Polyethylene glycolated il-2 for inhibiting adaptive immune responses to gene therapy

Abstract

The present 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 the subject by administering to the subject the gene therapy agent and an IL-2 conjugate. At least one amino acid residue of the IL-2 conjugate is replaced with a non-natural amino acid linked to a conjugation moiety. The present disclosure further relates to methods of selecting a subject for treatment with a gene therapy agent and an IL-2 conjugate.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 625,113, filed January 25, 2024, and U.S. Provisional Patent Application No. 63 / 641,189, filed May 1, 2024, each of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0002] This disclosure provides methods for delivering gene therapy agents to the cells of a subject, methods for treating individuals in need with gene therapy agents, methods for increasing the expression of gene therapy agents, methods for reducing the immune response to gene therapy agents, and methods for preventing immune-related adverse events in subjects receiving IL-2 conjugates. In some embodiments, the IL-2 conjugate expands Treg cells, reduces the immune response to gene therapy agents, promotes increased expression of gene therapy agents, and / or prevents immune-related adverse events in subjects receiving gene therapy agents. sequence list

[0003] This application is submitted together with an electronic sequence list. The sequence list is provided as a file entitled “01183-0317-00PCT.xml”, created on January 22, 2025, and is 40,250 bytes in size. The information in the electronic sequence list is incorporated herein by reference in its entirety. Background Technology and Summary of the Invention

[0004] One of the current challenges of adeno-associated virus (AAV)-based and lipid nanoparticle (LNP)-based gene therapies is the host immune response, which leads to immune-related adverse events (irAEs) and reduced therapeutic efficacy. Adaptive immune responses following exposure to the AAV capsid and its transgenes evolve into cytotoxic T-cell responses, and transduced cells are destroyed by activated cytotoxic T-cells (Ertl, 2022), ultimately resulting in irAEs and loss of transgene expression. Similarly, expression of LNP-delivered transgenes (ceDNA or RNA) can trigger cytotoxic T-cell responses to therapeutic proteins, leading to the death of transduced cells and associated toxicity. Regulatory T cells play a crucial role in regulating the activation of immune cells, including cytotoxic T cells. Therefore, it is hypothesized that selective and transient expansion of regulatory T cells could inhibit the activation of cytotoxic T cells by AAV and LNPs, thereby promoting better safety and transgene expression. IL-2 is a key cytokine for regulatory T-cell proliferation. However, expanding regulatory T cells in vivo using recombinant human IL-2 (rhIL-2) is challenging because other lymphocytes, such as cytotoxic T cells, also express the IL-2 receptor (IL-2R). Although regulatory T cells bind IL-2 more strongly than other lymphocytes due to their constitutive expression of IL-2Rα (Hernandez et al., 2022), the therapeutic window for selective expansion of regulatory T cells is very narrow. Furthermore, rhIL-2 exhibits a very poor half-life (<90 min) in vivo, requiring multiple intravenous (IV) infusions to achieve therapeutic goals (see, for example, www.accessdata.fda.gov / drugsatfda_docs / label / 2012 / 103293s5130lbl.pdf). Therefore, there is a need for improved methods for delivering gene therapy agents to the cells of subjects, methods for treating individuals in need with gene therapy agents, methods for increasing the expression of gene therapy agents, methods for reducing the immune response to gene therapy agents, and methods for preventing immune-related adverse events in subjects. This disclosure is intended to address one or more of these needs, provide additional benefits, or at least offer the public a useful option.

[0005] The inventors have explored the use of IL-2 conjugates containing the conjugated portion in the methods described above. As described herein, IL-2 conjugates can prevent or reduce their binding to IL-2Rβ and can lead to selective expansion of regulatory T cells because IL-2 conjugates can have reduced binding to other lymphocytes such as cytotoxic T cells (i.e., cytotoxic T lymphocytes (CTLs)). Furthermore, compared to IL-2, IL-2 conjugates as described herein can exhibit a longer serum half-life, which allows for administration routes other than IV infusion and can achieve better patient compliance. A mouse study has been conducted that applied regulatory T cells to AAV gene therapy to modulate the immune response (Arjomandnejad M, Sylvia K, Blackwood M, Nixon T, Tang Q, Muhuri M, Gruntman AM, Gao G, FlotteTR, Keeler AM. Mol Ther Methods Clin Dev. [Molecular Therapy - Methods and Clinical Developments] 2021 Oct 28;23:490-506.), but the method of applying regulatory T cells in this study involved adoptive cell transfer, which increases complexity and regulatory issues compared to the adoptive cell transfer-free method described in this article.

[0006] The inventors have selectively expanded regulatory T cells using compound A as a pretreatment or as a combination therapy when administering AAV-based or LNP-based gene therapies to alleviate irAE and enhance transgene expression in various animal models, such as mice, rats, and non-human primates.

[0007] The following examples are covered. Example 1 is a method of delivering a gene therapy agent to the cells of a subject, the method comprising administering an IL-2 conjugate to the subject, wherein the gene therapy agent is administered to the subject before, simultaneously with or after the IL-2 conjugate, wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate portion, and the non-natural amino acid linked to the conjugate portion is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or located in reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

[0008] Example 2 is a method of treating an individual in need with a gene therapy agent, the method comprising administering an IL-2 conjugate to the subject, wherein the gene therapy agent is administered to the subject before, simultaneously with or after the IL-2 conjugate, and the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or located in reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

[0009] Example 3 is a method for increasing the expression of a gene therapy agent, the method comprising: IL-2 conjugates were administered to the subjects; The gene therapy agent was administered to the subject before, simultaneously with, or after the IL-2 conjugate, and The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

[0010] Example 4 is a method for reducing the immune response to gene therapy agents, the method comprising: IL-2 conjugates were administered to the subjects; The gene therapy agent was administered to the subject before, simultaneously with, or after the IL-2 conjugate, and The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

[0011] Example 5 is a method for preventing immune-related adverse events in subjects, the method comprising: IL-2 conjugates were administered to the subjects; The gene therapy agent was administered to the subject before, simultaneously with, or after the IL-2 conjugate, and The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

[0012] Example 6 is a method as described in any of the preceding examples, wherein the method further includes, prior to administering 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 expression of one or more activated biomarkers or an increase in the expression of one or more activated biomarkers in these immune cells, wherein the expression or increase in the expression of the one or more activated biomarkers after incubation with the gene therapy agent identifies the subject as requiring the IL-2 conjugate.

[0013] Example 7 is a method for selecting subjects to be treated 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 expression of one or more activated biomarkers or an increase in the expression of one or more activated biomarkers in these immune cells, wherein the expression or increase in the expression of the one or more activated biomarkers after incubation with the gene therapy agent identifies subjects to be treated with the gene therapy agent and the IL-2 conjugate; and c) selecting the subjects to be treated with the gene therapy agent and the IL-2 conjugate identified in step b). The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

[0014] Example 8 is the method as described in the previous example, further comprising 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).

[0015] Example 9 is a method as described in any one of Examples 6 to 8, wherein the immune cell is a lymphocyte, T cell, CD8+ T cell, effector T cell, cytotoxic T cell or NK cell.

[0016] Example 10 is a method as described in any of the preceding examples, wherein the non-natural amino acid is connected to the conjugate via a linker.

