSV origin) that are flanked by HSV terminal repeat sequences. Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper functions. When a recombinantAttorney Docket No. 01183-0317-00PCT viral vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the recombinant viral vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of HSV packaging functions. A recombinant viral vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, for example, an HSV particle. A recombinant viral vector can be packaged into an HSV capsid to generate a “recombinant herpes simplex viral particle.”

[0145] “Solid lipid nanoparticles” (SLNs, sLNPs), or “lipid nanoparticles” (LNPs) as used herein refer to nanoparticles comprising lipids that can contain a payload. In some examples, there is only one phospholipid layer; furthermore, in some embodiments, the bulk of the interior of the particle is composed substantially of lipophilic substance and a payload. Payloads such as nucleic acids can be embedded in the interior. In some examples, the lipid nanoparticle is a liposome, which comprises a lipid bilayer and may comprise a hydrophilic or aqueous interior comprising a payload.

[0146] As used herein, the term “improving” as it relates to gene therapy may refer to the act of boosting, heightening, lengthening or otherwise increasing the expression of the therapeutic gene payload of a gene therapy agent. In some embodiments, an improved gene therapy is one where expression of the therapeutic gene payload of the gene therapy agent administered with an IL-2 conjugate is increased by greater than any of about 10%, 25%, 50%, 75%, or 100% compared to gene therapy administered without the IL-2 conjugate. In some embodiments, an improved gene therapy is one where time of expression of the therapeutic gene payload of the gene therapy agent administered with an IL-2 conjugate is lengthened by greater than any of about 10%, 25%, 50%, 75%, or 100% compared to gene therapy administered without the IL-2 conjugate. In some examples, gene therapy is improved by decreasing an immune response (e.g., an adaptive immune response) to the gene therapy agent. In some embodiments, an improved gene therapy is one where an immune response to gene therapy agent administered with an IL-2 conjugate is decreased by greater than any of about 10%, 25%, 50%, 75%, or 100% compared to gene therapy administered without the IL-2 conjugate. In some embodiments, the decrease in an immune response to a gene therapy agent is measured as a decrease in a cytokine signature following exposure of the gene therapy agent to immune cells in the presence of an IL-2 conjugate compared to exposure of the gene therapy agent to immune cells in the absence of the IL-2 conjugate.Attorney Docket No. 01183-0317-00PCT

[0147] As used herein, the term “modulating” as it refers to gene therapy may refer to the act of changing, altering, varying, improving or otherwise modifying the presence, or an activity of, a gene therapy agent. For example, modulating an immune response to a gene therapy agent may refer to any act leading to changing, altering, varying, improving or otherwise modifying an immune response to the gene therapy agent (e.g., decreasing, delaying and / or eliminating an immune response (e.g., an adaptive immune response) to the gene therapy agent).

[0148] As used herein, the term “cytokine signature” as it relates to an immune response (e.g., adaptive immune response) to a gene therapy agent refers to altered (e.g., increased, decreased) expression of one or more cytokines following exposure of an adaptive immune cell to a gene therapy agent. In some examples, the cytokines of the cytokine signature are specific to an interleukin-6 (IL-6); tumor necrosis factor-alpha (TNF-α); tumor necrosis factor-beta (TNF-β); interferon alpha (IFN-α); interleukin-10 (IL-10); interleukin-8 (IL-8); Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES); Granulocyte- macrophage colony-stimulating factor (GM-CSF); interferon gamma (IFN-γ); interferon gamma- induced protein 10 (IP-10); interleukin-1beta (IL-1β); interleukin-2 (IL-2); and / or interleukin-4 (IL-4) pathway.

[0149] Adaptive immune cells are white blood cells that mediate adaptive immunity and include B cells, T cells, and NK cells. AAVs upon cell entry can evoke an immune response. The magnitude of this immune response may depend on AAV serotype and cell type. Once AAVs transduce a host immune cell they can engage immune receptors. Once these immune receptors are activated by viruses, they secrete cytokines that establish an anti-viral state within the infected cell and alert the neighboring cells.

[0150] Innate immune cells are white blood cells that mediate innate immunity and include basophils, dendritic cells, eosinophils, Langerhans cells, mast cells, monocytes and macrophages, neutrophils and NK cells. Different AAV capsids can enter these adaptive immune cells with different efficiencies often referred to as transduction efficiency. Some serotypes such as AAV1 are efficient at transducing certain immune cells like monocytes whereas other AAVs like AAV6 are efficient at transducing cells like dendritic cells (Grimm, D et al., J. Virol., 2008, 82(12):5887-5911). AAVs upon cell entry can evoke an immune response. The magnitude of this immune response is dependent on AAV serotype and cell type. Once AAVs transduce a host immune cell they can engage immune receptors such as TLRs (e.g., TLR9). Several studies using mouse models reveal that TLR9 is a key DNA sensor contributing to AAV immunogenicity (Zhu, J et al., J Clin Invest. 2009;119(8):2388-2398; Ashley SN et al., Cell. Immunol.2019, 346:103997). Once these TLRs are activated by viruses they secrete cytokinesAttorney Docket No. 01183-0317-00PCT that establish an anti-viral state within the infected cell and alert the neighboring cells. (Carty, M and Bowie, AG, Clin Exp Immunol, 2010, 161(3):397-406; Lester, SN and Li, K, J Mol Biol.2014; 426(6):1246–1264; Fitzgerald, KA and Kagan, JC, Cell, 2020180(6):1044-1066).

[0151] As used herein, the upregulation or downregulation of certain subset of cytokines is referred to as a “cytokine signature”. These cytokine signatures comprising three or more cytokines can be used as predictive markers for diseases and success of therapies. Examples of cytokine signatures are found in Zuniga, J et al., Int. J. Infect. Diseases, 2020, 94:4-11, Bergamaschi, C et al., Cell Reports, 2021, 36:109504; Del Valle, DM et al., Nat. Med. 2020, 26:1636-1643.

[0152] “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a nucleic acid introduced by genetic engineering techniques into a different cell type is a heterologous nucleic acid (and, when expressed, can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0153] The term “transgene” refers to a nucleic acid that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. In some embodiments, it confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as siRNA.

[0154] The terms “genome particles (gp),” “genome equivalents,” or “genome copies” as used in reference to a viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as described in the Examples herein, or for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0155] The terms “infection unit (iu),” “infectious particle,” or “replication unit,” as used in reference to a viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also known as replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0156] The term “transducing unit (tu)” as used in reference to a viral titer, refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product as measured in functional assays such as described in Examples herein, or forAttorney Docket No. 01183-0317-00PCT example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0157] An “inverted terminal repeat” or “ITR” sequence is a term well understood in the art and refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation.

[0158] An “AAV inverted terminal repeat (ITR)” sequence, a term well-understood in the art, is an approximately 145-nucleotide sequence that is present at both termini of the native single- stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contains several shorter regions of self-complementarity (designated A, A', B, B', C, C' and D regions), allowing intrastrand base-pairing to occur within this portion of the ITR.

[0159] A “terminal resolution sequence” or “trs” is a sequence in the D region of the AAV ITR that is cleaved by AAV rep proteins during viral DNA replication. A mutant terminal resolution sequence is refractory to cleavage by AAV rep proteins. “AAV helper functions” refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genes which aid in AAV replication and packaging. Other AAV helper functions are known in the art such as genotoxic agents.

[0160] “AAV helper functions” refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, helper virus or helper virus genes which aid in AAV replication and packaging. Other AAV helper functions are known in the art such as genotoxic agents.

[0161] A “helper virus” for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. A number of such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses such as vaccinia, and baculovirus. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non- human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.Attorney Docket No. 01183-0317-00PCT

[0162] “Percent (%) sequence identity” with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. A potential alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W / Z, where W is the number of nucleotides scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

[0163] An “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. For example, an effective amount of a gene therapy agent refers to an amount effective, at dosages and for periods of timeAttorney Docket No. 01183-0317-00PCT necessary, to achieve the desired gene therapeutic result. In another example, an effective amount of an IL-2 conjugate may refer to an amount effective, at dosages and for periods of time necessary, to achieve the desired result of improved gene therapy.

[0164] A “therapeutically effective amount” of a substance / molecule of the invention, (e.g., a gene therapy agent and / or an IL-2 conjugate) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects.

[0165] The term “suitable control” as it refers to a cytokine signature is the expression of the cytokines in the cytokine signature from adaptive immune cells that are not incubated with the gene therapy agent or the expression of the cytokines in the cytokine signature from adaptive immune cells prior to incubation with the gene therapy agent.

[0166] Administration “in combination with” as it related to a gene therapy agent and a modulator of an adaptive immune response (e.g., an IL-2 conjugate) includes simultaneous (concurrent), consecutive or sequential administration in any order of the gene therapy agent and the modulator of an adaptive immune response (e.g., an IL-2 conjugate).

[0167] The term “concurrently” is used herein to refer to administration of a gene therapy agent and a modulator of the adaptive immune response (e.g., an IL-2 conjugate), where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of a gene therapy agent or a modulator of an adaptive immune response (e.g., an IL-2 conjugate) continues after discontinuing the administration of the other agent / modulator.

[0168] As used herein, “in conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality (a gene therapy agent or a modulator of an adaptive immune response (e.g., an IL-2 conjugate)) before, during or after administration of the other treatment modality to the individual.

[0169] The terms “or a combination thereof” and “or combinations thereof” as used herein refers to any and all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC,Attorney Docket No. 01183-0317-00PCT CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0170] “Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise. II. Exemplary Methods A. Overview

[0171] The present disclosure provides methods of delivering a gene therapy agent to a cell of a subject, treating an individual in need thereof with a gene therapy agent, increasing expression of a gene therapy agent, reducing an immune response to a gene therapy agent, preventing immune- related adverse events in a subject, selecting a subject for treatment with a gene therapy agent and an IL-2 conjugate, using an IL-2 conjugate. The IL-2 conjugate can be capable of selectively upregulating distinct population(s) of lymphocytes (e.g., CD4+ T regulatory cells), e.g., through cytokine / cytokine receptor signaling. In some embodiments, the amino acid sequence of the IL-2 conjugate has at least one amino acid residue replaced by an unnatural amino acid linked to a conjugating moiety. In some embodiments, the unnatural amino acid that is linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0172] In some embodiments, also described herein is a method of selectively upregulating CD4+ T regulatory cell through IL-2 / IL-2R signaling. In some embodiments, the IL-2 conjugate suppresses CD8+ T cell proliferation in the subject. In some embodiments, the IL-2 conjugate suppresses effector memory CD8+ T cell proliferation in the subject. In some embodiments, IL-2 is an IL-2 conjugate, which interacts with the IL-2Rαβγ complex and with a weakened IL-2Rβγ interaction relative to wild-type IL-2. In some embodiments, further described herein are methods of delivering a gene therapy agent to a cell of a subject with use of an IL-2 conjugate described herein. In additional embodiments, described herein are pharmaceutical compositionsAttorney Docket No. 01183-0317-00PCT and kits which comprise one or more IL-2 conjugates and / or gene therapy agents described herein, e.g., for use in the disclosed methods.

[0173] As shown in the examples, gene therapy resulted in a significant increase in proliferating and effector CD8+ T cell proportion in the spleen with which was mitigated by treatment with an IL-2 conjugate according to this disclosure. The mitigation of CD8+ T cell responses were not meaningfully different when the IL-2 conjugate was administered as a pre-treatment or given as a co-treatment. In addition, upon ex vivo restimulation of splenocytes with a gene therapy agent peptide pool and a transgene peptide pool, there was a significant increase in the proportion of IFNγ secreting CD8+ T cells in response to each set of peptides, which were mitigated by IL-2 conjugate treatment. There was no meaningful difference in mitigation of AAV and / or transgene specific CD8+ T cell responses when the IL-2 conjugate was given as a pre-treatment or as a co- treatment. Without wishing to be bound by any particular theory, this suggests that administering AAV gene therapy within the timeframe of the expansion of Tregs mediated by the IL-2 conjugate maintains the therapeutic benefit of the IL-2 conjugate of suppressing adverse immune responses to the gene therapy agent and / or the transgene. B. Cytokine Conjugates

[0174] In some embodiments, the IL-2 conjugate used in the disclosed methods comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the IL-2 conjugate has at least one amino acid residue replaced by an unnatural amino acid linked to a conjugating moiety. In some embodiments, the unnatural amino acid that is linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL- 2Rαβγ. In some embodiments, the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.

[0175] Table 1 provides exemplary IL-2 sequences. The sequence of SEQ ID NO: 1 is aldesleukin, in which the first amino acid of wild-type IL-2 has been removed. An amino acid in SEQ ID NO: 1 or a sequence having a percentage identity thereto as described elsewhere hereinAttorney Docket No. 01183-0317-00PCT may be replaced with an unnatural amino acid. Exemplary sequences comprising an unnatural amino acid, indicated by X, are listed as SEQ ID NOs: 2-14. Table 1 Name Sequence SEQ ID NO: aldesleukin PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 1 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_K8X PTSSSTKXTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 2 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_H15X PTSSSTKKTQLQLEXLLLDLQMILNGINNYKNPKLTRMLTF 3 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_L18X PTSSSTKKTQLQLEHLLXDLQMILNGINNYKNPKLTRMLTF 4 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_D19X PTSSSTKKTQLQLEHLLLXLQMILNGINNYKNPKLTRMLTF 5 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_M22X PTSSSTKKTQLQLEHLLLDLQXILNGINNYKNPKLTRMLTF 6 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_N25X PTSSSTKKTQLQLEHLLLDLQMILXGINNYKNPKLTRMLTF 7 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_N87X PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 8 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISXINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_E99X PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 9 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSXTTFMCEYADETATIVEFLNRWITF SQSIISTLT IL-2_N118X PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 10 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLXRWITF SQSIISTLT IL-2_T122X PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 11 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPAttorney Docket No. 01183-0317-00PCT RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIXF SQSIISTLT IL-2_Q125X PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 12 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SXSIISTLT IL-2_S126X PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 13 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQXIISTLT IL-2_T130X PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF 14 KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP RDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF SQSIISXLT IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT 15 (homo sapiens) FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR (mature form) PRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FCQSIISTLT IL-2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDL 16 (homo sapiens) QMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE (precursor) ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTF NCBI Accession MCEYADETATIVEFLNRWITFCQSIISTLT No.: AAB46883.1 CTP Peptide (30 FQSSSSKAPPPSLPSPSRLPGPSDTPILPQ 17 amino acids) CTP Peptide (31 FQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ 18 amino acids) ITR sequence CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC 19 GGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGG GCG IL-2_C125S (in APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT 20 reference to SEQ FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR ID NO: 15) PRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FSQSIISTLT IL-2_H16X APTSSSTKKTQLQLEXLLLDLQMILNGINNYKNPKLTRMLT 21 FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FSQSIISTLT IL-2_L19X APTSSSTKKTQLQLEHLLXDLQMILNGINNYKNPKLTRMLT 22 FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FSQSIISTLT IL-2_N88X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT 23 FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISXINVIVLELKGSETTFMCEYADETATIVEFLNRWIT FSQSIISTLT IL- APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT 24 2_N88R_D109X FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRAttorney Docket No. 01183-0317-00PCT PRDLISRINVIVLELKGSETTFMCEYAXETATIVEFLNRWIT FSQSIISTLT

[0176] In some embodiments, the position of the at least one unnatural amino acid is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132, wherein the numbering of the amino acid residues is in reference to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108, wherein the numbering of the amino acid residues is in reference to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is L18, wherein the numbering of the amino acid residues is in reference to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is D19, wherein the numbering of the amino acid residues is in reference to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is H15, wherein the numbering of the amino acid residues is in reference to the sequence of SEQ ID NO: 1.

[0177] In some embodiments, IL-2 conjugates modified at an amino acid position are provided. In some embodiments, the modification is to an unnatural amino acid. In some embodiments, described herein is an isolated and IL-2 conjugate that comprises at least one unnatural amino acid. In some embodiments, the IL-2 polypeptide is an isolated and purified mammalian IL-2, for example, a rodent IL-2 protein, or a human IL-2 protein. In some embodiments, the IL-2 polypeptide is a human IL-2 protein. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 1. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 1. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 2. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 2. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 3. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 3. In someAttorney Docket No. 01183-0317-00PCT embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 4. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 4. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 5. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 6. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 7. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 7. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 8. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 8.

[0178] In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 9. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 9. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 10. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 10. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 11. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 11. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 12. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 12. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQAttorney Docket No. 01183-0317-00PCT ID NO: 14. In some embodiments, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 14.

[0179] In some embodiments, the IL-2 polypeptide is a truncated variant, e.g., relative to a wild- type IL-2, such as SEQ ID NO: 15 or 16. In some embodiments, the truncation is an N-terminal deletion. In some embodiments, the truncation is a C-terminal deletion. In some embodiments, the truncation comprises both N-terminal and C-terminal deletions. For example, the truncation can be a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from either the N-terminus or the C-terminus, or both termini. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 2 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 3 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 4 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 5 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 6 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 7 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 8 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 9 residues. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 10 residues.

[0180] In some embodiments, the IL-2 polypeptide comprises an amino acid addition in reference to, for example, SEQ ID NO: 1 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the addition is an N-terminal addition. In some embodiments, the addition is a C-terminal addition. In some embodiments, the addition comprises both N-terminal and C-terminal additions. In some embodiments, the IL-2 polypeptide comprises an alanine or methionine N-terminal addition to the first amino acid of the sequence having at least 80% sequence identity to SEQ ID NO: 1.

[0181] In some embodiments, the IL-2 polypeptide is a functionally active fragment. In some embodiments, the functionally active fragment comprises IL-2 region 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 10-133, wherein the residue positions are in reference to theAttorney Docket No. 01183-0317-00PCT positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 20-133, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 30-133, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 10-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 20-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 1-130, wherein the residue positions are in reference to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 1-125, wherein the residue positions are in reference to the positions in SEQ ID NO: 1.

[0182] In some embodiments, described herein is an IL-2 conjugate that comprises an isolated, purified, and IL-2 conjugate and a conjugating moiety. In some embodiments, the IL-2 conjugate has a decreased affinity to an IL-2 receptor βγ (IL-2Rβγ) subunit relative to a wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety is bound to an amino acid residue that interacts with IL-2Rβγ (e.g., at the IL-2 / IL-2Rβγ interface). In some embodiments, the conjugating moiety is bound to an amino acid residue that is proximal to the IL-2 / IL-2Rβγ interface (e.g., about 5Å, about 10Å, about 15Å, or about 20Å away from the IL-2 / IL-2Rβγ interface). As used herein, the residues involved in the IL-2 / IL-2Rβγ interface comprise IL-2 residues that form hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2Rβγ subunit.