[0017] Example 11 is the method as described in the previous example, wherein the connector includes a homobifunctional connector, a heterobifunctional connector, a cleavable or non-cleavable dipeptide connector, a maleimide group, a spacer, or a combination thereof.

[0018] Example 12 is a method as described in any of the preceding examples, wherein the non-natural amino acid is a substituted lysine, a substituted phenylalanine, a substituted histidine, a substituted cysteine, contains an azide group, contains an alkynyl group, contains an aldehyde group, contains an aromatic side chain, or contains a ketone group.

[0019] Example 13 is a method as described in any of the preceding examples, wherein the at least one non-natural amino acid includes N6-azidoethoxy-L-lysine, N6-((2-azidoethoxy)-carbonyl)-L-lysine, N6-propynylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, allyloxycarbonyl lysine, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, p-propynyl-phenylalanine. Acids, 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)seleno)propionic acid or 2-amino-3-(phenylseleno)propionic acid.

[0020] Example 14 is a method as described in any of the preceding examples, wherein the non-natural amino acid is azide-substituted lysine.

[0021] Example 15 is a method as described in any of the preceding examples, wherein the non-natural amino acid is N6-((2-azidoethoxy)-carbonyl)-L-lysine.

[0022] Example 16 is a method as described in any of the preceding examples, wherein the conjugated portion comprises a water-soluble polymer.

[0023] Example 17 is the method described in the previous example, wherein the water-soluble polymer includes polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

[0024] Example 18 is the method as described in the previous example, wherein the conjugation portion comprises PEG.

[0025] Example 19 is the method as described in the previous example, wherein the conjugated portion is PEG with a molecular weight of about 10-85 kDa or selected from about 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa and 85 kDa.

[0026] Example 20 is a method as described in any of the preceding examples, wherein the conjugated portion is PEG with a molecular weight of about 20-70 kDa or selected from about 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa and 70 kDa.

[0027] Example 21 is a method as described in any of the preceding examples, wherein the conjugated portion is PEG with a molecular weight of about 30-60 kDa or selected from about 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa and 60 kDa.

[0028] Example 22 is a method as described in any of the foregoing examples, wherein the amino acid linked to the conjugation portion has the structure of formula (I): Formula (I); in: Z is CH2 and Y is ; Y is CH2 and Z is ; Z is CH2 and Y is ;or Y is CH2 and Z is ; W is a PEG group; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the next amino acid residue.

[0029] Example 23 is the method as described in the previous example, wherein Z is CH2 and Y is Or Y is CH2 and Z is .

[0030] Example 24 is the method as described in the previous example, wherein the structure of formula (I) has the structure of formula (IV) or formula (V): Formula (IV); Formula (V); in: W is the PEG group, with a molecular weight of approximately 5-60 kDa or approximately 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa or 60 kDa.

[0031] Example 25 is the method as described in any of the preceding examples, wherein the IL-2 conjugate further comprises an alanine or methionine at the N-terminus of the first amino acid of the sequence having at least 80% sequence identity with SEQ ID NO: 1.

[0032] Example 26 is a method as described in any of the preceding examples, wherein the IL-2 conjugate comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.

[0033] Example 27 is a method as described in any of the preceding examples, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the positions are 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 are replaced by this non-natural amino acid.

[0034] Example 28 is the method as described in any of the preceding examples, wherein positions K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108 of the sequence referenced in SEQ ID NO: 1 are replaced by the non-natural amino acid.

[0035] Example 29 is the method as described in the previous example, wherein position L18 of the sequence referenced in SEQ ID NO: 1 is replaced by the non-natural amino acid.

[0036] Example 30 is the method as described in Example 28, wherein position H15 of the sequence referenced in SEQ ID NO: 1 is replaced by the non-natural amino acid.

[0037] Example 31 is a method as described in any of the preceding examples, wherein the IL-2 conjugate is capable of amplifying CD4+ T regulatory (Treg) cells.

[0038] Example 32 is a method as described in any of the preceding examples, wherein the non-natural amino acid and / or the conjugated portion impairs or blocks the receptor signaling efficacy of the IL-2 conjugate for IL-2Rβγ, or reduces the recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex.

[0039] Example 33 is a method as described in any of the preceding examples, wherein the IL-2 conjugate has lower receptor signaling efficacy for IL-2Rβγ than wild-type IL-2 has.

[0040] Example 34 is a method as described in any of the preceding examples, wherein the receptor signaling efficacy of the IL-2 conjugate for IL-2Rαβγ is greater than or equal to the receptor signaling efficacy of wild-type IL-2 for IL-2Rαβγ.

[0041] Example 35 is a method as described in any of the preceding examples, wherein the IL-2 conjugate amplifies the CD4+ Treg population in the subject.

[0042] Example 36 is a method as described in any of the preceding examples, wherein the IL-2 conjugate inhibits the proliferation of CD8+ T cells in the subject.

[0043] Example 37 is a method as described in any of the preceding examples, wherein the IL-2 conjugate inhibits the proliferation of effector memory CD8+ T cells in the subject.

[0044] Example 38 is a method as described in any of the preceding examples, wherein the gene therapy agent comprises a vector, and the IL-2 conjugate inhibits vector-specific IFNγ-secreting CD8+ T cells in the subject.

[0045] Example 39 is a method as described in any of the preceding examples, wherein the gene therapy agent encodes a transgenic product, and the IL-2 conjugate inhibits transgenic product-specific IFNγ-secreting CD8+ T cells in the subject.

[0046] Example 40 is a method as described in any of the preceding examples, wherein the gene therapy agent encodes a transgenic product, and the IL-2 conjugate inhibits the production of antibodies against the transgenic product.

[0047] Example 41 is a method as described in any of the preceding examples, wherein the gene therapy agent encodes a transgenic product, and the IL-2 conjugate inhibits the production of IgG1 antibodies against the transgenic product.

[0048] Example 42 is a method as described in any of the preceding examples, wherein the gene therapy agent encodes a transgenic product, and the IL-2 conjugate prolongs the expression of the transgenic product in a subject who is given the gene therapy agent but not the IL-2 conjugate.

[0049] Example 43 is the method as described in the previous example, wherein the extended expression of the transgenic product is for at least about 5 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 14 weeks, or about 16 weeks.

[0050] Example 44 is a method as described in any of the foregoing examples, wherein the gene therapy agent comprises a viral vector.

[0051] Example 45 is the method as described in Example 44, wherein the IL-2 conjugate inhibits the production of antibodies against the viral vector.

[0052] Example 46 is the method as described in Example 44 or Example 45, wherein the IL-2 conjugate inhibits the production of antibodies against the capsid protein of the viral vector.

[0053] Example 47 is a method as described in any one of Examples 44 to 46, wherein the viral vector is an adeno-associated virus (AAV) particle.