[0183] In some embodiments, the conjugating moiety is bound to an amino acid residue selected from an amino acid position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1. In some embodiments, the conjugating moiety is bound to an amino acid residue selected from an amino acid position K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108 in reference to the sequence of SEQ ID NO: 1. In some embodiments, the conjugating moiety is bound to an amino acid residue selected from an amino acid position L18 in reference to the sequence of SEQ ID NO: 1. In some embodiments, the conjugating moiety is bound to an amino acid residue selectedAttorney Docket No. 01183-0317-00PCT from an amino acid position H15 in reference to the sequence of SEQ ID NO: 1. In some embodiments, the conjugating moiety is bound to an amino acid residue selected from an amino acid position D19 in reference to the sequence of SEQ ID NO: 1.

[0184] In some embodiments, the IL-2 conjugate further comprises an additional mutation. In some embodiments, the additional mutation is at an amino acid position selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108 in reference to the sequence of SEQ ID NO: 1. In such cases, the amino acid is conjugated to an additional conjugating moiety for increase in serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to an unnatural amino acid prior to binding to the additional conjugating moiety.

[0185] In some embodiments, the receptor signaling potency is measured by an ED50 value. In some embodiments, the IL-2 conjugate provides a first ED50 value for activating IL-2βγ signaling complex and a second ED50 value for activating IL-2αβγ signaling complex, and wherein a difference between the first ED50 and the second ED50 value is less than 10-fold. In some embodiments, the IL-2 conjugate provides a first ED50 value for activating IL-2βγ signaling complex and a second ED50 value for activating IL-2αβγ signaling complex, and wherein a difference between the first ED50 and the second ED50 value is less than 5-fold. In some embodiments, the difference is less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, less than 75%, less than 50%, or less than 25%.

[0186] In some embodiments, the conjugating moiety is linked to the N-terminus or the C- terminus of an IL-2 polypeptide, either directly or indirectly through a linker peptide. In some embodiments, the conjugating moiety (e.g., a polymer, a protein, or a peptide) is genetically fused to the IL-2, at the N-terminus or the C-terminus of IL-2, and either directly or indirectly through a linker peptide. In some embodiments, the conjugating moiety is linked to the N- terminus or the C-terminus amino acid residue. In some embodiments, the conjugating moiety is linked to a reactive group that is bound to the N-terminus or C-terminus amino acid residue.

[0187] In some embodiments, the IL-2 conjugate with reduced binding affinity to IL-2Rβγ is capable of expanding CD4+ T regulatory cell populations. In some embodiments, the IL-2 conjugate with reduced binding affinity to IL-2Rβγ is capable of suppressing CD8+ T cell populations, including CD8+ T effector memory cell populations, vector-specific IFNγ-secreting CD8+ T cells, and transgene-product-specific IFNγ-secreting CD8+ T cells. In some embodiments, the IL-2 conjugate with reduced binding affinity to IL-2Rβγ is capable of suppressing the production of antibodies against the transgene product and the production ofAttorney Docket No. 01183-0317-00PCT IgG1 antibodies against the transgene product. In some embodiments, the conjugating moiety impairs or blocks binding of IL-2 with IL-2Rβγ.

[0188] In some embodiments, activation of CD4+ T regulatory cell population via the IL-2Rαβγ complex by the IL-2 conjugate retains significant potency of activation of said cell population relative to a wild-type IL-2 polypeptide. In some embodiments, the activation by the IL-2 conjugate is equivalent to that of the wild-type IL-2 polypeptide. In other instances, the activation by the IL-2 conjugate is higher than that of the wild-type IL-2 polypeptide. In some embodiments, the receptor signaling potency of the IL-2 conjugate to the IL-2Rαβγ complex is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2Rαβγ complex. In some embodiments, the receptor signaling potency of the IL-2 conjugate is at least 50% higher than the respective potency of the wild-type IL-2 polypeptide. In some embodiments, the receptor signaling potency of the IL-2 conjugate is about or at least 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1500%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, 15000%, 20000%, 30000%, 40000%, 50000%, 100000%, or higher than the respective potency of the wild-type IL-2 polypeptide. In such cases, the dose or concentration of the IL-2 conjugate used for achieving a similar level of activation of the CD4+ T regulatory cell population as a wild-type IL-2 polypeptide is lower than a dose or concentration used for the wild-type IL-2 polypeptide.

[0189] In some embodiments, activation of CD4+ T regulatory cell population via the IL-2Rαβγ complex by the IL-2 conjugate retains significant potency of activation of said cell population by a wild-type IL-2 polypeptide. In some embodiments, the receptor signaling potency of the IL-2 conjugate the IL-2Rβγ complex is lower than a receptor signaling potency of the wild-type IL-2 polypeptide the IL-2Rβγ complex. In some embodiments, the receptor signaling potency of the IL-2 conjugate is about or at least 25%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 50-fold lower than the respective potency of the wild-type IL-2 polypeptide.

[0190] In some embodiments, the IL-2 conjugate exhibits a first receptor signaling potency to IL-2Rαβγ and a second receptor signaling potency to IL-2Rβγ. In some embodiments, the first receptor signaling potency to IL-2Rαβγ is an improved potency relative to a wild-type IL-2 polypeptide. In some embodiments, the second receptor signaling potency to IL-2Rβγ is an impaired potency relative to the wild-type IL-2 polypeptide. In some embodiments, the IL-2 conjugate exhibits a first receptor signaling potency to IL-2Rαβγ and a second receptor signaling potency to IL-2Rβγ, and wherein the first receptor signaling potency is at least 50%, 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-Attorney Docket No. 01183-0317-00PCT fold, 500-fold, 1000-fold, or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 50% or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 50% higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 2-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 5-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 10-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 20-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 50-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 100-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 500-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 1000-fold or higher than the second receptor signaling potency. In some embodiments, the first receptor signaling potency of the modified IL-2 polypeptide is higher than a receptor signaling potency of the wild-type IL-2 polypeptide to the IL-2Rαβγ, and the second receptor signaling potency of the modified IL-2 polypeptide is lower than a receptor signaling potency of the wild- type IL-2 polypeptide to the IL-2Rβγ. In some embodiments, both receptor signaling potencies are lower than their respective potencies in a wild-type IL-2 polypeptide. In other cases, both receptor signaling potencies are higher than their respective potencies in a wild-type IL-2 polypeptide.

[0191] In some embodiments, the IL-2 conjugate decreases a toxic adverse event in a subject administered with the IL-2 conjugate. Exemplary toxic adverse events include eosinophilia, capillary leak, and vascular leak syndrome (VLS). In some embodiments, the IL-2 conjugate decreases the occurrence of a toxic adverse event in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin. In some embodiments, the IL-2 conjugate decreases the severity of a toxic adverse event in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.

[0192] In some embodiments, the toxic adverse event is eosinophilia. In some embodiments, the IL-2 conjugate decreases the occurrence of eosinophilia in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subjectAttorney Docket No. 01183-0317-00PCT administered with a wild-type IL-2 or aldesleukin. In some embodiments, the IL-2 conjugate decreases the severity of eosinophilia in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.

[0193] In some embodiments, the toxic adverse event is capillary leak. In some embodiments, the IL-2 conjugate decreases the occurrence of capillary leak in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin. In some embodiments, the IL-2 conjugate decreases the severity of capillary leak in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.

[0194] In some embodiments, the toxic adverse event is VLS. In some embodiments, the IL-2 conjugate decreases the occurrence of VLS in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin. In some embodiments, the IL-2 conjugate decreases the severity of VLS in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100%, relative to a second subject administered with a wild-type IL-2 or aldesleukin.

[0195] 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.

[0196] In some embodiments, the IL-2 conjugate has a plasma half-life of from about 1 hour to about 7 days, from about 12 hours to about 7 days, from about 18 hours to about 7 days, from about 24 hours to about 7 days, from about 1 hours to about 5 days, from about 12 hours to about 5 days, from about 24 hours to about 5 days, from about 2 days to about 5 days, or from about 2 days to about 3 days.

[0197] In some embodiments, the IL-2 conjugate has a plasma half-life of from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours toAttorney Docket No. 01183-0317-00PCT 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.

[0198] In some embodiments, the IL-2 conjugate has a plasma half-life that is capable of proliferating and / or expanding a CD4+ T regulatory cells but does not exert a deleterious effect such as apoptosis.

[0199] In some embodiments, the IL-2 conjugate has an extended plasma half-life, e.g., by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more relative to a wild-type IL-2. In some embodiments, the IL-2 conjugate has an extended plasma half-life, e.g., by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more relative to a wild-type IL-2 or aldesleukin.

[0200] In some embodiments, described herein is an IL-2 conjugate comprising an unnatural amino acid covalently attached to a conjugating moiety, wherein the unnatural amino acid is located in region 1-132, and wherein the region 1-132 is in reference to residues P1-T132 of the sequence of SEQ ID NO: 1 or the unnatural amino acid is located in region 8-108, and wherein the region 8-108 is in reference to residues K8-D108 of the sequence of SEQ ID NO: 1.

[0201] In some embodiments, the IL-2 conjugate comprises a mutation at L18 in reference to residue position 18 of the sequence of SEQ ID NO: 1, and comprises a conjugating moiety comprising a PEG having a molecular weight of about 2 kDa to about 60 kDa. In some embodiments, the molecular weight comprises about 30 kDa. In some embodiments, the molecular weight comprises about 35 kDa. In some embodiments, the molecular weight comprises about 40 kDa. In some embodiments, the molecular weight comprises about 45 kDa. In some embodiments, the molecular weight comprises about 50 kDa. In some embodiments, the molecular weight comprises about 55 kDa. In some embodiments, the molecular weight comprises about 60 kDa. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the IL-2 conjugate. In some embodiments, the PEG corresponds with a longer in vivo plasma half-life of the IL-2 conjugate, as compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, the PEG corresponds with a shorter in vivo plasma half-life of the IL-2 conjugate, as compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect, nor has minimal effect, on the receptor signaling potency of the IL-2 conjugate to the IL-2α or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect, or has minimal effect, on the desired reduced binding of the IL-2 conjugate to IL-2Rαβγ or theAttorney Docket No. 01183-0317-00PCT maintained binding with IL-2Rαβγ signaling complex, wherein the reduced binding to IL-2Rβγ is compared to binding between a wild-type IL-2 polypeptide and IL-2Rβγ. In some embodiments, the molecular weight of the PEG does not affect the formation of the modified IL- 2polypeptide / IL-2Rαβγ complex, wherein the reduced binding to IL-2Rβγ is compared to binding between a wild-type IL-2 polypeptide and IL-2Rβγ.

[0202] In some embodiments, the IL-2 conjugate comprises a mutation at H15 in reference to residue position 15 of the sequence of SEQ ID NO: 1, comprises a conjugating moiety comprising a PEG having a molecular weight of about 2 kDa to about 60 kDa.In some embodiments, the molecular weight comprises about 30 kDa. In some embodiments, the molecular weight comprises about 35 kDa. In some embodiments, the molecular weight comprises about 40 kDa. In some embodiments, the molecular weight comprises about 45 kDa. In some embodiments, the molecular weight comprises about 50 kDa. In some embodiments, the molecular weight comprises about 55 kDa. In some embodiments, the molecular weight comprises about 60 kDa

[0203] In some embodiments, the conjugating moiety is bound to an amino acid residue selected from an amino acid position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132, in which the numbering of the amino acid residues is in reference to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid position is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108. In some embodiments, the amino acid position is selected from L18 and H15. In some embodiments, the amino acid position is at K8. In some embodiments, the amino acid position is at L11. In some embodiments, the amino acid position is at E14. In some embodiments, the amino acid position is at H15. In some embodiments, the amino acid position is at L18. In some embodiments, the amino acid position is at D19. In some embodiments, the amino acid position is at M22. In some embodiments, the amino acid position is at N87. In some embodiments, the amino acid position is at E99. In some embodiments, the amino acid position is at D108.

[0204] In some embodiments, the IL-2 conjugate further comprises an additional mutation. In such cases, the amino acid is conjugated to an additional conjugating moiety for increase in serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to an unnatural amino acid prior to binding to the additional conjugating moiety.Attorney Docket No. 01183-0317-00PCT

[0205] In some embodiments, the IL-2 conjugate has a decreased binding affinity to IL-2 receptor β (IL-2Rβ) subunit, IL-2 receptor γ (IL-2Rγ) subunit, or a combination thereof, of the IL-2Rαβγ complex, relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity of the IL-2 conjugate to IL-2 receptor β (IL-2Rβ) subunit, IL-2 receptor γ (IL- 2Rγ) subunit, or a combination thereof, relative to a wild-type IL-2 polypeptide, is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99%.

[0206] In some embodiments, the decreased binding affinity of the IL-2 conjugate to IL-2 receptor β (IL-2Rβ) subunit, IL-2 receptor γ (IL-2Rγ) subunit, or a combination thereof, relative to a wild-type IL-2 polypeptide, is about 25%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000- fold, or more.

[0207] In some embodiments, the IL-2 conjugate has a reduced IL-2Rβγ subunit recruitment to the IL-2 / IL-2Rβγ complex. In some embodiments, the reduced recruitment is compared to an IL- 2Rβγ subunit recruitment by an equivalent IL-2 polypeptide without the unnatural amino acid (e.g., a wild-type IL-2 polypeptide). In some embodiments, the decrease in IL-2Rβγ subunit recruitment is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease relative to an equivalent IL-2 polypeptide without the unnatural amino acid modification (e.g., a wild-type IL-2 polypeptide).

[0208] In some embodiments, the decrease in IL-2Rβγ subunit recruitment is about 50%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold, or more relative to an equivalent IL-2 polypeptide without the unnatural amino acid modification (e.g., a wild-type IL-2 polypeptide). In some embodiments, the IL-2 conjugate further has an increase in IL-2Rαβγ recruitment.

[0209] In some embodiments, the IL-2 conjugate has an increase in IL-2Rαβγ recruitment to the IL-2 polypeptide. In some embodiments, the reduced recruitment is compared to an IL-2Rαβγ recruitment by an equivalent IL-2 polypeptide without the unnatural amino acid (e.g., a wild- type IL-2 polypeptide). In some embodiments, the increase in IL-2Rαβγ recruitment is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% increase relative to an equivalent IL-2 polypeptide without the unnatural amino acid modification. In some embodiments, the IL-2 conjugate further has a decrease in recruitment of an IL-2Rβγ.

[0210] In some embodiments, the increase in IL-2Rαβγ recruitment is about 50%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300- fold, 400-fold, 500-fold, 1,000-fold, or more relative to an equivalent IL-2 polypeptide without the unnatural amino acid modification (e.g., a wild-type IL-2 polypeptide). In someAttorney Docket No. 01183-0317-00PCT embodiments, the IL-2 conjugate further has a decrease in recruitment of an IL-2Rγ subunit to an IL-2 / IL-2Rβ complex.

[0211] In some embodiments, an IL-2 conjugate described herein has a decrease in receptor signaling potency to IL-2Rβγ. In some embodiments, the decrease in receptor signaling potency is about 50%, 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 a wild-type IL-2 polypeptide.

[0212] In some embodiments, the receptor signaling potency is measured by an EC50 value. In some embodiments, the decrease in receptor signaling potency is an increase in EC50. In some embodiments, the increase in EC50 is about 50%, 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 a wild-type IL-2 polypeptide.

[0213] In some embodiments, the receptor signaling potency is measured by an ED50 value. In some embodiments, the decrease in receptor signaling potency is an increase in ED50. In some embodiments, the increase in ED50 is about 50%, 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 a wild-type IL-2 polypeptide.

[0214] In some embodiments, an IL-2 conjugate described herein has an expanded therapeutic window compared to a therapeutic window of a wild-type IL-2 polypeptide. In some embodiments, the expanded therapeutic window is due to a decrease in binding between the IL-2 conjugate and interleukin 2 receptor βγ (IL-2Rβγ), a decrease in receptor signaling potency to IL-2Rβγ, a decrease in recruitment of an IL-2Rβγ subunit to the IL-2 / IL-2Rαβγ complex, or an increase in recruitment of an IL-2Rαβγ to the IL-2 polypeptide. In some embodiments, the IL-2 conjugate does not have an impaired activation of interleukin 2 αβγ receptor (IL-2Rαβγ).

[0215] In some embodiments, the IL-2 conjugate exhibits a first receptor signaling potency to an IL-2βγ signaling complex and a second receptor signaling potency to an IL-2αβγ signaling complex, and wherein a difference between the first receptor signaling potency and the second receptor signaling potency is at least 50%. In some embodiments, the difference is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some embodiments, the first receptor signaling potency is less than the second receptor signaling potency. In some embodiments, the first receptor signaling potency is at least 25%, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100- fold, 500-fold, 1000-fold, or lower than the second receptor signaling potency. In someAttorney Docket No. 01183-0317-00PCT embodiments, the IL-2 conjugate has a lower receptor signaling potency to an IL-2βγ signaling complex than a second receptor signaling potency to an IL-2αβγ signaling complex. In some embodiments, the first receptor signaling potency of the IL-2 conjugate is at least 25% lower than a receptor signaling potency of the wild-type IL-2 polypeptide. In some embodiments, the first receptor signaling potency of the IL-2 conjugate is at least 25%, 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold lower than a receptor signaling potency of the wild-type IL-2 polypeptide. In some embodiments, the first receptor signaling potency and the second receptor signaling potency are both lower that the respective potencies of the wild-type IL-2 polypeptide, but the first receptor signaling potency is lower than the second receptor signaling potency. In some embodiments, the difference between the first receptor signaling potency and the second receptor signaling potency increases the therapeutic window for the IL-2 conjugate.

[0216] In some embodiments, the conjugating moiety impairs or blocks the receptor signaling potency of IL-2 with IL-2Rβγ, or reduces recruitment of the IL-2Rβ subunit and / or the IL-2Rγ subunit to the IL-2 / IL-2Rβγ complex. In some embodiments, the IL-2 conjugate further has a decrease in recruitment of an IL-2Rγ subunit to an IL-2 / IL-2Rβ complex.

[0217] In some embodiments, the IL-2 conjugate with the decrease in receptor signaling potency to IL-2Rβγ is capable of expanding CD4+ T regulatory (Treg) cells.