[0054] Example 48 is the method described in the previous example, wherein the AAV particle comprises AAV1 capsule, AAV2 capsule, AAV3 capsule, AAV4 capsule, AAV5 capsule, AAV6 capsule, AAV7 capsule, AAV8 capsule, AAVrh8 capsule, AAV9 capsule, AAV10 capsule, AAVrh10 capsule, AAV11 capsule, AAV12 capsule, AAVrh32.33 capsule, AAV-XL32 capsule, AAV-XL32.1 capsule, AAV LK03 capsule, AAV2R471A capsule, AAV2 / 2-7m8 capsule, AAV DJ capsule, AAV DJ8 capsule, AAV2 N587A capsule, AAV2 E548A capsule, AAV2 N708A capsule, and AAV... V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or functional variants thereof.

[0055] Example 49 is the method as described in the previous example, wherein the AAV capsid contains a tyrosine mutation, a heparin-binding mutation, or an HBKO mutation.

[0056] Example 50 is the method as described in any one of Examples 47 to 49, wherein the AAV viral particle contains an AAV genome containing one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR, or AAV12 ITR.

[0057] Example 51 is the method as described in the previous example, wherein the one or more ITRs and the capsid of the AAV particle are derived from the same AAV serotype.

[0058] Example 52 is the method as described in the previous example, wherein the one or more ITRs and the capsid of the AAV particle are derived from different AAV serotypes.

[0059] Example 53 is the method as described in any one of Examples 44 to 46, wherein the viral vector is an adenovirus particle.

[0060] Example 54 is the method as described in the previous example, wherein the adenovirus particle comprises a capsid from adenovirus serotypes 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, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3, canine Ad 2, sheep Ad, or swine Ad 3 or functional variants thereof.

[0061] Example 55 is the method as described in any one of Examples 44 to 46, wherein the viral vector is a lentiviral particle.

[0062] Example 56 is the method described in the previous example, wherein the lentiviral particles are pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningovirus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114 or functional variants thereof.

[0063] Example 57 is a method as described in any one of Examples 44 to 46, wherein the viral vector is a herpes simplex virus (HSV) particle.

[0064] Example 58 is the method as described in the previous example, wherein the HSV particle is an HSV-1 particle or an HSV-2 particle or a functional variant thereof.

[0065] Example 59 is a method as described in any one of Examples 1 to 41, wherein the gene therapy agent comprises lipid nanoparticles.

[0066] Example 60 is a method as described in any of the preceding examples, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

[0067] Example 61 is the method as described in the previous example, wherein the heterologous transgene is operatively linked to a promoter.

[0068] Example 62 is the method as described in the previous example, wherein the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0069] Example 63 is a method as described in any one of Examples 60 to 62, wherein the nucleic acid comprises terminally closed DNA (ceDNA).

[0070] Example 64 is the method described in Example 60, wherein the nucleic acid comprises mRNA.

[0071] Example 65 is a method as described in any of the preceding examples, wherein the gene therapy agent and the IL-2 conjugate are administered simultaneously to the subject.

[0072] Example 66 is a method as described in any one of Examples 1 to 64, wherein the gene therapy agent is administered to the subject prior to the IL-2 conjugate.

[0073] Example 67 is the method as described in the previous example, wherein the gene therapy agent is administered less than 14 days or less than 7 days prior to the IL-2 conjugate.

[0074] Example 68 is a method as described in any one of Examples 1 to 64, wherein the gene therapy agent is administered to the subject after the IL-2 conjugate.

[0075] Example 69 is the method as described in the previous example, 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] Example 70 is a method as described in any one of Examples 1 to 64, wherein the IL-2 conjugate is administered before, simultaneously with or after the administration of the gene therapy agent.

[0077] Example 71 is a method as described in any of the foregoing examples, wherein the individual suffers from a disease or condition suitable for treatment by gene therapy.

[0078] Example 72 is the method as described in the previous example, wherein the disease or condition is a single-gene disease or condition.

[0079] Example 73 is a method as described in any of the preceding examples, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, intracranially, intracerebrally, intra-CSF, intra-DRG, intravenously, intraocularly, intracerebellomedullary cistern, or intrahepatically.

[0080] Example 74 is a method as described in any of the preceding examples, wherein the IL-2 conjugate is administered parenterally and / or systemically.

[0081] Example 75 is a method as described in any of the preceding examples, wherein the IL-2 conjugate is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, intracranially, intracerebrally, intracranially, intradermally, intravenously, intraocularly, intracerebellomedullary cistern, or intrahepatically.

[0082] Example 76 is a method as described in any of the foregoing examples, wherein the subject is a mammal.

[0083] Example 77 is a method as described in any of the foregoing examples, wherein the subject is a primate.

[0084] Example 78 is a method as described in any of the foregoing examples, wherein the subject is a human.

[0085] Example 79 is a method as described in any of the preceding examples, wherein the IL-2 conjugate is administered approximately 1, 2, 3, 4, 5, 6, or 7 days prior to the gene therapy agent.

[0086] Example 80 is the method as described in any one of Examples 1 to 78, wherein the IL-2 conjugate is administered approximately 1, 2, 3, or 4 days after the gene therapy agent.

[0087] Example 81 is the method as described in any one of Examples 1 to 78, wherein the IL-2 conjugate is administered on the same day as the gene therapy agent.

[0088] Example 82 is a method as described in any of the preceding examples, 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] Example 83 is the method as described in the previous example, wherein the IL-2 conjugate is administered at a dose of about 0.05 mg / kg.

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

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

[0092] Example 86 illustrates the use of the IL-2 conjugate in the preparation of a medicament used in any of the preceding examples.

[0093] Example 87 is an IL-2 conjugate used in any one of Examples 1 to 85. Attached Figure Description

[0094] Figure 1 Kinetic data on the amplification of CD4 Tregs by compound A in mice are provided, as measured by the percentage of CD4 Tregs. The figure illustrates the amplification of CD4 Tregs in peripheral blood of mice after subcutaneous administration of 0.3 mg / kg compound A.

[0095] Figure 2Aand 2B The following data were provided: Compound A effectively inhibited effector memory CD8 T cells (CD8 T cells) stimulated by AAVrh32.33-LacZ administration in mouse PBMCs. EM ; Figure 2A ) and LacZ-specific CD8 T cells ( Figure 2B The amplification of ).

[0096] Figure 3A and 3B The following data were provided: Compound A effectively inhibited the secretion of AAV capsid-specific IFNγ-type CD8 T cells in the spleen of mice. Figure 3A ) and LacZ-specific IFNγ-secreting CD8 T cells ( Figure 3B Both.

[0097] Figure 4A and 4B The following data were provided: Compound A enhanced the expression of the OVA gene delivered by the AAVrh32.33 vector in mice ( Figure 4A And inhibit the production of anti-OVA IgG1 ( Figure 4B This indicates an inverse correlation between serum OVA and anti-OVA IgG1 levels. The results show that compound A enhances OVA expression while inhibiting anti-OVA IgG1 production.

[0098] Figure 5A , 5B The graph shown in Figure 5C demonstrates that compound A significantly increases the CD4 Treg population in rat PBMCs ( Figure 5A It effectively inhibited the proliferation of CD8 T cells stimulated by AAVrh10-EGFP administration. Figure 5B ) and effector memory CD8 T cell expansion (CD8 T EM ; Figure 5C ).

[0099] Figure 6A and 6B The following data were shown: Administration of compound A significantly increased the CD4 Treg population in non-human primates ( Figure 6A ), as measured by the percentage of CD4 Treg cells in monomorphic cells, and enhances the expression of the OVA gene delivered by the AAVrh32.33 vector in serum (measured by [method / indication]). Figure 6B ).