[0218] In some embodiments, CD4+ Treg cell proliferation by the modified IL-2 / IL-2Rαβγ complex is equivalent or greater to that of a wild-type IL-2 polypeptide.

[0219] In some embodiments, the IL-2 / IL-2Rαβγ complex induces proliferation of the CD4+ Treg cells to a population that is sufficient to modulate a disease course in an animal model. 1. Natural and Unnatural Amino acids

[0220] In some embodiments, an unnatural amino acid is not conjugated with a conjugating moiety. In some embodiments, a cytokine described herein comprises an unnatural amino acid, wherein the cytokine is conjugated to the protein, wherein the point of attachment is not the unnatural amino acid.

[0221] In some embodiments, an amino acid residue described herein (e.g., within a cytokine such as IL-2) is mutated to an unnatural amino acid prior to binding to a conjugating moiety. In some embodiments, the mutation to an unnatural amino acid prevents or minimizes a self- antigen response of the immune system. As used herein, the term "unnatural amino acid" or “non-canonical amino acid” refers to an amino acid other than the 20 amino acids that occur naturally in protein. Non-limiting examples of unnatural amino acids include: p-acetyl-L- phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-Attorney Docket No. 01183-0317-00PCT propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl- phenylalanine, O- 4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, o-azido-L-phenylalanine, m-azido-L- phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p- Boronophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl- L-phenylalanine, N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK), an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a lysine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid; an α, α disubstituted amino acid; a β-amino acid; a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan. In some embodiments, the unnatural amino acid is an azido-substituted lysine.

[0222] In some embodiments, the unnatural amino acid comprises a selective reactive group, or a reactive group for site-selective labeling of a target polypeptide. In some embodiments, the chemistry is a biorthogonal reaction (e.g., biocompatible and selective reactions). In some embodiments, the chemistry is a Cu(I)-catalyzed or “copper-free” alkyne-azide triazole-forming reaction, the Staudinger ligation, inverse-electron-demand Diels-Alder (IEDDA) reaction, “photo-click” chemistry, or a metal-mediated process such as olefin metathesis and Suzuki- Miyaura or Sonogashira cross-coupling.

[0223] In some embodiments, the unnatural amino acid comprises a photoreactive group, which crosslinks, upon irradiation with, e.g., UV.

[0224] In some embodiments, the unnatural amino acid comprises a photo-caged amino acid.Attorney Docket No. 01183-0317-00PCT

[0225] In some embodiments, the unnatural amino acid is a para-substituted, meta-substituted, or an ortho-substituted amino acid derivative.

[0226] In some embodiments, the unnatural amino acid comprises p-acetyl-L-phenylalanine, o- azidomethyl-L-phenylalanine, m-azidomethyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine, p- propargyloxyphenylalanine, o-propargyl-phenylalanine, m-propargyl-phenylalanine, p- propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O- 4-allyl-L- tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L- phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, or isopropyl-L-phenylalanine.

[0227] In some embodiments, the unnatural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4- dihydroxy-phenylalanine, or 3-iodotyrosine.

[0228] In some embodiments, the unnatural amino acid is phenylselenocysteine.

[0229] In some embodiments, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide containing phenylalanine derivative.

[0230] In some embodiments, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide containing lysine derivative.

[0231] In some embodiments, the unnatural amino acid comprises an aromatic side chain.

[0232] In some embodiments, the unnatural amino acid does not comprise an aromatic side chain.

[0233] In some embodiments, the unnatural amino acid comprises an azido group.

[0234] In some embodiments, the unnatural amino acid comprises a Michael-acceptor group. In some embodiments, Michael-acceptor groups comprise an unsaturated moiety capable of forming a covalent bond through a 1,2-addition reaction. In some embodiments, Michael- acceptor groups comprise electron-deficient alkenes or alkynes. In some embodiments, Michael- acceptor groups include but are not limited to alpha,beta unsaturated: ketones, aldehydes, sulfoxides, sulfones, nitriles, imines, or aromatics.

[0235] In some embodiments, the unnatural amino acid is dehydroalanine.

[0236] In some embodiments, the unnatural amino acid comprises an aldehyde or ketone group.

[0237] In some embodiments, the unnatural amino acid is a lysine derivative comprising an aldehyde or ketone group.

[0238] In some embodiments, the unnatural amino acid is a lysine derivative comprising one or more O, N, Se, or S atoms at the beta, gamma, or delta position. In some embodiments, theAttorney Docket No. 01183-0317-00PCT unnatural amino acid is a lysine derivative comprising O, N, Se, or S atoms at the gamma position.

[0239] In some embodiments, the unnatural amino acid is a lysine derivative wherein the epsilon N atom is replaced with an oxygen atom.

[0240] In some embodiments, the unnatural amino acid is a lysine derivative that is not naturally-occurring post-translationally modified lysine.

[0241] In some embodiments, the unnatural amino acid is an amino acid comprising a side chain, wherein the sixth atom from the alpha position comprises a carbonyl group. In some embodiments, the unnatural amino acid is an amino acid comprising a side chain, wherein the sixth atom from the alpha position comprises a carbonyl group, and the fifth atom from the alpha position is a nitrogen. In some embodiments, the unnatural amino acid is an amino acid comprising a side chain, wherein the seventh atom from the alpha position is an oxygen atom.

[0242] In some embodiments, the unnatural amino acid is a serine derivative comprising selenium. In some embodiments, the unnatural amino acid is selenoserine (2-amino-3- hydroselenopropanoic acid). In some embodiments, the unnatural amino acid is 2-amino-3-((2- ((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid. In some embodiments, the unnatural amino acid is 2-amino-3-(phenylselanyl)propanoic acid. In some embodiments, the unnatural amino acid comprises selenium, wherein oxidation of the selenium results in the formation of an unnatural amino acid comprising an alkene.

[0243] In some embodiments, the unnatural amino acid comprises a cyclooctynyl group.

[0244] In some embodiments, the unnatural amino acid comprises a transcycloctenyl group.

[0245] In some embodiments, the unnatural amino acid comprises a norbornenyl group.

[0246] In some embodiments, the unnatural amino acid comprises a cyclopropenyl group.

[0247] In some embodiments, the unnatural amino acid comprises a diazirine group.

[0248] In some embodiments, the unnatural amino acid comprises a tetrazine group.

[0249] In some embodiments, the unnatural amino acid is a lysine derivative, wherein the side- chain nitrogen is carbamylated. In some embodiments, the unnatural amino acid is a lysine derivative, wherein the side-chain nitrogen is acylated. In some embodiments, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is N6-Boc-N6-methyllysine. In some embodiments, the unnatural amino acid is N6-acetyllysine. In some embodiments, the unnatural amino acid is pyrrolysine. In some embodiments, the unnatural amino acid is N6-trifluoroacetyllysine. In some embodiments, the unnatural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoicAttorney Docket No. 01183-0317-00PCT acid. In some embodiments, the unnatural amino acid is 2-amino-6-{[(p- iodobenzyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is N6-prolyllysine. In some embodiments, the unnatural amino acid is 2- amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine. In some embodiments, the unnatural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some embodiments, the unnatural amino acid is N6- (3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some embodiments, the unnatural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some embodiments, the unnatural amino acid is 2- amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is N6-(2-azidoethoxy)-carbonyl-lysine. In some embodiments, the unnatural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is 2-amino-6-{[(2- cyclooctynyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. In some embodiments, the unnatural amino acid is 2- amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid. In some embodiments, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some embodiments, the unnatural amino acid is N6- (butenyl-4-oxycarbonyl)lysine. In some embodiments, the unnatural amino acid is N6-(pentenyl- 5-oxycarbonyl)lysine. In some embodiments, the unnatural amino acid is N6-((but-3-yn-1- yloxy)carbonyl)-lysine. In some embodiments, the unnatural amino acid is N6-((pent-4-yn-1- yloxy)carbonyl)-lysine. In some embodiments, the unnatural amino acid is N6-(thiazolidine-4- carbonyl)lysine. In some embodiments, the unnatural amino acid is 2-amino-8-oxononanoic acid. In some embodiments, the unnatural amino acid is 2-amino-8-oxooctanoic acid. In some embodiments, the unnatural amino acid is N6-(2-oxoacetyl)lysine.

[0250] In some embodiments, the unnatural amino acid is N6-propionyllysine. In some embodiments, the unnatural amino acid is N6-butyryllysine, In some embodiments, the unnatural amino acid is N6-(but-2-enoyl)lysine, In some embodiments, the unnatural amino acid is N6- ((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some embodiments, the unnatural amino acid is N6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl)lysine. In some embodiments, the unnatural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1- yl)benzyl)oxy)carbonyl)lysine. In some embodiments, the unnatural amino acid is N6- ((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)lysine. In some embodiments, the unnatural amino acid is cysteinyllysine. In some embodiments, the unnatural amino acid is N6-((1-(6- nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine. In some embodiments, the unnaturalAttorney Docket No. 01183-0317-00PCT amino acid is N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some embodiments, the unnatural amino acid is N6-((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In some embodiments, the unnatural amino acid is N6-((meta nitrobenyloxy)N6-methylcarbonyl)lysine. In some embodiments, the unnatural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9- ylmethoxy)carbonyl)-lysine. In some embodiments, the unnatural amino acid is N6-((cyclohept- 3-en-1-yloxy)carbonyl)-L-lysine.

[0251] In some embodiments, the unnatural amino acid is 2-amino-3- (((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.

[0252] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a repurposed amber, opal, or ochre stop codon.

[0253] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a 4-base codon.

[0254] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a repurposed rare sense codon.

[0255] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a synthetic codon comprising an unnatural nucleic acid.

[0256] In some embodiments, the unnatural amino acid is incorporated into the cytokine by an orthogonal, modified synthetase / tRNA pair. Such orthogonal pairs comprise an unnatural synthetase that is capable of charging the unnatural tRNA with the unnatural amino acid, while minimizing charging of a) other endogenous amino acids onto the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs. Such orthogonal pairs comprise tRNAs that are capable of being charged by the unnatural synthetase, while avoiding being charged with a) other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, Archaea, or human sources. In some embodiments, an orthogonal synthetase / tRNA pair comprises components from a single organism. In some embodiments, an orthogonal synthetase / tRNA pair comprises components from two different organisms. In some embodiments, an orthogonal synthetase / tRNA pair comprising components that prior to modification, promote translation of two different amino acids. In some embodiments, an orthogonal synthetase is a modified alanine synthetase. In some embodiments, an orthogonal synthetase is a modified arginine synthetase. In some embodiments, an orthogonal synthetase is a modified asparagine synthetase. In some embodiments, an orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, an orthogonal synthetase is a modified cysteine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamine synthetase. In some embodiments, an orthogonal synthetaseAttorney Docket No. 01183-0317-00PCT is a modified glutamic acid synthetase. In some embodiments, an orthogonal synthetase is a modified alanine glycine. In some embodiments, an orthogonal synthetase is a modified histidine synthetase. In some embodiments, an orthogonal synthetase is a modified leucine synthetase. In some embodiments, an orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, an orthogonal synthetase is a modified lysine synthetase. In some embodiments, an orthogonal synthetase is a modified methionine synthetase. In some embodiments, an orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, an orthogonal synthetase is a modified proline synthetase. In some embodiments, an orthogonal synthetase is a modified serine synthetase. In some embodiments, an orthogonal synthetase is a modified threonine synthetase. In some embodiments, an orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, an orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, an orthogonal synthetase is a modified valine synthetase. In some embodiments, an orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, an orthogonal tRNA is a modified alanine tRNA. In some embodiments, an orthogonal tRNA is a modified arginine tRNA. In some embodiments, an orthogonal tRNA is a modified asparagine tRNA. In some embodiments, an orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, an orthogonal tRNA is a modified cysteine tRNA. In some embodiments, an orthogonal tRNA is a modified glutamine tRNA. In some embodiments, an orthogonal tRNA is a modified glutamic acid tRNA. In some embodiments, an orthogonal tRNA is a modified alanine glycine. In some embodiments, an orthogonal tRNA is a modified histidine tRNA. In some embodiments, an orthogonal tRNA is a modified leucine tRNA. In some embodiments, an orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, an orthogonal tRNA is a modified lysine tRNA. In some embodiments, an orthogonal tRNA is a modified methionine tRNA. In some embodiments, an orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, an orthogonal tRNA is a modified proline tRNA. In some embodiments, an orthogonal tRNA is a modified serine tRNA. In some embodiments, an orthogonal tRNA is a modified threonine tRNA. In some embodiments, an orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, an orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, an orthogonal tRNA is a modified valine tRNA. In some embodiments, an orthogonal tRNA is a modified phosphoserine tRNA.

[0257] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pairs, E. coli TyrRS (Ec-Tyr) / B. stearothermophilus tRNACUA pairs, E. coli LeuRSAttorney Docket No. 01183-0317-00PCT (Ec-Leu) / B. stearothermophilus tRNACUA pairs, and pyrrolysyl-tRNA pairs. In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Mj-TyrRS / tRNA pair. Exemplary UAAs that can be incorporated by a Mj- TyrRS / tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronophenylalanine; and o-nitrobenzyltyrosine.

[0258] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Ec-Tyr / tRNACUA or a Ec-Leu / tRNACUA pair. Exemplary UAAs that can be incorporated by a Ec-Tyr / tRNACUA or a Ec-Leu / tRNACUA pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O-propargyltyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3-(naphthalene-2-ylamino)-2-amino-propanoic acid.

[0259] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a pyrrolysyl-tRNA pair. In some embodiments, the PylRS is obtained from an archaebacterial, e.g., from a methanogenic archaebacterial. In some embodiments, the PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated by a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate substituted lysines such as 2-amino-6-((R)- tetrahydrofuran-2-carboxamido)hexanoic acid, N-ε-D-prolyl-L-lysine, and N-ε- cyclopentyloxycarbonyl-L-lysine; N-ε-Acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5- yl)ethoxy)carbonyl]-L-lysine; and N-ε-(1-methylcyclopro-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein may be prepared by use of M. mazei Pyl tRNA which is selectively charged with a non-natural amino acid such as N6-(2-azidoethoxy)- carbonyl-L-lysine (AzK) by the M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS). Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647.

[0260] In some embodiments, an unnatural amino acid is incorporated into a cytokine described herein (e.g., the IL polypeptide) by a synthetase disclosed in US 9,988,619 and US 9,938,516. Exemplary UAAs that can be incorporated by such synthetases include para-methylazido-L- phenylalanine, aralkyl, heterocyclyl, heteroaralkyl unnatural amino acids, and others. In some embodiments, such UAAs comprise pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycle. Such amino acids in some embodiments comprise azides, tetrazines, or other chemical group capable of conjugation to a coupling partner, such as a water-Attorney Docket No. 01183-0317-00PCT soluble moiety. In some embodiments, such synthetases are expressed and used to incorporate UAAs into cytokines in-vivo. In some embodiments, such synthetases are used to incorporate UAAs into cytokines using a cell-free translation system.

[0261] In some embodiments, an unnatural amino acid is incorporated into a cytokine described herein (e.g., the IL polypeptide) by a naturally occurring synthetase. In some embodiments, an unnatural 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 the auxotrophic amino acid are capable of charging the corresponding tRNA with an unnatural amino acid. In some embodiments, the unnatural amino acid is selenocysteine, or a derivative thereof. In some embodiments, the unnatural amino acid is selenomethionine, or a derivative thereof. In some embodiments, the unnatural amino acid is an aromatic amino acid, wherein the aromatic amino acid comprises an aryl halide, such as an iodide. In embodiments, the unnatural amino acid is structurally similar to the auxotrophic amino acid.

[0262] In some embodiments, the unnatural amino acid comprises an unnatural amino acid described and illustrated in, for example, in International Publication Number WO 2021 / 050554 A1, which is hereby incorporate by reference in its entirety.

[0263] In some embodiments, the unnatural amino acid comprises a lysine or phenylalanine derivative or analogue. In some embodiments, the unnatural amino acid comprises a lysine derivative or a lysine analogue. In some embodiments, the unnatural amino acid comprises a pyrrolysine (Pyl). In some embodiments, the unnatural amino acid comprises a phenylalanine derivative or a phenylalanine analogue. In some embodiments, the unnatural amino acid is an unnatural amino acid described in Wan, et al., “Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool,” Biochim Biophys Acta 1844(6): 1059-4070 (2014).

[0264] In some embodiments, an unnatural amino acid incorporated into a cytokine described herein (e.g., the IL polypeptide) is disclosed in US 9,840,493; US 9,682,934; US 2017 / 0260137; US 9,938,516; or US 2018 / 0086734. Exemplary UAAs that can be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl, and lysine derivative unnatural amino acids. In some embodiments, such UAAs comprise pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycle. Such amino acids in some embodiments comprise azides, tetrazines, or other chemical group capable of conjugation to a coupling partner, such as a water soluble moiety. In some embodiments, a UAA comprises an azide attached to an aromatic moiety via an alkyl linker. In some embodiments, an alkyl linker is a C1-C10 linker. In some embodiments, a UAA comprisesAttorney Docket No. 01183-0317-00PCT a tetrazine attached to an aromatic moiety via an alkyl linker. In some embodiments, a UAA comprises a tetrazine attached to an aromatic moiety via an amino group. In some embodiments, a UAA comprises a tetrazine attached to an aromatic moiety via an alkylamino group. In some embodiments, a UAA comprises an azide attached to the terminal nitrogen (e.g., N6 of a lysine derivative, or N5, N4, or N3 of a derivative comprising a shorter alkyl side chain) of an amino acid side chain via an alkyl chain. In some embodiments, a UAA comprises a tetrazine attached to the terminal nitrogen of an amino acid side chain via an alkyl chain. In some embodiments, a UAA comprises an azide or tetrazine attached to an amide via an alkyl linker. In some embodiments, the UAA is an azide or tetrazine-containing carbamate or amide of 3- aminoalanine, serine, lysine, or 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. 2. Conjugating Moieties

[0265] In some embodiments, disclosed herein are conjugating moieties that are bound to one or more cytokines (e.g., interleukins, IFNs, or TNFs) described supra. In some embodiments, the conjugating moiety is a molecule that perturbs the interaction of a cytokine with its receptor. In some embodiments, the conjugating moiety is any molecule that when bound to the cytokine, enables the cytokine conjugate to modulate an immune response. In some embodiments, the conjugating moiety is bound to the cytokine through a covalent bond. In some embodiments, a cytokine described herein is attached to a conjugating moiety with a triazole group. In some embodiments, a cytokine described herein is attached to a conjugating moiety with a dihydropyridazine or pyridazine group. In some embodiments, the conjugating moiety comprises a water-soluble polymer. In other instances, the conjugating moiety comprises a protein or a binding fragment thereof. In additional instances, the conjugating moiety comprises a peptide. In additional instances, the conjugating moiety comprises a nucleic acid. In additional instances, the conjugating moiety comprises a small molecule. In additional instances, the conjugating moiety comprises a bioconjugate (e.g., a TLR agonist such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or Flagellin). In some embodiments, the conjugating moiety increases serum half-life, and / or improves stability. In some embodiments, the conjugating moiety reduces cytokine interaction with one or more cytokine receptor domains or subunits. In additional cases, the conjugating moiety blocks cytokine interaction with one or more cytokine domains or subunits with its cognate receptor(s). In some embodiments, cytokine conjugates described herein compriseAttorney Docket No. 01183-0317-00PCT multiple conjugating moieties. In some embodiments, a conjugating moiety is attached to an unnatural amino acid in the cytokine peptide. In some embodiments, a cytokine conjugate is attached to an unnatural amino acid in the cytokine peptide. In some embodiments, a conjugating moiety is attached to the N or C terminal amino acid of the cytokine peptide. Various combinations sites are disclosed herein, for example, a first conjugating moiety is attached to an unnatural amino acid in the cytokine peptide, and a second conjugating moiety is attached to the N or C terminal amino acid of the cytokine peptide. In some embodiments, a single conjugating moiety is attached to multiple residues of the cytokine peptide (e.g. a staple). In some embodiments, a conjugating moiety is attached to both the N and C terminal amino acids of the cytokine peptide.