[0100] Figure 7A and 7B An exemplary IL-2 variant is shown for pSTAT5 signaling in primary human LRS cells. Figure 7A ) and the proliferation response in the mouse CTLL-2 population ( Figure 7B The dose-response curve of ).

[0101] Figure 8 Plasma concentration profiles of IL-2 conjugates K9_30kD, L19_30kD, N88R / D109_30kD, H16_30kD, Q126_30kD, and N88_30kD (all administered at 0.9 mg / kg) after administration to C57 / BL6 mice from Example 5 are shown.

[0102] Figure 9 The mean fold change (% in singlet) of Tregs after administration of the IL-2 conjugate to C57 / BL6 mice from Example 5 is shown.

[0103] Figure 10 The proportion of Treg (CD3+ CD4+ CD25+ FoxP3+) cells in the total cell population (single-state) of the IL-2 conjugate is shown in C57 / BL6 mice from Example 5.

[0104] Figure 11A and 11B The proportion of CD8+ T cells (CD3+ CD4- CD8+) in the total cell population (single-state) is shown after a single dose of the IL-2 conjugate. Figure 11A The proportion of CD8+ T cells (CD3+CD4-CD8+) in the total cell population (single-state) is shown in C57 / BL6 mice from Example 5 after single doses of IL-2 conjugates K9_30kD, L19_30kD, Q126_30kD, and H16_30kD. Figure 11B The proportion of CD8+ T cells (CD3+ CD4- CD8+) in the total cell population (single-state) is shown in C57 / BL6 mice from Example 5 after single doses of IL-2 conjugates E100_30kD, N88R / D109_30kD, T123_30kD, N88_30kD and V91_30kD.

[0105] Figure 12 The plasma concentration curves of the IL-2 conjugate after administration to cynomolgus monkeys from Example 5 are shown.

[0106] Figure 13 The proportion of Treg cells in the total blood cell population (single state) of cynomolgus monkeys is shown after administration of the IL-2 conjugate from Example 5.

[0107] Figure 14 The proportion of CD8+ T cells in the total blood cell population (single-state) of cynomolgus monkeys after administration of the IL-2 conjugate from Example 5 is shown.

[0108] Figure 15 The plasma concentration versus time of the H16_30kD variant in non-human primates at doses of 0.12 mg / kg and 0.67 mg / kg from Example 5 is shown in graphs, with the 0.12 mg / kg dose shown as the lower trace and the 0.67 mg / kg dose shown as the upper trace.

[0109] Figure 16 The graph shows the plasma concentrations of the H16_30kDa and H16_50kDa variants 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, with the 30 kDa variant shown as a lower trace (square) and the 50 kDa variant shown as an upper trace (triangle).

[0110] Figure 17 The graph shows the percentage of Tregs in the singlet versus time after administration 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 in non-human primates from Example 5, where the trace of the mediator is the lower trace (square), the trace of the 30 kDa variant is shown in the middle trace, and the trace of the 50 kDa variant is shown in the upper trace.

[0111] Figure 18 The study design of Example 5 is shown to evaluate the effect of H16_50kD on delayed-type hypersensitivity (DTH) in C57BL / 6 mice. DTH in mice was induced with keyforaminifera hemocyanin (KLH) (sensitization on day 1 followed by challenge on day 7), while H16_50kD was administered at doses of 0.03 mg / kg, 0.1 mg / kg, and 0.3 mg / kg from Example 5 (days 0 and 3).

[0112] Figure 19A , 19B 19C shows changes in ear thickness measurements and blood immunophenotype from C57BL / 6 mice in Example 5. Figure 19A The area under the curve (AUC) shows the increased ear thickness compared to mice stimulated with KLH only on day 7. Figure 19B The changes in middle ear thickness measurements in C57BL / 6 mice are shown before KLH stimulation (on day 7) and subsequently on days 8, 9, and 10. Figure 19CThe relative percentage of CD4+ T cells within the CD25+FoxP3+ cell population over time is shown in whole blood samples from mice. "KLH only" indicates KLH challenge performed only on day 7 (no sensitization on day 1) with only the vehicle administered. "Vehicle" indicates KLH sensitization (day 1) and challenge (day 7) with only the vehicle administered. "0.03" indicates KLH sensitization and challenge with H16_50kD administered at a dose of 0.03 mg / kg. "0.1" indicates KLH sensitization and challenge with H16_50kD administered at a dose of 0.1 mg / kg. "0.3" indicates KLH sensitization and challenge with H16_50kD administered at a dose of 0.3 mg / kg. "CsA" indicates KLH sensitization and challenge with cyclosporine A administration. See also Table 10 in Example 5.

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

[0114] Figure 21 The absolute count of CD4+CD25+FoxP3+ cells on day 10 is shown in a whole blood sample from mice in Example 5.

[0115] Figure 22A and 22B Data on the amplification of Treg, CD8+ T cells, CD4+ T cells, NK cells, and B cells by compound A in mice are provided. Treg amplification was measured at specified time points following subcutaneous administration of 0.3 mg / kg compound A to mice, as measured by the percentage of CD4+ T cells in peripheral blood. Figure 22A ). The expansion of CD4 Tregs, CD8+ T cells, CD4+ T cells, NK cells, and B cells was measured at specified time points after subcutaneous administration of 0.3 mg / kg compound A to mice, as measured by fold changes in peripheral blood ( Figure 22B ).

[0116] Figure 23A , 23B Data provided by 23C showed that compound A effectively inhibited effector memory CD8 T cells (CD8 T cells) that would normally occur after AAV administration. EM ; Figure 23AThe expansion of AAV capsid-specific IFNγ-secreting CD8 T cells was observed. Following ex vivo spleen restimulation on day 21, compound A effectively inhibited the proliferation of AAV capsid-specific IFNγ-secreting CD8 T cells. Figure 23B ) and LacZ-specific IFNγ-secreting CD8 T cells ( Figure 23C Both.

[0117] Figure 24A and 24B The diagram illustrates how compound A reduces CD8+ T cell responses and enhances transgene levels after gene therapy. Compound A enhances transgene levels in mice using the AAVrh32.33 vector (…). Figure 24A The expression of the OVA gene delivered via the AAVrh32.33 vector was measured, as indicated by serum OVA concentration. Following delivery of the OVA gene via the AAVrh32.33 vector, compound A significantly inhibited the expansion of effector memory CD8 T cells (CD8 T cells) in mice. EM ; Figure 24B ).

[0118] Figure 25A , 25B The graph shown in Figure 25C demonstrates that compound A significantly increases the CD4 Treg population in rat PBMCs ( ). Figure 25A It effectively inhibited the expansion of effector memory CD8 T cells (CD8 T cells) stimulated by AAVrh10-EGFP administration. EM ; Figure 25B ) and CD8 T cell proliferation ( Figure 25C ).

[0119] Figure 26A , 26B And 26C showed the following data: Compound A administration significantly increased the CD4 Treg population ( Figure 26A ), as measured by the percentage of CD4 Treg cells in monomorphic cells, and relative to serum expression of the OVA gene delivered by the AAVrh32.33 vector without compound A in non-human primates (measured in serum). Figure 26B This enhances the expression of the OVA gene delivered by the AAVrh32.33 vector in non-human primates, as measured in serum. Figure 26C ). Figure 26B and 26C Each line in the diagram represents the serum OVA level of an individual animal.

[0120] Figure 27A , 27B Figures 27C and 27D show a longitudinal analysis of serum levels of anti-AAV and anti-OVA IgG. Figure 27A and 27BIn the presence or absence of a single subcutaneous dose of 0.3 mg / kg compound A, rAAV1-OVA 2 × 10 11 Abdominal and gluteal muscles (VG) or AAVrh32.33-OVA 5 × 10 10 VG was administered intramuscularly to wild-type C57BL / 6 mice (N ≥ 5 per group). Serum samples were collected via continuous in vivo bleeding at different time points throughout the 16 weeks of the study. Serum anti-AAV1 IgG levels were measured by ELISA. Figure 27A ) and anti-OVA IgG ( Figure 27B ) level. Figure 27C and 27D In the presence or absence of a single subcutaneous dose of 0.08 mg / kg compound A, rAAV1-OVA 3 × 10 12 VG intramuscularly treated cynomolgus monkeys (N ≥ 4 per group). Serum samples were collected via continuous in vivo bleeding at different time points throughout the 12 weeks of the study. Serum anti-AAV1 IgG levels were measured by ELISA. Figure 27C ) and anti-OVA IgG ( Figure 27D The data are expressed as mean ± SEM. The dashed line indicates the lower limit of detection. Statistical analysis was performed using two-way ANOVA and the test of repeatability. p < 0.05, p < 0.01, p < 0.0001.

[0121] Figure 28 Data from cynomolgus monkeys treated intramuscularly with AAVrh32.33 or rAAV1 expressing the OVA transgene, with or without a single subcutaneous dose of compound A (up to 0.08 mg / kg), are presented. Immunocytic cellular analysis and / or serum transgene level measurements were performed sequentially via live hemorrhage. Serum OVA protein concentrations in non-human primates (NHPs) were compared. Kaplan-Meier plots were generated for combined data from both studies, comparing the duration of OVA expression once expressed following AAV gene therapy. Statistical analysis of the combined data was performed using Cox proportional regression to determine the hazard ratio of the AAV treatment group relative to the AAV + compound A treatment group.

[0122] Figure 29A , 29B 29C and 29D indicate that, with or without a single subcutaneous dose of 0.3 mg / kg compound A, the use of AAVrh32.33-LacZ 2 × 1011 Wild-type C57BL / 6 mice were treated intramuscularly with the gluteal muscle (VG). A single dose of compound A was administered 4 days prior to AAV gene therapy (pretreatment) or concurrently with AAV gene therapy (co-treatment). Mice from each group were sacrificed 21 days after AAV gene therapy treatment, and the proportion of proliferating CD8+ T cells was analyzed. Figure 29A ) and CD8+ T-effect memory (CD8+ T EM ) cell ratio ( Figure 29B CD8+ T cell immune responses were measured in spleen cells. Proliferating CD8+ T cells were identified by expressing the proliferative marker Ki67. EM Cells were identified as CD62L+ and CD44+ cells within the CD8+ T cell population. Figure 29C and Figure 29D Spleen cells were restimulated in vitro with a 1 µg / ml AAVrh32.33 peptide pool for 6 hours to determine AAV capsid-specific T cell responses. Figure 29C ), or use a 1 µg / ml β-galactosidase peptide pool for in vitro restimulation for 6 hours to determine transgene-specific T cell responses ( Figure 29D The response of CD8+ T cells expressing IFNγ in spleen cells was measured by intracellular cytokine staining. Data are expressed as mean ± SD. Statistical analysis was performed using two-way ANOVA and multiple comparison tests. p < 0.05, p < 0.01, p < 0.0001. Detailed Implementation

[0123] This specification and exemplary embodiments should not be considered restrictive. For the purposes of this specification and the appended claims, unless otherwise indicated, all figures representing quantities, percentages, or proportions, as well as other numerical values ​​used in the specification and claims, should in all cases be understood to be modified by the term "about" (unless such figures and values ​​are so modified). "About" indicates a degree of difference that does not substantially affect the nature of the described subject matter, for example, within 10%, 5%, 2%, or 1%. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At least, and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant digits reported 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 should be understood that they are not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the appended claims.

[0125] Before describing this instruction in detail, it should be understood that this disclosure is not limited to specific compositions or process steps and is therefore subject to variation. It should be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used in this specification and the appended claims include plural indicators. Thus, for example, a reference to “nucleic acid” includes multiple nucleic acids, and a reference to “cell” includes multiple cells, etc.

[0126] Numerical ranges include numbers within a defined range. Taking into account significant figures and measurement-related errors, measured and measurable values ​​should be understood as approximate values. Furthermore, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be restrictive. It should be understood that the foregoing general and detailed descriptions are exemplary and explanatory only, and not intended to limit the content taught.

[0127] Unless otherwise specified in the foregoing description, embodiments in the description that "comprise" various components are also considered to be "consisting of the listed components" or "substantially composed of the listed components"; embodiments in the description that "comprise various components" are also considered to "include the listed components" or "substantially composed of the listed components"; and embodiments in the description that "substantially consist of various components" are also considered to "comprise the listed components" or "include the listed components" (this interchangeability does not apply to the use of these terms in the claims).

[0128] The chapter headings used herein are for organizational purposes and should not be construed as limiting the subject matter in any way. In the event of any conflict between any cited or incorporated references and any express content (including definitions) of this specification, this specification shall prevail. I. Definition

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

[0130] As used herein, "isolated" means that a biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated, separately produced, or purified from other components (e.g., other components in a sample, cell, or organism in which the component is naturally present). "Isolated" nucleic acid molecules, proteins, or cells include nucleic acid molecules, proteins, or cells purified using standard purification methods. The terms "isolated" or "purified" do not require absolute purity; rather, they are intended as relative terms. Thus, for example, an isolated biological component is a biological component in which the biological component is more enriched in the formulation than in its natural environment within cells, organisms, samples, or production containers (e.g., cell culture systems). For example, the isolated biological component may constitute at least 50%, such as at least 70%, at least 80%, at least 90%, at least 95%, or higher, of the total biological component content of the formulation.

[0131] As used herein, “subject” refers to an animal, such as a member of a mammalian species (e.g., human) or a avian species (e.g., bird), or other organisms such as plants. More specifically, a subject can be a vertebrate, such as a mammal (e.g., a mouse), a primate, an ape, or a human. Animals include farm animals (e.g., cattle, dairy cows, poultry, horses, pigs, etc.), grazing animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual who has or is suspected of having a disease or is susceptible to a disease, or an individual who needs or is suspected of needing treatment. The terms “individual” or “patient” are intended to be used interchangeably with “subject.” For example, a subject can be an individual who needs gene therapy, for example, due to an autoimmune disease or a developmental, neurological, or other genetic condition. As another example, a subject can be a pregnant or planning-to-be-pregnant woman who may have been diagnosed with or is suspected of having a disease such as cancer or an autoimmune disease.