[0266] In some embodiments, a conjugating moiety described herein is a water-soluble polymer. In some embodiments, the water-soluble polymer is a nonpeptidic, nontoxic, and biocompatible. As used herein, a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as a cytokine moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist. In some embodiments, a water-soluble polymer is further non- immunogenic. In some embodiments, a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician, e.g., a physician, a toxicologist, or a clinical development specialist.

[0267] In some embodiments, the water-soluble polymer is characterized as having from about 2 to about 300 termini. Exemplary water soluble polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol (“PEG”), poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazolines (“POZ”) (which are described in WO 2008 / 106186), poly(N-acryloylmorpholine), and combinations of any of the foregoing.

[0268] In some embodiments, the water-soluble polymer is not limited to a particular structure. In some embodiments, the water-soluble polymer is linear (e.g., an end capped, e.g., alkoxy PEGAttorney Docket No. 01183-0317-00PCT or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages. Moreover, the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.

[0269] In some embodiments, the water-soluble polymer is represented by a length of repeating polymeric units, for example, a number n of polyethylene glycol units. In some embodiments, the water-soluble polymer has the structure:

[0270] , wherein the wavy line indicates attachment to a linker, reactive group, or unnatural amino acid, and n is 1-5000. In some embodiments, the water-soluble polymer has the structure:

[0271] , wherein the wavy line indicates attachment to a linker, reactive group, or unnatural amino acid, “Cap” indicates a capping group (for example, such as –OCH3, -O(C1-C6 alkyl), - SMe, -S(C1-C6 alkyl), -CO2H, -CO2(C1-C6 alkyl), -CONH2, -CONH(C1-C6 alkyl), -CON(C1- C6 alkyl)2, -NH2, -SH, or OH) and n is 1-5000. In some embodiments, n is 100-2000, 200-1000, 300-750, 400-600, 450-550, 400-2000, 750-3000, or 100-750. In some embodiments, n is about 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000. In some embodiments, n is at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or at least 1000. In some embodiments, n is no more than 100, 200, 300, 400, 500, 600, 700, 800, 900, or no more than 1000. In some embodiments, the n is represented as an average length of the water-soluble polymer.

[0272] 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 kDa. Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5 kDa to about 100 kDa, in the range of from about 6 kDa to about 90 kDa, in the range of from about 10 kDa to about 85 kDa, in the range of greater than 10 kDa to about 85 kDa, in the range of from about 20 kDa to about 85 kDa, in the range of from about 53 kDa to about 85 kDa, in the range of from about 25 kDa to about 120 kDa, in the range of from about 29 kDa to about 120 kDa, in the range of from about 35 kDa to about 120 kDa, and in the range of from about 40 kDa to about 120 kDa.

[0273] Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1 kDa, about 1.5 kDa, about 2 kDa, about 2.2 kDa, about 2.5 kDa, about 3 kDa, about 4 kDa, about 4.4 kDa, about 4.5 kDa, about 5 kDa, about 5.5 kDa, about 6 kDa, about 7 kDa, aboutAttorney Docket No. 01183-0317-00PCT 7.5 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 20 kDa, about 22.5 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, and about 75 kDa. Branched versions of the water-soluble polymer (e.g., a branched 40 kDa water-soluble polymer comprised of two 20 kDa polymers) having a total molecular weight of any of the foregoing can also be used. In one or more embodiments, the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight average molecular weight of less than about 6 kDa.

[0274] PEGs will typically comprise a number of (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how the PEG is defined]. As used herein, the number of repeating units is identified by the subscript “n” in “(OCH2CH2)n.” Thus, the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For any given polymer in which the molecular weight is known, it is possible to determine the number of repeating units (i.e., “n”) by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0275] In some embodiments, the water-soluble polymer is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower C1-6 alkoxy group, or a hydroxyl group. When the polymer is PEG, for example, a methoxy-PEG (commonly referred to as mPEG) may be used, which is a linear form of PEG wherein one terminus of the polymer is a methoxy (—OCH3) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified.

[0276] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of from about 100 Daltons to about 150 kDa. Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5 kDa to about 100 kDa, in the range of fromAttorney Docket No. 01183-0317-00PCT about 6 kDa to about 90 kDa, in the range of from about 10 kDa to about 85 kDa, in the range of greater than 10 kDa to about 85 kDa, in the range of from about 20 kDa to about 85 kDa, in the range of from about 53 kDa to about 85 kDa, in the range of from about 25 kDa to about 120 kDa, in the range of from about 29 kDa to about 120 kDa, in the range of from about 35 kDa to about 120 kDa, and in the range of from about 40 kDa to about 120 kDa. Exemplary weight- average molecular weights for the PEG group include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1 kDa, about 1.5 kDa, about 2 kDa, about 2.2 kDa, about 2.5 kDa, about 3 kDa, about 4 kDa, about 4.4 kDa, about 4.5 kDa, about 5 kDa, about 5.5 kDa, about 6 kDa, about 7 kDa, about 7.5 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 20 kDa, about 22.5 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 95 kDa, and about 100 kDa. In some embodiments, the PEG group is a linear PEG group having an average molecular weight as disclosed above. In some embodiments, the PEG group is a branched PEG group having an average molecular weight as disclosed above. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group having a defined molecular weight ± 10%, or 15% or 20% or 25%. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da ± 3000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 6,000 Da.

[0277] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group having an average molecular weight of from about 5 kDa to about 60 kDa. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5 kDa, about 5.5 kDa, about 6 kDa, about 7 kDa, about 7.5 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 20 kDa, about 22.5 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 95 kDa, and about 100 kDa. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a linear PEG group having an average molecular of aboutAttorney Docket No. 01183-0317-00PCT 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a branched PEG group having an average molecular weight of about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa.

[0278] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear methoxy PEG group having an average molecular weight of from about 5 kDa to about 60 kDa. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5 kDa, about 5.5 kDa, about 6 kDa, about 7 kDa, about 7.5 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 20 kDa, about 22.5 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 95 kDa, and about 100 kDa. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 30 kDa. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 50 kDa. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular of about 60 kDa. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear methoxy PEG group having a defined molecular weight ± 10%, or 15% or 20% or 25%. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a linear methoxy PEG group having a molecular weight of 30,000 Da ± 3000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 6,000 Da.

[0279] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a branched methoxy PEG group having an average molecular weight of from about 5 kDa to about 60 kDa. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5 kDa, about 5.5 kDa, about 6 kDa, about 7 kDa, about 7.5 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 20 kDa, about 22.5 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 95 kDa, and about 100 kDa. InAttorney Docket No. 01183-0317-00PCT some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular of about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 50 kDa, or about 60 kDa. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a branched methoxy PEG group having a defined molecular weight ± 10%, or 15% or 20% or 25%. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a branched methoxy PEG group having a molecular weight of 30,000 Da ± 3000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 6,000 Da.

[0280] In some embodiments, exemplary water-soluble polymers include, but are not limited to, linear or branched discrete PEG (dPEG) from Quanta Biodesign, Ltd; linear, branched, or forked PEGs from Nektar Therapeutics; and Y-shaped PEG derivatives from JenKem Technology.

[0281] 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(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α- hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene, polyoxazolines (“POZ”), poly(N-acryloylmorpholine), and a combination thereof. In some embodiments, the IL-2 polypeptide is conjugated to PEG (e.g., PEGylated). In some embodiments, the IL-2 polypeptide is conjugated to PPG. In some embodiments, the IL-2 polypeptide is conjugated to POZ. In some embodiments, the IL-2 polypeptide is conjugated to PVP.

[0282] In some embodiments, a water-soluble polymer comprises a polyglycerol (PG). In some embodiments, the polyglycerol is a hyperbranched PG (HPG) (e.g., as described by Imran, et al. “Influence of architecture of high molecular weight linear and branched polyglycerols on their biocompatibility and biodistribution,” Biomaterials 33:9135–9147 (2012)). In other cases, the polyglycerol is a linear PG (LPG). In additional cases, the polyglycerol is a midfunctional PG, a linear-block-hyperbranched PG (e.g., as described by Wurm et. Al., “Squaric acid mediatedAttorney Docket No. 01183-0317-00PCT synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)−protein conjugates,” Biomacromolecules 13:1161–1171 (2012)), or a side-chain functional PG (e.g., as described by Li, et. al., “Synthesis of linear polyether polyol derivatives as new materials for bioconjugation,” Bioconjugate Chem. 20:780–789 (2009).

[0283] In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide described herein is conjugated to a PG, e.g., a HPG, a LPG, a midfunctional PG, a linear-block- hyperbranched PG, or a side-chain functional PG. In some embodiments, the cytokine is an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is conjugated to a PG, a midfunctional PG, a linear-block-hyperbranched PG.

[0284] In some embodiments, a water-soluble polymer is a degradable synthetic PEG alternative. Exemplary degradable synthetic PEG alternatives include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; PEG derivatives comprising comonomers comprising degradable linkage such as poly[(ethylene oxide)-co-(methylene ethylene oxide)][P(EO-co-MEO)], cyclic ketene acetals such as 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene-1,3- dioxepane (MDO), and 2-methylene-4-phenyl-1,3-dioxolane (MPDL) copolymerized with OEGMA; or poly-(ε- caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

[0285] In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide described herein is conjugated to a degradable synthetic PEG alternative, such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di- end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO. In some embodiments, the cytokine is an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is conjugated to a degradable synthetic PEG alternative, such as for example, POEGM; backbone modified PEG derivatives generated by polymerization of telechelic, or di-end-functionalized PEG-based macromonomers; P(EO-co-MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.

[0286] In some embodiments, a water-soluble polymer comprises a poly(zwitterions). Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methyacryloyloxyethyl phosphorylcholine) (PMPC). In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide described herein is conjugated to a poly(zwitterion) such as PSBMA, PCBMA, or PMPC. In some embodiments, the cytokine is an IL-2 polypeptide. In someAttorney Docket No. 01183-0317-00PCT embodiments, the IL-2 polypeptide is conjugated to a poly(zwitterion) such as PSBMA, PCBMA, or PMPC.

[0287] In some embodiments, a water-soluble polymer comprises a polycarbonate. Exemplary polycarbones include, but are not limited to, pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5- carboxylate (MTC-OC6F5). In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide described herein is conjugated to a polycarbonate such as MTC-OC6F5. In some embodiments, the cytokine is an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is conjugated to a polycarbonate such as MTC-OC6F5.

[0288] In some embodiments, a water-soluble polymer comprises 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., a hydroxyl containing and / or zwitterionic derivatized PEG polymer). In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide described herein is conjugated to a polymer hybrid such as a polycarbonate / PEG polymer hybrid, a peptide / protein-polymer conjugate, or a hydroxyl containing and / or zwitterionic derivatized polymer (e.g., a hydroxyl containing and / or zwitterionic derivatized PEG polymer). In some embodiments, the cytokine is an IL-2 polypeptide. In some embodiments, 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).

[0289] In some embodiments, a water-soluble polymer comprises a polysaccharide. Exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES). In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a polysaccharide. In some embodiments, an IL-2 polypeptide is conjugated to dextran. In some embodiments, an IL-2 polypeptide is conjugated to PSA. In some embodiments, an IL-2 polypeptide is conjugated to HA. In some embodiments, an IL-2 polypeptide is conjugated to amylose. In some embodiments, an IL-2 polypeptide is conjugated to heparin. In some embodiments, an IL-2 polypeptide is conjugated to HS. In some embodiments, an IL-2 polypeptide is conjugated to dextrin. In some embodiments, an IL-2 polypeptide is conjugated to HES.

[0290] In some embodiments, a water-soluble polymer comprises a glycan. Exemplary classes of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF)Attorney Docket No. 01183-0317-00PCT polypeptide is conjugated to a glycan. In some embodiments, an IL-2 polypeptide is conjugated to N-linked glycans. In some embodiments, an IL-2 polypeptide is conjugated to O-linked glycans. In some embodiments, an IL-2 polypeptide is conjugated to glycolipids. In some embodiments, an IL-2 polypeptide is conjugated to O-GlcNAc. In some embodiments, an IL-2 polypeptide is conjugated to glycosaminoglycans.

[0291] In some embodiments, a water-soluble polymer comprises a polyoxazoline polymer. A polyoxazoline polymer is a linear synthetic polymer, and similar to PEG, comprises a low polydispersity. In some embodiments, a polyoxazoline polymer is a polydispersed polyoxazoline polymer, characterized with an average molecule weight. In some embodiments, the average molecule weight of a polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. In some embodiments, a polyoxazoline polymer comprises poly(2-methyl 2-oxazoline) (PMOZ), poly(2- ethyl 2-oxazoline) (PEOZ), or poly(2-propyl 2-oxazoline) (PPOZ). In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a polyoxazoline polymer. In some embodiments, an IL-2 polypeptide is conjugated to a polyoxazoline polymer. In some embodiments, an IL-2 polypeptide is conjugated to PMOZ. In some embodiments, an IL-2 polypeptide is conjugated to PEOZ. In some embodiments, an IL-2 polypeptide is conjugated to PPOZ.

[0292] In some embodiments, a water-soluble polymer comprises a polyacrylic acid polymer. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a polyacrylic acid polymer. In some embodiments, an IL-2 polypeptide is conjugated to a polyacrylic acid polymer.

[0293] In some embodiments, a water-soluble polymer comprises polyamine. Polyamine is an organic polymer comprising two or more primary amino groups. In some embodiments, a polyamine includes a branched polyamine, a linear polyamine, or cyclic polyamine. In some embodiments, a polyamine is a low-molecular-weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a polyamine. In some embodiments, an IL-2 polypeptide is conjugated to polyamine. In some embodiments, an IL-2 polypeptide is conjugated to putrescine, cadaverine, spermidine, spermine, ethylene diamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, or piperazine.Attorney Docket No. 01183-0317-00PCT

[0294] In some embodiments, a water-soluble polymer is described in US Patent Nos. 7,744,861, 8,273,833, and 7,803,777. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a linker described in US Patent No. 7,744,861, 8,273,833, or 7,803,777. In some embodiments, an IL-2 polypeptide is conjugated to a linker described in US Patent No. 7,744,861, 8,273,833, or 7,803,777.

[0295] In some embodiments, a conjugating moiety described herein is a lipid. In some embodiments, the lipid is a fatty acid. In some embodiments, the fatty acid is a saturated fatty acid. In other cases, the fatty acid is an unsaturated fatty acid. Exemplary fatty acids include, but are not limited to, fatty acids comprising from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some embodiments, the lipid binds to one or more serum proteins, thereby increasing serum stability and / or serum half-life.

[0296] In some embodiments, the lipid is conjugated to IL-2. In some embodiments, the lipid is a fatty acid, e.g., a saturated fatty acid or an unsaturated fatty acid. In some embodiments, the fatty acid is from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some embodiments, the fatty acid comprises about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length. In some embodiments, the fatty acid comprises caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylic acid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic acid), heneicosylic acid (heneicosanoic acid), behenic acid (docosanoic acid), tricosylic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosylic acid (pentacosanoic acid), or cerotic acid (hexacosanoic acid).

[0297] In some embodiments, the IL-2 lipid conjugate enhances serum stability and / or serum half-life.Attorney Docket No. 01183-0317-00PCT

[0298] In some embodiments, a conjugating moiety described herein is a protein or a binding fragment thereof. Exemplary proteins include albumin, transferrin, or transthyretin. In some embodiments, the protein or a binding fragment thereof comprises an antibody, or its binding fragments thereof. In some embodiments, a cytokine conjugate comprises a protein or a binding fragment thereof. In some embodiments, an IL-2 conjugate comprising a protein or a binding fragment thereof has an increased serum half-life, and / or stability. In some embodiments, an IL-2 conjugate comprising a protein or a binding fragment thereof has a reduced IL-2 interaction with one or more IL-2R subunits. In additional cases, the protein or a binding fragment thereof blocks IL-2 interaction with one or more IL-2R subunits.

[0299] In some embodiments, the conjugating moiety is albumin. Albumin is a family of water- soluble globular proteins. It is commonly found in blood plasma, comprising about 55-60% of all plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide in which the tertiary structure is divided into three domains, domain I (amino acid residues 1-195), domain II (amino acid residues 196-383), and domain III (amino acid residues 384-585). Each domain further comprises a binding site, which can interact either reversibly or irreversibly with endogenous ligands such as long- and medium-chain fatty acids, bilirubin, or hemin, or exogenous compounds such as heterocyclic or aromatic compounds.

[0300] In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to albumin. In some embodiments, the cytokine polypeptide is conjugated to human serum albumin (HSA). In additional cases, the cytokine polypeptide is conjugated to a functional fragment of albumin.

[0301] In some embodiments, an IL-2 polypeptide is conjugated to albumin. In some embodiments, the IL-2 polypeptide is conjugated to human serum albumin (HSA). In additional cases, the IL-2 polypeptide is conjugated to a functional fragment of albumin.