[0132] As used herein, the term "potency" refers to the amount of cytokine (e.g., IL-2 peptide) required to produce a target effect. In some embodiments, the term "potency" refers to the amount of cytokine (e.g., IL-2 peptide) required to activate a target cytokine receptor (e.g., IL-2 receptor). In other embodiments, the term "potency" refers to the amount of cytokine (e.g., IL-2 peptide) 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 the maximum effect. In other embodiments, 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 that is attached (e.g., via a linker) to a conjugate moiety (e.g., containing a PEG group); an IL-2 conjugate may be, but is not necessarily, in the form of a pharmaceutically acceptable salt, solvate, or hydrate. As described elsewhere herein, an IL-2 polypeptide may contain a non-natural amino acid that may act as an attachment site to the conjugate moiety.

[0134] As used herein, the terms “operably linked” and “functionally linked” with respect to promoters refer to the relationship between a coding sequence and a promoter element. A promoter is operably or functionally linked to a coding sequence when the expression of a coding sequence via transcription is regulated or controlled by a promoter element. Regarding promoter elements, the terms “operably linked” and “functionally linked” are used interchangeably herein.

[0135] As used herein, the term "gene therapy agent" refers to a nucleic acid (e.g., expression construct, miRNA, antisense, shRNA, siRNA) or a combination of nucleic acid and a drug agent used to deliver nucleic acids to an individual or cell to modify or manipulate the expression of one or more nucleic acids (e.g., genes, mRNA) in the individual or cell to alter the biological properties of living cells. Examples of gene therapy agents include, but are not limited to, viral vectors (e.g., adeno-associated virus, adenovirus, lentivirus, herpes simplex virus, baculovirus), bacterial vectors, and non-viral vectors (e.g., lipid nanoparticles encapsulating therapeutic nucleic acids or plasmid DNA (e.g., terminally blocked DNA) containing therapeutic nucleic acids and / or encoding therapeutic peptides).

[0136] As used in this article, "vector" refers to a recombinant plasmid or virus containing nucleic acid to be delivered to a host cell in vitro or in vivo.

[0137] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, including ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases. The backbone of a nucleic acid may contain sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a nucleic acid may contain polymers of synthetic subunits (such as phosphoramides), and thus may be oligodeoxynucleoside phosphoramides (P-NH2) or mixed phosphoramide-phosphodiester oligomers. Furthermore, double-stranded nucleic acids can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing it under appropriate conditions, or by using a DNA polymerase to synthesize the complementary strand de novo with appropriate primers.

[0138] The terms "peptide" and "protein" are used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues can contain native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Full-length proteins and fragments thereof are included in this definition. The term also includes post-translational modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this invention, "peptide" refers to a protein that includes modifications to its native sequence, such as deletions, additions, and substitutions (which may be conserved in nature), provided that the protein maintains its desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations in the host that produces these proteins or by errors due to PCR amplification.

[0139] "Recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterologous sequences (nucleic acid sequences that are not naturally present in the virus from which the vector is derived, e.g., sequences not of viral origin or derived 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 (ITR) sequences.

[0140] “Recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV), flanked by at least one (e.g., two) AAV inverted terminal repeat (ITR) sequences. When such rAAV vectors are present in host cells infected with a suitable helper virus (or expressing a suitable helper function) and expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), they can replicate and be packaged into infectious viral particles. When an rAAV vector is incorporated into a larger polynucleotide (e.g., into a chromosome or into another vector such as a plasmid used for cloning or transfection), it can be referred to as a “pro-vector”, which can be “rescued” by replication and capsidation in the presence of AAV packaging function and a suitable helper function. rAAV vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, lipid complexes, encapsulated in liposomes, and, in the examples, capsidated within viral particles (particularly AAV particles). The rAAV vector can be packaged in the capsid of an AAV virus to generate "recombinant adeno-associated virus particles (rAAV particles)".

[0141] "rAAV virus" or "rAAV virus particle" refers to a viral particle composed of at least one AAV capsid protein and a capsidized rAAV vector genome.

[0142] A “recombinant adenovirus vector” is a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from adenovirus), flanked by at least one adenovirus inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeats (ITRs). Such recombinant viral vectors can be replicated and packaged into infectious viral particles when present in host cells expressing essential adenovirus genes missing from the recombinant viral genome (e.g., E1, E2, E4, etc.). When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., into a chromosome or into another vector such as a plasmid used for cloning or transfection), the recombinant viral vector can be referred to as the “original vector,” which can be “rescued” by replication and capsidation in the presence of adenovirus packaging function. Recombinant viral vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and capsidated in viral particles (e.g., adenovirus particles). Recombinant viral vectors can be packaged into adenovirus capsids to generate “recombinant adenovirus particles.”

[0143] A “recombinant lentiviral vector” is a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from lentiviruses), flanked by at least one lentiviral terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral LTRs. When present in host cells that have been infected with appropriate helper functions, such recombinant viral vectors can be replicated and packaged into infectious viral particles. Recombinant lentiviral vectors can be packaged into lentiviral capsids to generate “recombinant lentiviral particles.”

[0144] "Recombinant herpes simplex virus vector (recombinant HSV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from HSV), flanked by HSV terminal repeat sequences. When present in host cells already infected with suitable helper functions, such recombinant viral vectors can be replicated and packaged into infectious viral particles. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., into a chromosome or another vector used for cloning or transfection, such as a plasmid), the recombinant viral vector can be referred to as the "original vector," which can be "rescued" in the presence of HSV packaging function through replication and capsidation. Recombinant viral vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, lipid complexes, encapsulated in liposomes, and capsidated within viral particles (e.g., HSV particles). Recombinant viral vectors can be packaged into HSV capsids to generate "recombinant herpes simplex virus particles."

[0145] As used herein, “solid lipid nanoparticles” (SLN, sLNP) or “lipid nanoparticles” (LNP) refer to nanoparticles containing lipids that may contain a payload. In some instances, only a single phospholipid layer is present; furthermore, in some embodiments, the interior of the particle is largely composed of a lipophilic substance and a payload. The payload, such as nucleic acids, may be embedded within the interior. In some instances, lipid nanoparticles are liposomes, which contain a lipid bilayer and may contain a hydrophilic or aqueous interior containing a payload.

[0146] As used herein, the term "improvement" in relation to gene therapy can refer to the act of enhancing, increasing, prolonging, or otherwise increasing the expression of the therapeutic gene payload of a gene therapy agent. In some embodiments, an improved gene therapy is one in which the expression of the therapeutic gene payload of a gene therapy agent administered with an IL-2 conjugate is increased by more than any one of about 10%, 25%, 50%, 75%, or 100% compared to gene therapy not administered with an IL-2 conjugate. In some embodiments, an improved gene therapy is one in which the expression duration of the therapeutic gene payload of a gene therapy agent administered with an IL-2 conjugate is prolonged by more than any one of about 10%, 25%, 50%, 75%, or 100% compared to gene therapy not administered with an IL-2 conjugate. In some instances, gene therapy is improved by reducing the immune response to the gene therapy agent (e.g., adaptive immune response). In some embodiments, the improved gene therapy is a gene therapy in which the immune response to the gene therapy agent administered with the IL-2 conjugate is reduced by more than any one of about 10%, 25%, 50%, 75%, or 100% compared to gene therapy not administered with the IL-2 conjugate. In some embodiments, the reduction in the immune response to the gene therapy agent is measured as a decrease in cytokine signatures after exposure of the gene therapy agent to immune cells in the presence of the IL-2 conjugate compared to exposure of the gene therapy agent to immune cells in the absence of the IL-2 conjugate.

[0147] As used herein, the term “modification” when referring to gene therapy can refer to actions that alter, change, modify, improve, or otherwise modify the presence or activity of a gene therapy agent. For example, modulating the immune response to a gene therapy agent can refer to any action that results in altering, changing, modifying, improving, or otherwise modifying the immune response to a gene therapy agent (e.g., reducing, delaying, and / or eliminating the immune response to a gene therapy agent (e.g., adaptive immune response)).

[0148] As used herein, the term "cytokine signature" in relation to an immune response to a gene therapy agent (e.g., an adaptive immune response) refers to altered (e.g., increased, decreased) expression of one or more cytokines following exposure of adaptive immune cells to a gene therapy agent. In some instances, the cytokine signature is specific to the following: interleukin-6 (IL-6); tumor necrosis factor-α (TNF-α); tumor necrosis factor-β (TNF-β); interferon-α (IFN-α); interleukin-10 (IL-10); interleukin-8 (IL-8); factors regulating the expression and secretion of activated normal T cells (RANTES); granulocyte-macrophage colony-stimulating factor (GM-CSF); interferon-γ (IFN-γ); interferon-γ-inducible protein 10 (IP-10); interleukin-1β (IL-1β); interleukin-2 (IL-2); and / or the interleukin-4 (IL-4) pathway.

[0149] Adaptive immune cells are leukocytes that mediate adaptive immunity and include B cells, T cells, and NK cells. Once infected, AAV can elicit an immune response. The magnitude of this response depends on the AAV serotype and cell type. After AAV transduces host immune cells, they can bind to immune receptors. Once these receptors are activated by the virus, they secrete cytokines that establish an antiviral state within the infected cells and send alarm signals to neighboring cells.

[0150] Innate immune cells are leukocytes 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 varying efficiencies (often referred to as transduction efficiencies). Some serotypes (such as AAV1) are effective at transducing certain immune cells, such as monocytes, while others (such as AAV6) are effective at transducing cells, such as dendritic cells (Grimm, D et al., J. Virol. [Journal of Virology], 2008, 82(12):5887-5911). Once cells enter, the AAVs can elicit an immune response. The extent of this immune response depends on the AAV serotype and cell type. Once AAVs transduce host immune cells, they can bind to immune receptors, such as TLRs (e.g., TLR9). Several studies using mouse models have revealed that TLR9 is a key DNA sensor contributing to AAV immunogenicity (Zhu, J et al., JClin Invest. 2009;119(8):2388-2398; Ashley SN et al., Cell. Immunol. 2019, 346:103997). Once these TLRs are activated by the virus, they secrete cytokines that establish an antiviral state within infected cells and alert 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, 2020 180(6):1044-1066).

[0151] As used in this article, the upregulation or downregulation of certain subsets of cytokines is termed a “cytokine signature.” These cytokine signatures, which contain three or more cytokines, can be used as predictive biomarkers for disease and therapeutic success. Examples of cytokine signatures can be 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 originating from an entity that is genotype different from the entity being compared to, introduced into, or incorporated into. For example, nucleic acids introduced into different cell types through genetic engineering are heterologous nucleic acids (and can encode heterologous polypeptides when expressed). Similarly, cellular sequences (e.g., genes or portions thereof) incorporated into viral vectors are nucleotide sequences heterologous to the vector.

[0153] The term "transgenic" refers to a nucleic acid introduced into a cell that can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In some embodiments, it confers desired properties to the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic outcome. On the other hand, it can be transcribed into molecules that mediate RNA interference, such as siRNA.

[0154] The terms “genomic particles (gp),” “genomic equivalent,” or “genomic copy” used in reference to viral titers refer to the number of virions containing the recombinant AAV DNA genome, and are unrelated to infectivity or functionality. The number of genomic particles in a particular vector formulation can be measured using procedures such as those described in the examples herein or in, for example, the following literature: Clark et al. (1999) Hum. Gene Ther. [Human Gene Therapy], 10:1031-1039; Veldwijk et al. (2002) Mol. Ther. [Molecular Therapy], 6:272-278.

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

[0156] The term “transduction unit (tu)” as used in reference to viral titers refers to the number of infectious recombinant AAV vector particles that cause the production of functional transgenic products, as measured in functional assays such as those described in the examples herein or in, for example, the following literature: Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0157] "Inverted terminal repeat" or "ITR" sequence is a well-known term in the field, referring to a relatively short sequence with opposite orientation found at the end of a viral genome.

[0158] The term "AAV inverted terminal repeat (ITR)" is well-known in the art and refers to a sequence of approximately 145 nucleotides located at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. These outermost 125 nucleotides also contain several shorter, self-complementary regions (named A, A', B, B', C, C', and D regions), allowing intrastrand base pairing to occur within this portion of the ITR.

[0159] "Terminal parsing sequence" or "trs" is the sequence in the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. The mutant terminal parsing sequence is resistant to AAV rep protein cleavage. "AAV helper functions" refers to the functions that allow AAV to be replicated and packaged by the host cell. AAV helper functions can be provided in any of a variety of forms, including but not limited to helper viruses or helper viral genes that assist AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

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

[0161] The term "helper virus" in AAV refers to a virus that allows AAV (a defective parvovirus) to be replicated and packaged by host cells. Many such helper viruses have been identified, including adenoviruses, herpesviruses, poxviruses (such as vaccinia), and baculoviruses. Adenoviruses encompass many different subgroups, but subgroup C5 adenovirus (Ad5) is the most commonly used. Many adenoviruses of human, non-human mammalian, and avian origin are known and available from collections such as the ATCC. Herpesviruses also available from collections such as the ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Baculoviruses available from collections include *Autographa californica* nuclear polyhedrosis virus.

[0162] The “sequence identity percentage (%)” for 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 to those in a reference polypeptide or nucleic acid sequence after sequence alignment and the introduction of vacancies (if necessary) to obtain the maximum sequence identity percentage, and without considering any conserved substitutions as part of the sequence identity. Alignments for determining the amino acid or nucleic acid sequence identity percentage can be performed in various ways within the scope of the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (edited by Ausubel et al., 1987), Supplement 30, Chapter 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 alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For the purposes of this paper, the percentage of amino acid sequence identity between a given amino acid sequence A and, and, for a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity with, and, for a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues that are scored as identical matches in the alignment of A and B by a sequence alignment program, and where Y is the total number of amino acid residues in B. It should be understood that when the lengths of amino acid sequences A and B are not equal, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. For the purposes of this paper, the % nucleotide sequence identity of a given nucleic acid sequence C with, and or with respect to a given nucleic acid sequence D (which can be alternatively expressed as a given nucleic acid sequence C having or containing a certain % nucleotide sequence identity with, and or with respect to a given nucleic acid sequence D) is calculated as follows: 100 multiplied by the fraction W / Z, where W is the number of nucleotides that are identified as identical matches in the alignment of C and D by the sequence alignment program, and Z is the total number of nucleotides in D. It should be understood that when the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleotide sequence identity of C with D will not be equal to the % nucleotide sequence identity of D with C.