[0302] In some embodiments, the conjugating moiety is transferrin. Transferrin is a 679 amino acid polypeptide that is about 80 kDa in size and comprises two Fe3+ binding sites with one at the N-terminal domain and the other at the C-terminal domain. In some embodiments, human transferrin has a half-life of about 7-12 days.

[0303] In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to transferrin. In some embodiments, the cytokine polypeptide is conjugated to human transferrin. In additional cases, the cytokine polypeptide is conjugated to a functional fragment of transferrin.Attorney Docket No. 01183-0317-00PCT

[0304] In some embodiments, an IL-2 polypeptide is conjugated to transferrin. In some embodiments, the IL-2 polypeptide is conjugated to human transferrin. In additional cases, the IL-2 polypeptide is conjugated to a functional fragment of transferrin.

[0305] In some embodiments, the conjugating moiety is transthyretin (TTR). Transthyretin is a transport protein located in the serum and cerebrospinal fluid which transports the thyroid hormone thyroxine (T4) and retinol-binding protein bound to retinol.

[0306] In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to transthyretin (via one of its termini or via an internal hinge region). In some embodiments, the cytokine polypeptide is conjugated to a functional fragment of transthyretin.

[0307] In some embodiments, an IL-2 polypeptide is conjugated to transthyretin (via one of its termini or via an internal hinge region). In some embodiments, the IL-2 polypeptide is conjugated to a functional fragment of transthyretin.

[0308] In some embodiments, the conjugating moiety is an antibody, or its binding fragments thereof. In some embodiments, an antibody or its binding fragments thereof comprise a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)'3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.

[0309] In some embodiments, the conjugating moiety comprises a scFv, bis-scFv, (scFv)2, dsFv, or sdAb. In some embodiments, the conjugating moiety comprises a scFv. In some embodiments, the conjugating moiety comprises a bis-scFv. In some embodiments, the conjugating moiety comprises a (scFv)2. In some embodiments, the conjugating moiety comprises a dsFv. In some embodiments, the conjugating moiety comprises a sdAb.

[0310] In some embodiments, the conjugating moiety comprises an Fc portion of an antibody, e.g., of IgG, IgA, IgM, IgE, or IgD. In some embodiments, the moiety comprises an Fc portion of IgG (e.g., IgG1, IgG3, or IgG4).

[0311] In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to an antibody, or its binding fragments thereof. In some embodiments, the cytokine polypeptide is conjugated to a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)'3 fragments, single-Attorney Docket No. 01183-0317-00PCT chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof. In additional cases, the cytokine polypeptide is conjugated to an Fc portion of an antibody. In additional cases, the cytokine polypeptide is conjugated to an Fc portion of IgG (e.g., IgG1, IgG3, or IgG4).

[0312] In some embodiments, an IL-2 polypeptide is conjugated to an antibody, or its binding fragments thereof. In some embodiments, the IL-2 polypeptide is conjugated to a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)'3 fragments, single-chain variable fragment (scFv), bis- scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof. In additional cases, the IL-2 polypeptide is conjugated to an Fc portion of an antibody. In additional cases, the IL-2 polypeptide is conjugated to an Fc portion of IgG (e.g., IgG1, IgG3, or IgG4).

[0313] In some embodiments, an IL-2 polypeptide is conjugated to a water-soluble polymer (e.g., PEG) and an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragments thereof comprises a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)'3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, humabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the antibody or binding fragments thereof comprises a scFv, bis-scFv, (scFv)2, dsFv, or sdAb. In some embodiments, the antibody or binding fragments thereof comprises a scFv. In some embodiments, the antibody or binding fragment thereof guides the IL-2 conjugate to a target cell of interest and the water-soluble polymer enhances stability and / or serum half-life.

[0314] In some embodiments, one or more IL-2 polypeptide – water-soluble polymer (e.g., PEG) conjugates are further bound to an antibody or binding fragments thereof. In some embodiments, the ratio of the IL-2 conjugate to the antibody is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. In some embodiments, the ratio of the IL-2 conjugate to the antibody is aboutAttorney Docket No. 01183-0317-00PCT 1:1. In other cases, the ratio of the IL-2 conjugate to the antibody is about 2:1, 3:1, or 4:1. In additional cases, the ratio of the IL-2 conjugate to the antibody is about 6:1 or higher.

[0315] In some embodiments, the one or more IL-2 polypeptide – water-soluble polymer (e.g., PEG) conjugates are directly bound to the antibody or binding fragments thereof. In other instances, the IL-2 conjugate is indirectly bound to the antibody or binding fragments thereof with a linker. Exemplary linkers include homobifunctional linkers, heterobifunctional linkers, maleimide-based linkers, zero-trace linkers, self-immolative linkers, spacers, and the like.

[0316] In some embodiments, the antibody or binding fragments thereof is bound either directly or indirectly to the IL-2 polypeptide portion of the IL-2 polypeptide – water-soluble polymer (e.g., PEG) conjugate. In such cases, the conjugation site of the antibody to the IL-2 polypeptide is at a site that will not impede binding of the IL-2 polypeptide with the IL-2Rβγ. In additional cases, the conjugation site of the antibody to the IL-2 polypeptide is at a site that partially blocks binding of the IL-2 polypeptide with the IL-2Rβγ. In additional cases, the conjugation site of the antibody to the IL-2 polypeptide is at a site that will impede or further impede binding of the IL- 2 polypeptide with the IL-2Rα. In other embodiments, the antibody or binding fragments thereof is bound either directly or indirectly to the water-soluble polymer portion of the IL-2 polypeptide – water-soluble polymer (e.g., PEG) conjugate.

[0317] In some embodiments, a conjugating moiety described herein is a peptide. In some embodiments, the peptide is a non-structured peptide. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a peptide. In some embodiments, the IL-2 conjugate comprising a peptide has an increased serum half-life, and / or stability. In some embodiments, the IL-2 conjugate comprising a peptide has a reduced IL-2 interaction with one or more IL-2R subunits. In additional cases, the peptide blocks IL-2 interaction with one or more IL-2R subunits.

[0318] In some embodiments, the conjugating moiety is a XTEN™ peptide (Amunix Operating Inc.) and the modification is referred to as XTENylation. XTENylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a XTEN™ peptide (Amunix Operating Inc.), a long unstructured hydrophilic peptide comprising different percentage of six amino acids: Ala, Glu, Gly, Ser, and Thr. In some embodiments, a XTEN™ peptide is selected based on properties such as expression, genetic stability, solubility, aggregation resistance, enhanced half-life, increased potency, and / or increased in vitro activity in combination with a polypeptide of interest. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a XTEN peptide. In some embodiments, an IL-2 polypeptide is conjugated to a XTEN peptide.Attorney Docket No. 01183-0317-00PCT

[0319] In some embodiments, the conjugating moiety is a glycine-rich homoamino acid polymer (HAP) and the modification is referred to as HAPylation. HAPylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a glycine-rich homoamino acid polymer (HAP). In some embodiments, the HAP polymer comprises a (Gly4Ser)n repeat motif and sometimes are about 50, 100, 150, 200, 250, 300, or more residues in length. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to HAP. In some embodiments, an IL-2 polypeptide is conjugated to HAP.

[0320] In some embodiments, the conjugating moiety is a PAS polypeptide and the modification is referred to as PASylation. PASylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding a PAS polypeptide. A PAS polypeptide is a hydrophilic uncharged polypeptide consisting of Pro, Ala and Ser residues. In some embodiments, the length of a PAS polypeptide is at least about 100, 200, 300, 400, 500, or 600 amino acids. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to a PAS polypeptide. In some embodiments, an IL-2 polypeptide is conjugated to a PAS polypeptide.

[0321] In some embodiments, the conjugating moiety is an elastin-like polypeptide (ELP) and the modification is referred to as ELPylation. ELPylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest with a nucleic acid encoding an elastin-like polypeptide (ELPs). An ELP comprises a VPGxG repeat motif in which x is any amino acid except proline. In some embodiments, a cytokine (e.g., an interleukin, IFN, or TNF) polypeptide is conjugated to ELP. In some embodiments, an IL-2 polypeptide is conjugated to ELP.

[0322] In some embodiments, the conjugating moiety is a CTP peptide. A CTP peptide comprises a 30 or 31 amino acid residue peptide (FQSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 17) or FQDSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 18)) in which the S* denotes O-glycosylation sites (OPKO). In some embodiments, a CTP peptide is genetically fused to a cytokine polypeptide (e.g., an IL-2 polypeptide). In some embodiments, a cytokine polypeptide (e.g., an IL-2 polypeptide) is conjugated to a CTP peptide.

[0323] In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modified by glutamylation. Glutamylation (or polyglutamylation) is a reversible posttranslational modification of glutamate, in which the γ-carboxy group of glutamate forms a peptide-like bond with the amino group of a free glutamate in which the α-carboxy group extends into a polyglutamate chain.Attorney Docket No. 01183-0317-00PCT

[0324] In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modified by a gelatin-like protein (GLK) polymer. In some embodiments, the GLK polymer comprises multiple repeats of Gly-Xaa-Yaa wherein Xaa and Yaa primarily comprise proline and 4-hydroxyproline, respectively. In some embodiments, the GLK polymer further comprises amino acid residues Pro, Gly, Glu, Qln, Asn, Ser, and Lys. In some embodiments, the length of the GLK polymer is about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150 residues or longer.

[0325] In some embodiments, the conjugating moiety comprises an extracellular biomarker. In some embodiments, the extracellular biomarker is a tumor antigen. In some embodiments, 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 embodiments, the conjugating moiety is bond or conjugated to the cytokine (e.g., IL-2). In some embodiments, the conjugating moiety is genetically fused, for example, at the N-terminus or the C-terminus, of the cytokine (e.g., IL-2).

[0326] In some embodiments, the conjugating moiety comprises a molecule from a post- translational modification. In some embodiments, examples of post-translational modification include myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypiation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., additional of alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation. In some embodiments, the cytokine (e.g., IL-2) is modified by a post-translational modification such as myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypiation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., additional of alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation. 3. Conjugation

[0327] In some embodiments, useful functional reactive groups for conjugating or binding a conjugating moiety to a cytokine polypeptide (e.g., an IL-2 polypeptide) described herein include, for example, zero or higher-order linkers. In some embodiments, an unnatural amino acid incorporated into an interleukin described herein comprises a functional reactive group. In some embodiments, a linker comprises a functional reactive group that reacts with an unnatural amino acid incorporated into an interleukin described herein. In some embodiments, aAttorney Docket No. 01183-0317-00PCT conjugating moiety comprises a functional reactive group that reacts with an unnatural amino acid incorporated into an interleukin described herein. In some embodiments, a conjugating moiety comprises a functional reactive group that reacts with a linker (optionally pre-attached to a cytokine peptide) described herein. In some embodiments, a linker comprises a reactive group that reacts with an unnatural amino acid in a cytokine peptide described herein. In some embodiments, higher-order linkers comprise bifunctional linkers, such as homobifunctional linkers or heterobifunctional linkers. Exemplary homobifuctional linkers include, but are not limited to, Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3′3′- dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′-dithiobispropionimidate (DTBP), 1,4-di-3′- (2′-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide- containing compound (DFDNB), such as 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, benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene- bis(iodoacetamide), or N,N′-hexamethylene-bis(iodoacetamide).

[0328] In some embodiments, the bifunctional linker comprises a heterobifunctional linker. Exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long- chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N- succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-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-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4- iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ- maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimideAttorney Docket No. 01183-0317-00PCT 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-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1- carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs- AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl- (4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(ρ- azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4′- azido-2′-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′- nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB- NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N- succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP), N-sulfosuccinimidyl(4- azidophenyl)-1,3′-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′- dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo- sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3- trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers such as1-(ρ- Azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′- pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4- maleimide carbonyl-reactive and photoreactive cross-linkers such as ρ-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(ρ- azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as ρ-azidophenyl glyoxal (APG).

[0329] In some embodiments, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety (e.g., on a conjugating moiety or on IL-2). Exemplary electrophilic groups include carbonyl groups—such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. In some embodiments, the reactive functional group is aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In someAttorney Docket No. 01183-0317-00PCT embodiments, an unnatural amino acid incorporated into an interleukin described herein comprises an electrophilic group.

[0330] In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker is a dipeptide 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.

[0331] In some embodiments, the linker is a peptide linker comprising, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some embodiments, the peptide linker comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, or less amino acids. In additional cases, the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0332] In some embodiments, the linker comprises a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety comprises p-aminobenzyl alcohol (PAB), p- aminobenzyoxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker comprises a dipeptide linker moiety and a self-immolative linker moiety. In some embodiments, the self-immolative linker moiety is such as described in U.S. Patent No. 9089614 and WIPO Application No. WO2015038426.

[0333] In some embodiments, the cleavable linker is glucuronide. In some embodiments, the cleavable linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker is hydrazine. In some embodiments, the cleavable linker is a reducible linker.

[0334] In some embodiments, the linker comprises a maleimide group. In some embodiments, the maleimide group is also referred to as a maleimide spacer. In some embodiments, the maleimide group further comprises a caproic acid, forming maleimidocaproyl (mc). In some embodiments, the linker comprises maleimidocaproyl (mc). In some embodiments, linker is maleimidocaproyl (mc). In other instances, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) described above.

[0335] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an eliminationAttorney Docket No. 01183-0317-00PCT reaction through a retro-Michael reaction. In some embodiments, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some embodiments, the linker comprises a self-stabilizing maleimide. In some embodiments, the linker is a self-stabilizing maleimide.

[0336] Described herein are IL-2 conjugates having the structure of Formula (I):W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, and 50kDa; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and

[0337] X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, X is an amino acid position of a recombinant human IL-2, wherein the amino acid position is in reference to the positions in SEQ ID NO: 1; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments of an IL-2 conjugate of Formula (I), Z is CH2and Y . In some embodiments of an IL-2 conjugate ofAttorney Docket No. 01183-0317-00PCTFormula (I), Y is CH2 and Z . Further provided herein are IL-2conjugates wherein Z is CH2 and Y is, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further provided herein are IL-2 conjugates whereingroup having an average molecular weight selected from 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, and 60kDa. Further provided herein are IL-2 conjugates wherein Y is CH2and Z is. In some embodiments of an IL-2 conjugate of Formula (I), the PEG group has an average molecular weight selected from 5kDa, 10kDa, 30kDa, 40kDa, 45 kDa, 50kDa, 55kDa, and 60kDa. Here and throughout, embodiments of Z and Y also encompass a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 30kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 35kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 40kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 45kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 50kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 55kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 60kDa. Further provided herein are IL-2 conjugates wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132. Further described herein are IL-2 conjugates, wherein the position of the structure of Formula (VI) or (VII), or a mixture of (VI) and (VII), in the amino acid sequence of the IL-2 conjugate is selected from K8, L11, E14, H15,Attorney Docket No. 01183-0317-00PCT L18, D19, M22, N87, E99, or D108. In some embodiments of an IL-2 conjugate of Formula (I), X is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132. In some embodiments of an IL-2 conjugate of Formula (I), X is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108. In some embodiments an IL-2 conjugate of Formula (I) comprises the sequence of any one of SEQ ID NOs: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments an IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 1.

[0338] Described herein are IL-2 conjugates having the structure of Formula (II):Formula (II); wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, and 60kDa; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, X is an amino acid position having the structure:recombinant human IL-2 selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108, wherein the amino acid position is in reference to the positions of the sequence of SEQ ID NO: 1.Attorney Docket No. 01183-0317-00PCT

[0339] Described herein are IL-2 conjugates having the structure of Formula (III):Formula (III); wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, and 60kDa; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, X is an amino acid position having the structure:recombinant human IL-2 selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108, wherein the amino acid is in reference to the positions of the sequence of SEQ ID NO: 1.

[0340] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1, wherein [AzK_PEG50kDa] has the structure of Formula (II) or Formula (III), or a mixture of Formula (II) and Formula (III). Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III) comprising the total amount of [AzK_PEG50kDa] in the IL-2 conjugate is about 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III) comprising the total amount of [AzK_PEG50kDa] in the IL-2 conjugate is greater than 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III) comprising the total amount of [AzK_PEG50kDa] in the IL-2Attorney Docket No. 01183-0317-00PCT conjugate is less than 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III) comprising the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is about 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III) comprising the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is greater than 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III) comprising the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is less than 1:1.

[0341] In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. Here and throughout, embodiments of Formula (II) and / or (III) also encompass a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further described herein are IL-2 conjugates wherein the [AzK_PEG] is a mixture of Formula (II) and Formula (III). In some embodiments of an IL-2 conjugate of Formula (II) and / or Formula (III), X is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132 of recombinant human IL-2. In some embodiments of an IL-2 conjugate of Formula (II) and / or Formula (III), X is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108 of recombinant human IL-2. In some embodiments of an IL-2 conjugate of Formula (II) and / or Formula (III), the PEG group has an average molecular weight of about 10 kDa, about 20 kDa, about 30 kDa, about 35 kDa, about 50 kDa, about 60 kDa, and X is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108 of recombinant human IL-2.

[0342] Further described herein are IL-2 conjugates wherein the IL-2 conjugate has the amino acid sequence of any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, or 60kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, or 60kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight of 30kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight ofAttorney Docket No. 01183-0317-00PCT 35kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 40kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 45kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 50kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 55kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 60kDa. Further described herein are IL-2 conjugates wherein the IL-2 conjugate has the amino acid sequence of any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. Further described herein are IL-2 conjugates wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, or 60kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 50kDa and 30kDa.

[0343] Described herein are IL-2 conjugates having the structure of Formula (IV):Formula (IV); wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, and 60kDa; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, X is an amino acid position having the structure:Attorney Docket No. 01183-0317-00PCTrecombinant human IL-2 selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108, wherein the amino acid position is in reference to the positions of the sequence of SEQ ID NO: 1. Described herein are IL-2 conjugates having the structure of Formula (V):Formula (V); wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, and 60kDa; and X is an amino acid position having the structurerecombinant human IL-2 selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108, wherein the amino acid is in reference to the positions of the sequence of SEQ ID NO: 1. Here and throughout, embodiments of Formula (IV) and / or (V) also encompass a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further described herein are IL-2 conjugates wherein the [AzK_L1_PEG] is a mixture of Formula (IV) and Formula (V). Further described herein are IL-2 conjugates wherein the [AzK_L1_PEG] has the structure of Formula (IV):Formula (IV)

[0345] Here and throughout, the structure of Formula (IV) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Further described herein are IL-2 conjugates wherein the IL-2 conjugate has the amino acid sequence of any one of SEQ ID NOs: 2-14 or aAttorney Docket No. 01183-0317-00PCT sequence having at least 80% sequence identity to SEQ ID NO: 1. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, or 60kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 50kDa and 30kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight of 5kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight of 30kDa. Further described herein are IL-2 conjugates wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, or 60kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 5kDa and 30kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight of 30 kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 35 kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 40kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 45kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 50kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 55kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 60kDa. Further described herein are IL-2 conjugates wherein the [AzK_L1_PEG] has the structure of Formula (V)Formula (V)

[0346] Here and throughout, the structure of Formula (V) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Further described herein are IL-2 conjugates wherein the IL-2 conjugate has the amino acid sequence of any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected fromAttorney Docket No. 01183-0317-00PCT 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, or 60kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight selected from 50kDa and 30kDa. Further described herein are IL-2 conjugates wherein W is a PEG group having an average molecular weight of 30 kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 35 kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 40kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 45kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 50kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 55kDa. Further provided herein are IL-2 conjugates wherein the PEG group has an average molecular weight of 60kDa. Further described herein are IL-2 conjugates wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0347] Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG] in the IL-2 conjugate is about 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG] in the IL-2 conjugate is greater than 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG] in the IL-2 conjugate is less than 1:1. Further described herein are IL-2 conjugates wherein W is a linear or branched PEG group. Further described herein are IL-2 conjugates wherein W is a linear PEG group. Further described herein are IL-2 conjugates wherein W is a branched PEG group. Further described herein are IL-2 conjugates wherein W is a methoxy PEG group. Further described herein are IL-2 conjugates wherein the methoxy PEG group is linear or branched. Further described herein are IL-2 conjugates wherein the methoxy PEG group is linear. Further described herein are IL-2 conjugates wherein the methoxy PEG group is branched. Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1 [AzK_L1_PEG50kDa] has the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V):Attorney Docket No. 01183-0317-00PCTFormula (V); wherein: W is a PEG group having an average molecular weight of 50kDa; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_ PEG50kDa] in the IL-2 conjugate is about 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG50kDa] in the IL-2 conjugate is greater than 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG50kDa] in the IL-2 conjugate is less than 1:1. Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1 [AzK_L1_PEG30kDa] has the structure of Formula (IV) or Formula (V), or is a mixture of the structures of Formula (IV) and Formula (V):Attorney Docket No. 01183-0317-00PCTFormula (V); wherein: W is a PEG group having an average molecular weight of 30kDa; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.

[0348] Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is about 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is greater than 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is less than 1:1. Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1 [Azk_L1_PEG] is a mixture of the structures of Formula (IV) and Formula (V):Attorney Docket No. 01183-0317-00PCTFormula (V); wherein: W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, and 60kDa; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.

[0349] In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG] in the IL-2 conjugate is about 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG] in the IL-2 conjugate is greater than 1:1. Further described herein are IL-2 conjugates wherein the ratio of the amount of the structure of Formula (IV) to the amount of the structure of Formula (V) comprising the total amount of [AzK_L1_PEG] in the IL-2 conjugate is less than 1:1. Further described herein are IL-2 conjugates wherein W is a linear or branched PEG group. Further described herein are IL-2 conjugates wherein W is a linear PEG group. Further described herein are IL-2 conjugates wherein W is a branched PEG group. Further described herein are IL-2Attorney Docket No. 01183-0317-00PCT conjugates wherein W is a methoxy PEG group. Further described herein are IL-2 conjugates wherein the methoxy PEG group is linear or branched. Further described herein are IL-2 conjugates wherein the methoxy PEG group is linear. Further described herein are IL-2 conjugates wherein the methoxy PEG group is branched.

[0350] In some embodiments of an IL-2 conjugate of Formula (IV) and / or Formula (V), X is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132 of recombinant human IL-2. In some embodiments of an IL-2 conjugate of Formula (IV) and / or Formula (V), X is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108 of recombinant human IL-2. In some embodiments of an IL-2 conjugate of Formula (IV) and / or Formula (V), the PEG group has an average molecular weight of about 10 kDA, about 20 kDA, about 30 kDa, about 35 kDa, about 50 kDa, about 60 kDa, and X is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108 of recombinant human IL-2.

[0351] In some embodiments an IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of any one of SEQ ID NOs: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1.

[0352] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 1 in which at least one amino acid residue in the IL-2 conjugate is replaced by the structure of Formula (VI) or (VII), or a mixture of (VI) and (VII):Formula (VII);Attorney Docket No. 01183-0317-00PCT wherein: n is an integer in the range from about 2 to about 5000; and X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and

[0353] X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. Here and throughout, embodiments of Formula (VI) and / or (VII) also encompass a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (VI) or (VII), or a mixture of (VI) and (VII), in the amino acid sequence of the IL-2 conjugate is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132. Further described herein are IL-2 conjugates, wherein the position of the structure of Formula (VI) or (VII), or a mixture of (VI) and (VII), in the amino acid sequence of the IL-2 conjugate is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (VI) or (VII), or a mixture of (VI) and (VII), in the amino acid sequence of the IL-2 conjugate is selected from H15 and L18.

[0354] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 1 in which at least one amino acid residue in the IL-2 conjugate is replaced by the structure of Formula (VIII) or (IX), or a mixture of (VIII) and (IX):Formula (VIII);Attorney Docket No. 01183-0317-00PCTFormula (IX); wherein: n is an integer in the range from about 2 to about 5000; and X has the structure:indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. Here and throughout, embodiments of Formula (VIII) and / or (IX) also encompass a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (VIII) or (IX), or a mixture of (VIII) and (IX), in the amino acid sequence of the IL-2 conjugate is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132. Further described herein are IL-2 conjugates, wherein the position of the structure of Formula (VIII) or (IX), or a mixture of (VIII) and (IX), in the amino acid sequence of the IL-2 conjugate is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (VIII) or (IX), or a mixture of (VIII) and (IX), in the amino acid sequence of the IL-2 conjugate is selected from H15 and L18.

[0355] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 1 in which at least one amino acid residue in the IL-2 conjugate is replaced by the structure of Formula (X) or (XI), or a mixture of (X) and (XI):Attorney Docket No. 01183-0317-00PCTFormula (XI); wherein: n is an integer in the range from about 2 to about 5000; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. Here and throughout, embodiments of Formula (X) and / or (XI) also encompass a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (X) or (XI), or a mixture of (X) and (XI), in the amino acid sequence of the IL-2 conjugate is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132. Further described herein are IL-2 conjugates, wherein the position of the structure of Formula (X) or (XI), or a mixture of (X) and (XI), in the amino acid sequence of the IL-2 conjugate is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (X) or (XI), or a mixture of (X) and (XI), in the amino acid sequence of the IL-2 conjugate is selected from H15 and L18.

[0356] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 1 in which at least one amino acid residue in the IL-2 conjugate is replaced by the structure of Formula (XII) or (XIII), or a mixture of (XII) and (XIII):Attorney Docket No. 01183-0317-00PCTFormula (XIII); wherein: n is an integer in the range from about 2 to about 5000; and

[0357] the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. Here and throughout, embodiments of Formula (XII) and / or (XIII) also encompass a pharmaceutically acceptable salt, solvate, or hydrate thereof. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (XII) or (XIII), or a mixture of (XII) and (XIII), in the amino acid sequence of the IL-2 conjugate is selected from P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, and T132. Further described herein are IL-2 conjugates, wherein the position of the structure of Formula (XII) or (XIII), or a mixture of (XII) and (XIII), in the amino acid sequence of the IL-2 conjugate is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. Further described herein are IL-2 conjugates wherein the position of the structure of Formula (XII) or (XIII), or a mixture of (XII) and (XIII), in the amino acid sequence of the IL-2 conjugate is selected from H15 and L18.

[0358] Described herein are pharmaceutical compositions of Formula (I), Formula (IV), or Formula (V). In some embodiments, a pharmaceutical compositions of Formula (I), FormulaAttorney Docket No. 01183-0317-00PCT (IV), or Formula (V) comprises a sequence comprising any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, a pharmaceutical compositions of Formula (I), Formula (IV), or Formula (V) comprises a sequence comprising any one of SEQ ID NO: 4. Described herein are pharmaceutical compositions of Formula (I), Formula (VI), or Formula (VII). In some embodiments, a pharmaceutical compositions of Formula (I), Formula (VI), or Formula (VII) comprises a sequence comprising any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, a pharmaceutical compositions of Formula (I), Formula (VI), or Formula (VII) comprises a sequence comprising any one of SEQ ID NO: 4. Described herein are pharmaceutical compositions of Formula (I), Formula (VIII), or Formula (IX). In some embodiments, a pharmaceutical compositions of Formula (I), Formula (VIII), or Formula (IX) comprises a sequence comprising any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, a pharmaceutical compositions of Formula (I), Formula (VIII), or Formula (IX) comprises a sequence comprising any one of SEQ ID NO: 4. Described herein are pharmaceutical compositions of Formula (I), Formula (X), or Formula (XI). In some embodiments, a pharmaceutical compositions of Formula (I), Formula (X), or Formula (XI) comprises a sequence comprising any one of SEQ ID NOS: 2- 14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, a pharmaceutical compositions of Formula (I), Formula (X), or Formula (XI) comprises a sequence comprising any one of SEQ ID NO: 4. Described herein are pharmaceutical compositions of Formula (XII), Formula (XIII), or Formula (V). In some embodiments, a pharmaceutical compositions of Formula (I), Formula (XII), or Formula (XIII) comprises a sequence comprising any one of SEQ ID NOS: 2-14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, a pharmaceutical compositions of Formula (I), Formula (XII), or Formula (XIII) comprises a sequence comprising any one of SEQ ID NO: 4.

[0359] In some embodiments described herein, a conjugation reaction described herein comprises an inverse-electron demand cycloaddition reaction comprising a diene and a dienophile. In some embodiments, the diene comprises a tetrazine. In some embodiments, the dienophile comprises an alkene. In some embodiments, the dienophile comprises an alkyne. In some embodiments, the alkyne is a strained alkyne. In some embodiments, the alkene is a strained diene. In some embodiments, the alkyne is a trans-cyclooctyne. In some embodiments, the alkyne is a cyclooctene. In some embodiments, the alkene is a cyclopropene. In some embodiments, the alkene is a fluorocyclopropene. In some embodiments, a conjugation reactionAttorney Docket No. 01183-0317-00PCT described herein results in the formation of a cytokine peptide attached to a linker or conjugation moiety via a 6-membered ring heterocycle comprising two nitrogen atoms in the ring.

[0360] In some embodiments described herein, a conjugation reaction described herein comprises an olefin metathesis reaction. In some embodiments, a conjugation reaction described herein comprises reaction of an alkene and an alkyne with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reaction of two alkenes with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reaction of two alkynes with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reaction of an alkene or alkyne with a ruthenium catalyst and an amino acid comprising an allyl group. In some embodiments, a conjugation reaction described herein comprises reaction of an alkene or alkyne with a ruthenium catalyst and an amino acid comprising an allyl sulfide or selenide. In some embodiments, a ruthenium catalyst is Hoveda- Grubbs 2ndgeneration catalyst. In some embodiments, an olefin metathesis reaction comprises reaction of one or more strained alkenes or alkynes.

[0361] In some embodiments described herein, a conjugation reaction described herein comprises a cross-coupling reaction. In some embodiments, cross-coupling reactions comprise transition metal catalysts, such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or other transition metal catalyst and one or more ligands. In some embodiments, transition metal catalysts are water-soluble. In some embodiments described herein, a conjugation reaction described herein comprises a Suzuki-Miyaura cross-coupling reaction. In some embodiments described herein, a conjugation reaction described herein comprises reaction of an aryl halide (or triflate, or tosylate), an aryl or alkenyl boronic acid, and a palladium catalyst. In some embodiments described herein, a conjugation reaction described herein comprises a Sonogashira cross-coupling reaction. In some embodiments described herein, a conjugation reaction described herein comprises reaction of an aryl halide (or triflate, or tosylate), an alkyne, and a palladium catalyst. In some embodiments, cross-coupling reactions result in attachment of a linker or conjugating moiety to a cytokine peptide via a carbon-carbon bond.

[0362] In some embodiments described herein, a conjugation reaction described herein comprises a deprotection or “uncaging” reaction of a reactive group prior to conjugation. In some embodiments, a conjugation reaction described herein comprises uncaging of a reactive group with light, followed by a conjugation reaction. In some embodiments, a reactive group is protected with an aralkyl moiety comprising one or more nitro groups. In some embodiments, uncaging of a reactive group results in a free amine, sulfide, or other reactive group. In someAttorney Docket No. 01183-0317-00PCT embodiments, a conjugation reaction described herein comprises uncaging 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, a reactive group is protected with an allyl moiety. In some embodiments, a reactive group is protected with an allylic carbamate. In some embodiments, a reactive group is protected with a propargylic moiety. In some embodiments, a reactive group is protected with a propargyl carbamate. In some embodiments, a reactive group is protected with a dienophile, wherein exposure to a diene (such as a tetrazine) results in deprotection of the reactive group.

[0363] In some embodiments described herein, a conjugation reaction described herein comprises a ligand-directed reaction, wherein a ligand (optionally) attached to a reactive group) facilitates the site of conjugation between the reactive group and the cytokine peptide. In some embodiments, the ligand is cleaved during or after reaction of the cytokine peptide with the reactive group. In some embodiments, the conjugation site of the cytokine peptide is an unnatural amino acid described herein. In some embodiments the reactive group comprises a leaving group, such as an electron-poor aryl or heteroaryl group. In some embodiments the reactive group comprises a leaving group, such as an electron-poor alkyl group that is displaced by the cytokine peptide. In some embodiments, a conjugation reaction described herein comprises a reaction of a radical trapping agent with a radical species. In some embodiments, a conjugation reaction described herein comprises an oxidative radical addition reaction. In some embodiments, a radical trapping agent is an arylamine. In some embodiments, a radical species is a tyrosyl radical. In some embodiments, radical species are generated by a ruthenium catalyst (such as [Ru(bpy)3]) and light.

[0364] Enzymatic reactions are optionally used for conjugation reactions described herein. Exemplary enzymatic conjugations include SortA-mediated conjugation, a TGs-mediated conjugation, or an FGE-mediated conjugation. In some embodiments, a conjugation reaction described herein comprises native protein ligation (NPL) of a terminal 1-amino-2-thio group with a thioester to form an amide bond.

[0365] Various conjugation reactions are described herein for reacting a linker or conjugating moiety with a cytokine peptide, wherein the reaction occurs with an unnatural amino acid in the cytokine peptide. In some embodiments, a conjugation reaction comprises formation of a disulfide bond at an unnatural amino acid residue. In some embodiments, a conjugation reaction comprises a 1,4 Michael addition reaction of an unnatural amino acid. In some embodiments, a conjugation reaction comprises a cyanobenzothiazole ligation of an unnatural amino acid. In some embodiments, a conjugation reaction comprises crosslinking with an acetone moiety, suchAttorney Docket No. 01183-0317-00PCT as 1,3-dichloro-2-propionone. In some embodiments, a conjugation reaction comprises a 1,4 Michael addition to a dehydroalanine, formed by reaction of an unnatural amino acid with O- mesitylenesulfonylhydroxylamine. In some embodiments a conjugation reaction comprises reaction of an unnatural amino acid with a triazolinedione (TAD), or TAD derivative. In some embodiments a conjugation reaction comprises reaction of an unnatural amino acid with a rhodium carbenoid.

[0366] Various conjugation reactions are used to conjugate linkers, conjugation moieties, and unnatural amino acids incorporated into cytokine peptides described herein. Such conjugation reactions are often compatible with aqueous conditions, such as “bioorthogonal” reactions. In some embodiments, conjugation reactions are mediated by chemical reagents such as catalysts, light, or reactive chemical groups found on linkers, conjugation moieties, or unnatural amino acids. In some embodiments, conjugation reactions are mediated by enzymes. In some embodiments, a conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, a conjugation reaction used herein is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417.

[0367] In some embodiments described herein, a conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises reaction of an azide and a phosphine (“Click” reaction). In some embodiments, the conjugation reaction is catalyzed by copper. In some embodiments, a conjugation reaction described herein results in cytokine peptide comprising a linker or conjugation moiety attached via a triazole. In some embodiments, a conjugation reaction described herein comprises reaction of an azide with a strained olefin. In some embodiments, a conjugation reaction described herein comprises reaction of an azide with a strained alkyne. In some embodiments, a conjugation reaction described herein comprises reaction of an azide with a cycloalkyne, for example DBCO.

[0368] In some embodiments described herein, a conjugation reaction described herein comprises:, wherein X is the position in the IL-2 conjugate comprising an unnatural amino acid, such as inAttorney Docket No. 01183-0317-00PCT any one of SEQ ID NOS: 2 to 14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the conjugating moiety comprises water soluble polymer. In some embodiments, a reactive group comprises an alkyne or azide. In some embodiments described herein, a conjugation reaction described herein comprises:, wherein X is the position in the IL-2 conjugate comprising an unnatural amino acid, such as in any one of SEQ ID NOs: 2 to 14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments described herein, a conjugation reaction described herein comprises:, wherein X is the position in the IL-2 conjugate comprising an unnatural amino acid, such as in any one of SEQ ID NOS: 2 to 14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments described herein, a conjugation reaction described herein comprises:, wherein X is the position in the IL-2 conjugate comprising an unnatural amino acid, such as inAttorney Docket No. 01183-0317-00PCT any one of SEQ ID NOS: 2 to 14 or a sequence having at least 80% sequence identity to SEQ ID NO: 1.

[0369] In some embodiments described herein, a conjugation reaction described herein comprises are cycloaddition reaction between an azide moiety, such as that contained in a protein containing an amino acid residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as that derived from DBCO, which is a chemical moiety comprising a dibenzocyclooctyne group. PEG groups comprising a DBCO moiety are commercially available or may be prepared by methods known to those of ordinary skill in the art.Attorney Docket No. 01183-0317-00PCTCytokine Azk_PEG variant proteinsAttorney Docket No. 01183-0317-00PCT

[0370] Conjugation reactions such as a click reaction described herein may generate a single regioisomer, or a mixture of regioisomers. In some embodiments, the ratio of regioisomers is about 1:1. In some embodiments the ratio of regioisomers is about 2:1. In some embodiments the ratio of regioisomers is about 1.5:1. In some embodiments the ratio of regioisomers is about 1.2:1. In some embodiments the ratio of regioisomers is about 1.1:1. In some embodiments the ratio of regioisomers is greater than 1:1.Attorney Docket No. 01183-0317-00PCT 4. Cytokine Polypeptide Production

[0371] In some embodiments, the IL-2 conjugates described herein, either containing an unnatural amino acid mutation, are generated recombinantly or are synthesized chemically. In some embodiments, IL-2 conjugates described herein are generated recombinantly, for example, either by a host cell system, or in a cell-free system.

[0372] In some embodiments, IL-2 conjugates are generated recombinantly through a host cell system. In some embodiments, the host cell is a eukaryotic cell (e.g., mammalian cell, insect cells, yeast cells or plant cell) or a prokaryotic cell (e.g., gram-positive bacterium or a gram- negative bacterium). In some embodiments, a eukaryotic host cell is a mammalian host cell. In some embodiments, a mammalian host cell is a stable cell line, or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division. In other cases, a mammalian host cell is a transient cell line, or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division.

[0373] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells , 293 H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™- Jurkat cell line, FreeStyle™ 293-F cells, 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.

[0374] In some embodiments, a eukaryotic host cell is an insect host cell. Exemplary insect host cell include Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0375] In some embodiments, a 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 INVSc1.

[0376] In some embodiments, a eukaryotic host cell is a plant host cell. In some embodiments, the plant cells comprise a cell from algae. Exemplary plant cell lines include strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.

[0377] In some embodiments, a host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F’, INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.Attorney Docket No. 01183-0317-00PCT

[0378] In some embodiments, suitable polynucleic acid molecules or vectors for the production of an IL-2 polypeptide described herein include any suitable vectors derived from either a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors from bacteria (e.g., E. coli), insects, yeast (e.g., Pichia pastoris), algae, or mammalian source. Bacterial vectors include, for example, pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0379] Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.

[0380] Yeast vectors include, for example, Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA, B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, & C yeast vector, or pYES3 / CT yeast vector.

[0381] Algae vectors include, for example, pChlamy-4 vector or MCS vector.

[0382] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG- Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0383] In some embodiments, a cell-free system is used for the production of a cytokine (e.g., IL-2) polypeptide described herein. In some embodiments, a cell-free system comprises a mixture of cytoplasmic and / or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some embodiments, a cell-free system utilizes prokaryotic cell components. In other instances, a cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis isAttorney Docket No. 01183-0317-00PCT obtained in a cell-free system based on, for example, Drosophila cell, Xenopus egg, Archaea, or HeLa cells. Exemplary cell-free systems include E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.

[0384] Cell-free translation systems variously comprise components such as plasmids, mRNA, DNA, tRNAs, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and / or unnatural amino acids, and / or other components used for protein expression. Such components are optionally modified to improve yields, increase synthesis rate, increase protein product fidelity, or incorporate unnatural amino acids. In some embodiments, cytokines described herein are synthesized using cell-free translation systems described in US 8,778,631; US 2017 / 0283469; US 2018 / 0051065; US 2014 / 0315245; or US 8,778,631. In some embodiments, cell-free translation systems comprise modified release factors, or even removal of one or more release factors from the system. In some embodiments, cell-free translation systems comprise a reduced protease concentration. In some embodiments, cell-free translation systems comprise modified tRNAs with re-assigned codons used to code for unnatural amino acids. In some embodiments, the synthetases described herein for the incorporation of unnatural amino acids are used in cell-free translation systems. In some embodiments, tRNAs are pre-loaded with unnatural amino acids using enzymatic or chemical methods before being added to a cell-free translation system. In some embodiments, components for a cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organism.

[0385] In some embodiments, a cytokine (e.g., IL-2) polypeptide is generated as a circularly permuted form, either via an expression host system or through a cell-free system.

[0386] An orthogonal or expanded genetic code can be used in the present disclosure, in which one or more specific codons present in the nucleic acid sequence of a cytokine (e.g., IL-2) polypeptide are allocated to encode the unnatural amino acid so that it can be genetically incorporated into the cytokine (e.g., IL-2) by using an orthogonal tRNA synthetase / tRNA pair. The orthogonal tRNA synthetase / tRNA pair is capable of charging a tRNA with an unnatural amino acid and is capable of incorporating that unnatural amino acid into the polypeptide chain in response to the codon.

[0387] In some embodiments, the codon is the codon amber, ochre, opal or a quadruplet codon. In some embodiments, the codon corresponds to the orthogonal tRNA which will be used to carry the unnatural amino acid. In some embodiments, the codon is amber. In other cases, the codon is an orthogonal codon.Attorney Docket No. 01183-0317-00PCT

[0388] In some embodiments, the codon is a quadruplet codon, which can be decoded by an orthogonal ribosome ribo-Q1. In some embodiments, the quadruplet codon is as illustrated in Neumann, et al., “Encoding multiple unnatural amino acids via evolution of a quadruplet- decoding ribosome,” Nature, 464(7287): 441-444 (2010).

[0389] In some embodiments, a codon used in the present disclosure is a recoded codon, e.g., a synonymous codon or a rare codon that is replaced with alternative codon. In some embodiments, the recoded codon is as described in Napolitano, et al., “Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli,” PNAS, 113(38): E5588- 5597 (2016). In some embodiments, the recoded codon is as described in Ostrov et al., “Design, synthesis, and testing toward a 57-codon genome,” Science 353(6301): 819-822 (2016).

[0390] In some embodiments, unnatural nucleic acids are utilized leading to incorporation of one or more unnatural amino acids into the cytokine (e.g., IL-2). Exemplary unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifiuoromethyl 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-deazaadenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5- propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size- expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl,Attorney Docket No. 01183-0317-00PCT other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2- amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine( [5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g. 9- (2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5- b]indol-2-one), pyridoindole cytidine (H- pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced with other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2- pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6- dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5- bromouracil, 5- chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8- azaguanine, 5-hydroxycytosine, 2’-deoxyuridine, 2-amino-2’-deoxyadenosine, and those described in U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5,093,232; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096; WO 99 / 62923; Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858- 859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Additional base modifications can be found, for example, in U.S. Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993.

[0391] Unnatural nucleic acids comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acids in some embodiments include one or several heterocyclic bases other than the principal five base components of naturally- occurring nucleic acids. For example, the heterocyclic base includes, in some embodiments, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4- aminopyrrolo [2.3-d] pyrimidin-5-yl, 2-amino-4-oxopyrolo [2, 3-d] pyrimidin-5-yl, 2- amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the nucleic acid via the 9-position, the pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7- position and the pyrazolopyrimidines via the 1-position.Attorney Docket No. 01183-0317-00PCT

[0392] In some embodiments, nucleotide analogs are also modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those with modification at the linkage between two nucleotides and contains, for example, a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkage between two nucleotides are through a 3’-5’ linkage or a 2’-5’ linkage, and the linkage contains inverted polarity such as 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0393] In some embodiments, unnatural nucleic acids include 2’,3’-dideoxy-2’,3’-didehydro- nucleosides (PCT / US2002 / 006460), 5’-substituted DNA and RNA derivatives (PCT / US2011 / 033961; Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020; PCT / JP2000 / 004720; PCT / JP2003 / 002342; PCT / JP2004 / 013216; PCT / JP2005 / 020435; PCT / JP2006 / 315479; PCT / JP2006 / 324484; PCT / JP2009 / 056718; PCT / JP2010 / 067560), or 5’-substituted monomers made as the monophosphate with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

[0394] In some embodiments, unnatural nucleic acids include modifications at the 5’-position and the 2’-position of the sugar ring (PCT / US94 / 02993), such as 5’-CH2-substituted 2’-O- protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some embodiments, unnatural nucleic acids include amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3’ linked nucleoside in the dimer (5’ to 3’) comprises a 2’-OCH3 and a 5’-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Unnatural nucleic acids can include 2’-substituted 5’-CH2(or O) modified nucleosides (PCT / US92 / 01020). Unnatural nucleic acids can include 5’-Attorney Docket No. 01183-0317-00PCT methylenephosphonate 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). Unnatural nucleic acids can include 5’-phosphonate monomers having a 2’-substitution (US2006 / 0074035) and other modified 5’-phosphonate monomers (WO1997 / 35869). Unnatural nucleic acids can include 5’-modified methylenephosphonate monomers (EP614907 and EP629633). Unnatural nucleic acids can include analogs of 5’ or 6’-phosphonate ribonucleosides comprising a hydroxyl group at the 5’ and / or 6’-position (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033). Unnatural nucleic acids can include 5’-phosphonate deoxyribonucleoside monomers and dimers having a 5’-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Unnatural nucleic acids can include nucleosides having a 6’-phosphonate group wherein the 5’ or / and 6’-position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. 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).

[0395] In some embodiments, unnatural nucleic acids also include modifications of the sugar moiety. In some embodiments, nucleic acids contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, nucleic acids comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, without limitation, addition of substituent groups (including 5’ and / or 2’ substituent groups; bridging of two ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, C1-C12alkyl or a protecting group); and combinations thereof. Examples of chemically modified sugars can be found in WO2008 / 101157, US2005 / 0130923, and WO2007 / 134181.

[0396] In some embodiments, a modified nucleic acid comprises modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group. The sugar can be in a pyranosyl or furanosyl form. The sugar moiety may be the furanoside of ribose, deoxyribose, arabinose or 2’-O-alkylribose, and the sugar can beAttorney Docket No. 01183-0317-00PCT attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2’-alkoxy-RNA analogs, 2’-amino-RNA analogs, 2’-fluoro-DNA, and 2’-alkoxy- or amino-RNA / DNA chimeras. For example, a sugar modification may include 2’-O-methyl-uridine or 2’-O-methyl-cytidine. Sugar modifications include 2’-O-alkyl-substituted deoxyribonucleosides and 2’-O-ethyleneglycol like ribonucleosides. The preparation of these sugars or sugar analogs and the respective “nucleosides” wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known. Sugar modifications may also be made and combined with other modifications.

[0397] Modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2’ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10, alkyl or C2 to C10 alkenyl and alkynyl. 2’ sugar modifications also include but are not limited to -O[(CH2)nO]mCH3, -O(CH2)nOCH3, -O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)nONH2, and -O(CH2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10.

[0398] Other modifications at the 2’ position include but are not limited to: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3’ position of the sugar on the 3’ terminal nucleotide or in 2’-5’ linked oligonucleotides and the 5’ position of the 5’ terminal nucleotide. Modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH2and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures and which detail and describe a range of base modifications, such as U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,700,920, each of which is herein incorporated by reference in its entirety.Attorney Docket No. 01183-0317-00PCT

[0399] Examples of nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5’-vinyl, 5’-methyl (R or S), 4’-S, 2’-F, 2’-OCH3, and 2’- O(CH2)2OCH3 substituent groups. The substituent at the 2’ position can also be selected from allyl, amino, azido, thio, O-allyl, O-(C1-C1Oalkyl), OCF3, O(CH2)2SCH3, O(CH2)2-O-N(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rmand Rnis, independently, H or substituted or unsubstituted C1-C10 alkyl.

[0400] In certain embodiments, nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4’ and the 2’ ribosyl ring atoms. In certain embodiments, nucleic acids provided herein include one or more bicyclic nucleic acids wherein the bridge comprises a 4’ to 2’ bicyclic nucleic acid. Examples of such 4’ to 2’ bicyclic nucleic acids include, but are not limited to, one of the formulae: 4’-(CH2)-O-2’ (LNA); 4’-(CH2)-S-2’; 4’-(CH2)2-O-2’ (ENA); 4’-CH(CH3)-O-2’ and 4’-CH(CH2OCH3)-O-2’, and analogs thereof (see, U.S. Patent No. 7,399,845); 4’- C(CH3)(CH3)-O-2’and analogs thereof, (see WO2009 / 006478, WO2008 / 150729, US2004 / 0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134, and WO2008 / 154401). Also see, 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. U. S. A., 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; U.S. Patent Nos. 4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; International Publication Nos. WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, WO2007 / 090071, and WO2007 / 134181; U.S. Patent Publication Nos. US2004 / 0171570, US2007 / 0287831, and US2008 / 0039618; U.S. Provisional Application Nos. 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and International Applications Nos. PCT / US2008 / 064591, PCT US2008 / 066154, PCT US2008 / 068922, and PCT / DK98 / 00393.

[0401] In certain embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids can be linked together using any inter nucleic acid linkage. The two main classes of inter nucleic acid linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing inter nucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing inter nucleic acid linkingAttorney Docket No. 01183-0317-00PCT groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates. Unnatural nucleic acids can contain a single modification. Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.

[0402] Backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in any combination. Other non- phosphate linkages may also be used.

[0403] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and / or enhance their stability in vivo.

[0404] In some embodiments, a phosphorous derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like. Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found in Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; and Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview” in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, “Oligonucleoside Phosphorothioates” in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Patent No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem. 8: 1157-1179.

[0405] In some embodiments, backbone modification comprises replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group. Examples of such modifications include: anionic internucleoside linkage; N3’ to P5’ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligosAttorney Docket No. 01183-0317-00PCT (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). A modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g. a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages.

[0406] Substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). United States Patent Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O- hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651- 3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycolAttorney Docket No. 01183-0317-00PCT chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous United States patents teach the preparation of such conjugates and include, but are not limited to U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0407] In some embodiments, the unnatural nucleic acids further form unnatural base pairs. Exemplary unnatural nucleotides capable of forming an unnatural DNA or RNA base pair (UBP) under conditions in vivo includes, but is not limited to, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. Other examples of unnatural nucleotides capable of forming unnatural UBPs that may be used to prepare the IL-2 conjugates disclosed herein may be found in Dien et al., J Am Chem Soc., 2018, 140:16115–16123; Feldman et al., J Am Chem Soc, 2017, 139:11427–11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res. 2014, 42:10235-10244; Malyshev et al., Nature, 2014, 509:385- 388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc. 2013, 135:5408-5419; and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010. In some embodiments, unnatural nucleotides include:Attorney Docket No. 01183-0317-00PCT

[0408] In some embodiments, the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein may be derived from a compound of the formulawherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azido; and the wavy line indicates a bond to a ribosyl or 2’-deoxyribosyl, wherein the 5’-hydroxy group of the ribosyl or 2’-deoxyribosyl moiety is in free form, or is optionally bonded to a monophosphate, a diphosphate, or a triphosphate group.

[0409] In some embodiments, the unnatural nucleotides that may be used to prepare the IL-2conjugates disclosed herein may be derived from,embodiments, the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosedAttorney Docket No. 01183-0317-00PCT ,salts thereof.

[0410] In some embodiments, an unnatural base pair generates an unnatural amino acid described in Dumas et al., “Designing logical codon reassignment – Expanding the chemistry in biology,” Chemical Science, 6: 50-69 (2015).

[0411] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a synthetic codon comprising an unnatural nucleic acid. In some embodiments, the unnatural amino acid is incorporated into the cytokine by an orthogonal, modified synthetase / tRNA pair. Such orthogonal pairs comprise an unnatural synthetase that is capable of charging the unnatural tRNA with the unnatural amino acid, while minimizing charging of a) other endogenous amino acids onto the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs. Such orthogonal pairs comprise tRNAs that are capable ofAttorney Docket No. 01183-0317-00PCT being charged by the unnatural synthetase, while avoiding being charged with a) other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, Archaea, or human sources. In some embodiments, an orthogonal synthetase / tRNA pair comprises components from a single organism. In some embodiments, an orthogonal synthetase / tRNA pair comprises components from two different organisms. In some embodiments, an orthogonal synthetase / tRNA pair comprising components that prior to modification, promote translation of two different amino acids. In some embodiments, an orthogonal synthetase is a modified alanine synthetase. In some embodiments, an orthogonal synthetase is a modified arginine synthetase. In some embodiments, an orthogonal synthetase is a modified asparagine synthetase. In some embodiments, an orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, an orthogonal synthetase is a modified cysteine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamic acid synthetase. In some embodiments, an orthogonal synthetase is a modified alanine glycine. In some embodiments, an orthogonal synthetase is a modified histidine synthetase. In some embodiments, an orthogonal synthetase is a modified leucine synthetase. In some embodiments, an orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, an orthogonal synthetase is a modified lysine synthetase. In some embodiments, an orthogonal synthetase is a modified methionine synthetase. In some embodiments, an orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, an orthogonal synthetase is a modified proline synthetase. In some embodiments, an orthogonal synthetase is a modified serine synthetase. In some embodiments, an orthogonal synthetase is a modified threonine synthetase. In some embodiments, an orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, an orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, an orthogonal synthetase is a modified valine synthetase. In some embodiments, an orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, an orthogonal tRNA is a modified alanine tRNA. In some embodiments, an orthogonal tRNA is a modified arginine tRNA. In some embodiments, an orthogonal tRNA is a modified asparagine tRNA. In some embodiments, an orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, an orthogonal tRNA is a modified cysteine tRNA. In some embodiments, an orthogonal tRNA is a modified glutamine tRNA. In some embodiments, an orthogonal tRNA is a modified glutamic acid tRNA. In some embodiments, an orthogonal tRNA is a modified alanine glycine. In some embodiments, an orthogonal tRNA is a modified histidine tRNA. In some embodiments, an orthogonal tRNA is a modified leucine tRNA. In someAttorney Docket No. 01183-0317-00PCT embodiments, an orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, an orthogonal tRNA is a modified lysine tRNA. In some embodiments, an orthogonal tRNA is a modified methionine tRNA. In some embodiments, an orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, an orthogonal tRNA is a modified proline tRNA. In some embodiments, an orthogonal tRNA is a modified serine tRNA. In some embodiments, an orthogonal tRNA is a modified threonine tRNA. In some embodiments, an orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, an orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, an orthogonal tRNA is a modified valine tRNA. In some embodiments, an orthogonal tRNA is a modified phosphoserine tRNA.

[0412] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pairs, E. coli TyrRS (Ec-Tyr) / B. stearothermophilus tRNACUA pairs, E. coli LeuRS (Ec-Leu) / B. stearothermophilus tRNACUApairs, and pyrrolysyl-tRNA pairs. In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Mj-TyrRS / tRNA pair. Exemplary UAAs that can be incorporated by a Mj- TyrRS / tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronophenylalanine; and o-nitrobenzyltyrosine.

[0413] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Ec-Tyr / tRNACUA or a Ec-Leu / tRNACUA pair. Exemplary UAAs that can be incorporated by a Ec-Tyr / tRNACUA or a Ec-Leu / tRNACUA pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O- propargyltyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3- (naphthalene-2-ylamino)-2-amino-propanoic acid.

[0414] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a pyrrolysyl-tRNA pair. In some embodiments, the PylRS is obtained from an archaebacterial, e.g., from a methanogenic archaebacterial. In some embodiments, the PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated by a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate substituted lysines such as 2-amino-6-((R)- tetrahydrofuran-2-carboxamido)hexanoic acid, N-ε-D-prolyl-L-lysine, and N-ε- cyclopentyloxycarbonyl-L-lysine; N-ε-Acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-Attorney Docket No. 01183-0317-00PCT yl)ethoxy)carbonyl]-L-lysine; and N-ε-(1-methylcyclopro-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein may be prepared by use of M. mazei tRNA which is selectively charged with a non-natural amino acid such as N6-((2-azidoethoxy)- carbonyl)-L-lysine (AzK) by the M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS). Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647.

[0415] In some embodiments, an unnatural amino acid is incorporated into a cytokine described herein (e.g., the IL polypeptide) by a synthetase disclosed in US 9,988,619 and US 9,938,516.

[0416] The host cell into which the constructs or vectors disclosed herein are introduced is cultured or maintained in a suitable medium such that the tRNA, the tRNA synthetase and the protein of interest are produced. The medium also comprises the unnatural amino acid(s) such that the protein of interest incorporates the unnatural amino acid(s). In some embodiments, a nucleoside triphosphate transporter (NTT) from bacteria, plant, or algae is also present in the host cell. In some embodiments, the IL-2 conjugates disclosed herein are prepared by use of a host cell that expresses a NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter used in the host cell may be selected from TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some embodiments, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, the NTT is PtNTT1. In some embodiments, the NTT is PtNTT2. In some embodiments, the NTT is PtNTT3. In some embodiments, the NTT is PtNTT4. In some embodiments, the NTT is PtNTT5. In some embodiments, the NTT is PtNTT6. Other NTTs that may be used are disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; Malyshev et al. Nature 2014 (509(7500), 385-388; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317–1322.

[0417] The orthogonal tRNA synthetase / tRNA pair charges a tRNA with an unnatural amino acid and incorporates the unnatural amino acid into the polypeptide chain in response to the codon. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pairs, E. coli TyrRS (Ec-Tyr) / B. stearothermophilus tRNACUApairs, E. coli LeuRS (Ec-Leu) / B. stearothermophilus tRNACUA pairs, and pyrrolysyl-tRNA pairs. Other aaRS- tRNA pairs that may be used according to the present disclosure include those derived from M.Attorney Docket No. 01183-0317-00PCT mazei those described in Feldman et al., J Am Chem Soc., 2018140:1447–1454; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317–1322.

[0418] In some embodiments are provided methods of preparing the IL-2 conjugates disclosed herein in a cellular system that expresses a NTT and a tRNA synthetase. In some embodiments described herein, the NTT is selected from PtNTT1, PtNTT2, P...

Claims

Attorney Docket No. 01183-0317-00PCT CLAIMS What is claimed is:

1. A method of delivering a gene therapy agent to a cell of a subject, comprising administering an IL-2 conjugate to the subject, wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate, wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO:

1.

2. A method of treating an individual in need thereof with a gene therapy agent, comprising administering an IL-2 conjugate to the subject, wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate, and the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.Attorney Docket No. 01183-0317-00PCT 3. A method of increasing expression of a gene therapy agent, comprising: administering an IL-2 conjugate to a subject; wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate; and wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO:

1.

4. A method of reducing an immune response to a gene therapy agent, comprising: administering an IL-2 conjugate to a subject; wherein the gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate; and wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO: 1.Attorney Docket No. 01183-0317-00PCT 5. A method of preventing immune-related adverse events in a subject, comprising: administering an IL-2 conjugate to a subject; wherein a gene therapy agent is administered to the subject before, concurrently with, or after the IL-2 conjugate; and wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO:

1.

6. The method of any one of the preceding claims, wherein the method further comprises, before the administration of the gene therapy agent and the IL-2 conjugate to the subject, a) incubating immune cells from the subject with the gene therapy agent and b) analyzing the immune cells for the expression of one or more activation biomarkers or increased expression of one or more activation biomarkers, wherein expression or increased expression of the one or more activation biomarkers following incubation with the gene therapy agent identifies the subject as being in need of the IL-2 conjugate.

7. A method for selecting a subject for treatment with a gene therapy agent and an IL-2 conjugate, the method comprising a) incubating immune cells from the subject with the gene therapy agent, b) analyzing the immune cells for the expression of one or more activation biomarkers or increased expression of one or more activation biomarkers, wherein expression or increased expression of the one or more activation biomarkers following incubation with the gene therapy agent identifies the subject for treatment with the gene therapy agent and the IL-2 conjugate, and c) selecting the subject identified in step b) for treatment with the gene therapy agent and the IL-2 conjugate; wherein the IL-2 conjugate comprises an amino acid sequence having at least 80%Attorney Docket No. 01183-0317-00PCT sequence identity to SEQ ID NO: 1, in which at least one amino acid residue in the IL-2 conjugate is replaced by an unnatural amino acid linked to a conjugating moiety, and the unnatural amino acid linked to the conjugating moiety is positioned in the amino acid sequence so as to preferentially reduce binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is at position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 in reference to the sequence of SEQ ID NO:

1.

8. The method of the immediately preceding claim, further comprising steps of administering the IL-2 conjugate to the subject identified in step b), and administering the gene therapy agent to the subject identified in step b).

9. The method of any one of claims 6-8, wherein the immune cell is a lymphocyte, a T cell, a CD8+ T cell, an effector T cell, a cytotoxic T cell, or an NK cell.

10. The method of any one of the preceding claims, wherein the unnatural amino acid is linked to the conjugating moiety through a linker.

11. The method of the immediately preceding claim, wherein the linker comprises a homobifunctional linker, a heterobifunctional linker, a cleavable or a non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof.

12. The method of any one of the preceding claims, wherein the unnatural amino acid is a substituted lysine, is a substituted phenylalanine, is a substituted histidine, is a substituted cysteine, comprises an azido group, comprises an alkyne group, comprises an aldehyde group, comprises an aromatic side chain, or comprises a ketone group.

13. The method of any one of the preceding claims, wherein the at least one unnatural amino acid comprises N6-azidoethoxy-L-lysine, N6-((2-azidoethoxy)-carbonyl)-L-lysine, N6- propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine,Attorney Docket No. 01183-0317-00PCT methyltetrazine lysine, allyloxycarbonyllysine, p-acetyl-L-phenylalanine, p-azidomethyl- L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, p- propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl- 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, phosphonotyrosine, L-3-(2-naphthyl)alanine, 2- amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, or 2- amino-3-(phenylselanyl)propanoic acid.

14. The method of any one of the preceding claims, wherein the unnatural amino acid is an azido-substituted lysine.

15. The method of any one of the preceding claims, wherein the unnatural amino acid is N6- ((2-azidoethoxy)-carbonyl)-L-lysine.

16. The method of any one of the preceding claims, wherein the conjugating moiety comprises a water-soluble polymer.

17. The method of the immediately preceding claim, wherein the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof.

18. The method of the immediately preceding claim, wherein the conjugating moiety comprises PEG.

19. The method of the immediately preceding claim, wherein the conjugating moiety is PEG having a molecular weight of about 10-85 kDa or selected from about 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, 60kDa, 65kDa, 70kDa, 75 kDa, 80kDa, and 85 kDa.Attorney Docket No. 01183-0317-00PCT 20. The method of any one of the preceding claims, wherein the conjugating moiety is PEG having a molecular weight of about 20-70 kDa or selected from about 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, 60kDa, 65kDa, and 70kDa.

21. The method of any one of the preceding claims, wherein the conjugating moiety is PEG having a molecular weight of about 30-60 kDa or selected from about 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55 kDa, and 60kDa.

22. The method of any one of the preceding claims, wherein the amino acid linked to the conjugating moiety has the structure of Formula (I):Formula (I); wherein:W is a PEG group; andAttorney Docket No. 01183-0317-00PCT X has the structure:X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.

23. The method of the immediately preceding claim, wherein Z is CH2and Y is24. The method of the immediately preceding claim, wherein the structure of Formula (I) has the structure of Formula (IV) or Formula (V):Formula (V); wherein: W is a PEG group having a molecular weight of about 5-60 kDa or about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa.Attorney Docket No. 01183-0317-00PCT 25. The method of any one of the preceding claims, wherein the IL-2 conjugate further comprises an alanine or methionine N-terminal to the first amino acid of the sequence having at least 80% sequence identity to SEQ ID NO:

1.

26. The method of any one of the preceding claims, wherein the IL-2 conjugate comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

1.

27. The method of any one of the preceding claims, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1 in which position P1, T2, S3, S4, S5, T6, K7, K8, Q10, L11, E14, H15, L17, L18, D19, Q21, M22, N25, G26, N28, N29, Y30, K31, K34, T36, M45, P46, K47, A49, T50, E51, L52, K53, H54, Q56, E59, E66, N70, Q73, S74, K75, N76, F77, H78, R80, P81, R82, D83, S86, N87, I88, V90, I91, L93, E94, K96, G97, S98, E99, T100, T101, F102, M103, C104, E105, Y106, A107, D108, E109, T110, A111, T112, E115, N118, R119, T122, F123, S124, Q125, S126, S129, T130, L131, or T132 is replaced with the unnatural amino acid.

28. The method of any one of the preceding claims, wherein position K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108 in reference to the sequence of SEQ ID NO: 1 is replaced with the unnatural amino acid.

29. The method of the immediately preceding claim, wherein position L18 in reference to the sequence of SEQ ID NO: 1 is replaced with the unnatural amino acid.

30. The method of claim 28, wherein position H15 in reference to the sequence of SEQ ID NO: 1 is replaced with the unnatural amino acid.

31. The method of any one of the preceding claims, wherein the IL-2 conjugate is capable of expanding CD4+ T regulatory (Treg) cells.

32. The method of any one of the preceding claims, wherein the unnatural amino acid and / or the conjugating moiety impairs or blocks the receptor signaling potency of the IL-2 conjugate to IL-2Rβγ, or reduces recruitment of IL-2Rγ subunit to an IL-2 / IL-2Rβ complex.Attorney Docket No. 01183-0317-00PCT 33. The method of any one of the preceding claims, wherein the IL-2 conjugate has a receptor signaling potency to IL-2Rβγ that is lower than a receptor signaling potency of wild-type IL-2 to IL-2Rβγ.

34. The method of any one of the preceding claims, wherein the IL-2 conjugate has a receptor signaling potency to IL-2Rαβγ that is greater than or equal to a receptor signaling potency of wild-type IL-2 to IL-2Rαβγ.

35. The method of any one of the preceding claims, wherein the IL-2 conjugate expands a CD4+ Treg population in the subject.

36. The method of any one of the preceding claims, wherein the IL-2 conjugate suppresses CD8+ T cell proliferation in the subject.

37. The method of any one of the preceding claims, wherein the IL-2 conjugate suppresses effector memory CD8+ T cell proliferation in the subject.

38. The method of any one of the preceding claims, wherein the gene therapy agent comprises a vector and the IL-2 conjugate suppresses vector-specific IFNγ-secreting CD8+ T cells in the subject.

39. The method of any one of the preceding claims, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate suppresses transgene-product-specific IFNγ- secreting CD8+ T cells in the subject.

40. The method of any one of the preceding claims, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate suppresses production of antibodies against the transgene product.

41. The method of any one of the preceding claims, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate suppresses production of IgG1 antibodies against the transgene product.

42. The method of any one of the preceding claims, wherein the gene therapy agent encodes a transgene product and the IL-2 conjugate prolongs the expression of the transgeneAttorney Docket No. 01183-0317-00PCT product in the subject relative to a subject that is administered the gene therapy agent and without the IL-2 conjugate.

43. The method of the immediately preceding claim, wherein the prolonged expression of the transgene product is at least about 5 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 14 weeks, or about 16 weeks.

44. The method of any one of the preceding claims, wherein the gene therapy agent comprises a viral vector.

45. The method of claim 44, wherein the IL-2 conjugate suppresses production of antibodies against the viral vector.

46. The method of claim 44 or claim 45, wherein the IL-2 conjugate suppresses production of antibodies against a capsid protein of the viral vector.

47. The method of any one of claims 44-46, wherein the viral vector is an adeno-associated viral (AAV) particle.

48. The method of the immediately preceding claim, wherein the AAV particle comprises an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid, an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAV9 capsid, an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, an AAVrh32.33 capsid, an AAV-XL32 capsid, an AAV-XL32.1 capsid, an AAV LK03 capsid, an AAV2R471A capsid, an AAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV DJ8 capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a mouse AAV capsid rAAV2 / HBoV1 (chimeric AAV / human bocavirus virus 1), an AAV2HBKO capsid, an AAVPHP.B capsid or an AAVPHP.eB capsid, or a functional variant thereof.

49. The method of the immediately preceding claim, wherein the AAV capsid comprises a tyrosine mutation, a heparin binding mutation, or an HBKO mutation.Attorney Docket No. 01183-0317-00PCT 50. The method of any one of claims 47-49, wherein the AAV viral particle comprises an AAV genome comprising one or more inverted terminal repeats (ITRs), wherein the one or more ITRs is an AAV1 ITR, an AAV2 ITR, an AAV3 ITR, an AAV4 ITR, an AAV5 ITR, an AAV6 ITR, an AAV7 ITR, an AAV8 ITR, an AAVrh8 ITR, an AAV9 ITR, an AAV10 ITR, an AAVrh10 ITR, an AAV11 ITR, or an AAV12 ITR.

51. The method of the immediately preceding claim, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

52. The method of the immediately preceding claim, wherein the one or more ITRs and the capsid of the AAV particles are derived from different AAV serotypes.

53. The method of any one of claims 44-46, wherein the viral vector is an adenoviral particle.

54. The method of the immediately preceding claim, wherein the adenoviral particle comprises a capsid from Adenovirus serotype 2, 1, 5, 6, 19, 3, 11, 7, 14, 16, 21, 12, 18, 31, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24-30, 37, 40, 41, AdHu2, AdHu 3, AdHu4, , AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad type 3, canine Ad type 2, ovine Ad, or porcine Ad type 3, or a functional variant thereof.

55. The method of any one of claims 44-46, wherein the viral vector is a lentiviral particle.

56. The method of the immediately preceding claim, wherein the lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV), Ross river virus (RRV), Ebola virus, Marburg virus, Mokala virus, Rabies virus, RD114, or a functional variant thereof.

57. The method of any one of claims 44-46, wherein the viral vector is a Herpes simplex virus (HSV) particle.

58. The method of the immediately preceding claim, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle, or a functional variant thereof.

59. The method of any one of claims 1-41, wherein the gene therapy agent comprises a lipid nanoparticle.Attorney Docket No. 01183-0317-00PCT 60. The method of any one of the preceding claims, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

61. The method of the immediately preceding claim, wherein the heterologous transgene is operably linked to a promoter.

62. The method of the immediately preceding claim, wherein the promoter is a constitutive promoter, a tissue- specific promoter, or an inducible promoter.

63. The method of any one of claims 60-62, wherein the nucleic acid comprises closed-end DNA (ceDNA).

64. The method of claim 60, wherein the nucleic acid comprises mRNA.

65. The method of any one of the preceding claims, wherein the gene therapy agent is administered to the subject concurrently with the IL-2 conjugate.

66. The method of any one of claims 1-64, wherein the gene therapy agent is administered to the subject before the IL-2 conjugate.

67. The method of the immediately preceding claim, wherein the gene therapy agent is administered less than 14 days or less than 7 days before the IL-2 conjugate.

68. The method of any one of claims 1-64, wherein the gene therapy agent is administered to the subject after the IL-2 conjugate.

69. The method of the immediately preceding claim, wherein the gene therapy agent is administered less than 7 days, less than 3 days, or less than 1 day after the IL-2 conjugate.

70. The method of any one of claims 1-64, wherein the IL-2 conjugate is administered before, at the same time, or after administration of the gene therapy agent.

71. The method of any one of the preceding claims, wherein the individual has a disease or disorder suitable for treatment by gene therapy.Attorney Docket No. 01183-0317-00PCT 72. The method of the immediately preceding claim, wherein the disease or disorder is a monogenic disease or disorder.

73. The method of any one of the preceding claims, wherein the gene therapy agent is administered intravenously, intraperitoneally, intra-arterially, intramuscularly, subcutaneously, intracranially, intra-CSF, intra-DRG, intracerebroventricularly, intraocularly, intracisterna magna, or intrahepatically.

74. The method of any one of the preceding claims, wherein the IL-2 conjugate is administered parenterally and / or systemically.

75. The method of any one of the preceding claims, wherein the IL-2 conjugate is administered intravenously, intraperitoneally, intra-arterially, intramuscularly, subcutaneously, intracranially, intra-CSF, intra-DRG, intracerebroventricularly, intraocularly, intracisterna magna, or intrahepatically.

76. The method of any one of the preceding claims, wherein the subject is a mammal.

77. The method of any one of the preceding claims, wherein the subject is a primate.

78. The method of any one of the preceding claims, wherein the subject is a human.

79. The method of any one of the preceding claims, wherein the IL-2 conjugate is administered about 1, 2, 3, 4, 5, 6, or 7 days before the gene therapy agent.

80. The method of any one of claims 1-78, wherein the IL-2 conjugate is administered about 1, 2, 3, or 4 days after the gene therapy agent.

81. The method of any one of claims 1-78, wherein the IL-2 conjugate is administered on the same day as the gene therapy agent.

82. The method of any one of the preceding claims, wherein the IL-2 conjugate is administered at a dose of about 0.02-0.5 mg / kg about 0.03-0.4 mg / kg, about 0.04-0.1 mg / kg, or about 0.05-0.08 mg / kg.Attorney Docket No. 01183-0317-00PCT 83. The method of the immediately preceding claim, wherein the IL-2 conjugate is administered at a dose of about 0.05 mg / kg.

84. The method of claim 82, wherein the IL-2 conjugate is administered at a dose of about 0.08 mg / kg.

85. The method of claim 82, wherein the IL-2 conjugate is administered at a dose of about 0.3 mg / kg.

86. Use of an IL-2 conjugate for the manufacture of a medicament for use in the method of any one of the preceding claims.

87. An IL-2 conjugate for use in the method of any one of claims 1-85.

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