[0163] The "effective amount" of a drug refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dosage and time period. For example, the effective amount of a gene therapy agent refers to the amount that effectively achieves the desired gene therapy outcome within the necessary dosage and time period. In another instance, the effective amount of an IL-2 conjugate could refer to the amount that effectively achieves the desired outcome of improved gene therapy within the necessary dosage and time period.

[0164] The "therapeuticly effective amount" of the substance / molecule of the present invention (e.g., gene therapy agents and / or IL-2 conjugates) can vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to elicit the desired response in the individual. Therapeuticly effective amount is also the amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the substance / molecule.

[0165] The term "appropriate control" when referring to cytokine signatures refers to the expression of cytokines in the cytokine signature of adaptive immune cells that have not been incubated with the gene therapy agent, or the expression of cytokines in the cytokine signature of adaptive immune cells before incubation with the gene therapy agent.

[0166] When gene therapy agents and adaptive immune response modulators (e.g., IL-2 conjugates) are involved, "in combination with" administration includes administering gene therapy agents and adaptive immune response modulators (e.g., IL-2 conjugates) simultaneously (in parallel), sequentially, or in any order.

[0167] The term "simultaneously" is used herein to refer to the administration of a gene therapy agent and an adaptive immune response modulator (e.g., an IL-2 conjugate), wherein at least a portion of the administration overlaps in time. Therefore, concurrent administration includes a dosing regimen in which the administration of a gene therapy agent or an adaptive immune response modulator (e.g., an IL-2 conjugate) continues after the administration of another agent / modulator has been discontinued.

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

[0169] As used herein, the term "or a combination thereof" means any and all permutations and combinations of the terms listed prior to the term. For example, "A, B, C or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if the order is important in the particular context. Continuing this example, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0170] "Or" is used in an open sense, that is, equivalent to "and / or", unless the context requires otherwise. II. Exemplary Methods A. Overview

[0171] This disclosure provides methods for delivering gene therapy agents to the cells of a subject, treating individuals in need with gene therapy agents, increasing the expression of gene therapy agents, reducing the immune response to gene therapy agents, preventing immune-related adverse events in subjects, selecting subjects to be treated with gene therapy agents and IL-2 conjugates, and using IL-2 conjugates. IL-2 conjugates may be able to selectively upregulate different lymphocyte populations (e.g., CD4+ T regulatory cells), for example, through cytokine / cytokine receptor signaling. In some embodiments, at least one amino acid residue of the amino acid sequence of the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate. In some embodiments, the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

[0172] In some embodiments, this document also describes a method for selectively upregulating CD4+ T regulatory cells via IL-2 / IL-2R signaling. In some embodiments, IL-2 conjugates inhibit CD8+ T cell proliferation in a subject. In some embodiments, IL-2 conjugates inhibit effector memory CD8+ T cell proliferation in a subject. In some embodiments, IL-2 is an IL-2 conjugate that interacts with the IL-2Rαβγ complex and has attenuated IL-2Rβγ interaction relative to wild-type IL-2. In some embodiments, this document further describes a method for delivering a gene therapy agent to the cells of a subject using the IL-2 conjugates described herein. In other embodiments, this document describes pharmaceutical compositions and kits comprising one or more of the IL-2 conjugates and / or gene therapy agents described herein, which are used, for example, in the disclosed methods.

[0173] As illustrated in the examples, gene therapy resulted in a significant increase in the proportion of proliferative and effector CD8+ T cells in the spleen, which was mitigated by treatment with the IL-2 conjugate according to this disclosure. There was no significant difference in the reduction of CD8+ T cell responses when the IL-2 conjugate was administered as pretreatment or as cotreatment. Furthermore, upon in vitro restimulation of spleen cells with both gene therapy peptide pools and transgenic peptide pools, the proportion of IFNγ-secreting CD8+ T cells significantly increased in response to each peptide group, which was mitigated by treatment with the IL-2 conjugate. There was no significant difference in the reduction of AAV and / or transgenic-specific CD8+ T cell responses when the IL-2 conjugate was administered as pretreatment or as cotreatment. Without wishing to be bound by any particular theory, this suggests that administration of AAV gene therapy within the timeframe of IL-2 conjugate-mediated Treg amplification maintains the therapeutic benefit of IL-2 conjugate suppression of adverse immune responses to gene therapy agents and / or transgenes. B. Cytokine conjugates

[0174] In some embodiments, the IL-2 conjugate used in the disclosed method comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, at least one amino acid residue of the amino acid sequence of the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugation moiety. In some embodiments, the non-natural amino acid linked to the conjugation moiety is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ. In some embodiments, the non-natural amino acid linked to the conjugation moiety is located in the amino acid sequence, as shown in reference SEQ ID NO: 1. The positions of the sequence 1 are: 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, P 81, 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.

[0175] Table 1 provides exemplary IL-2 sequences. The sequence SEQ ID NO: 1 is adelleukin, wherein the first amino acid of wild-type IL-2 has been removed. Amino acids in SEQ ID NO: 1 or sequences having percentage identity with it as described elsewhere herein may be substituted with non-natural amino acids. Exemplary sequences containing non-natural amino acids indicated by X are listed as SEQ ID NO: 2-14. Table 1

[0176] In some embodiments, the position of the at least one non-natural 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 amino acid residues are numbered according to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108, wherein the amino acid residues are numbered according to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is L18, wherein the amino acid residues are numbered according to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is D19, wherein the amino acid residues are numbered according to the sequence of SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is H15, wherein the amino acid residues are numbered according to the sequence of SEQ ID NO: 1.

[0177] In some embodiments, IL-2 conjugates modified at amino acid positions are provided. In some embodiments, the modification is directed at a non-natural amino acid. In some embodiments, an isolated and purified IL-2 conjugate comprising at least one non-natural amino acid is described herein. In some embodiments, the IL-2 polypeptide is an isolated and purified mammalian IL-2, such as rodent IL-2 protein or human IL-2 protein. In some embodiments, the IL-2 polypeptide is human IL-2 protein. In some embodiments, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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 with 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 comprises the sequence of SEQ ID NO: 2. In some embodiments, the IL-2 polypeptide contains about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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 comprises the sequence of SEQ ID NO: 3. In some embodiments, the IL-2 polypeptide contains about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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 comprises the sequence of SEQ ID NO: 4. In some embodiments, the IL-2 polypeptide contains about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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 comprises the sequence of SEQ ID NO: 6. In some embodiments, the IL-2 polypeptide contains approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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 comprises 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 with 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 peptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 9. In some embodiments, the IL-2 peptide comprises the sequence of SEQ ID NO: 9. In some embodiments, the IL-2 peptide consists of the sequence of SEQ ID NO: 9. In some embodiments, the IL-2 peptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 10. In some embodiments, the IL-2 peptide comprises the sequence of SEQ ID NO: 10. In some embodiments, the IL-2 peptide consists of the sequence of SEQ ID NO: 10. In some embodiments, the IL-2 peptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises the sequence of SEQ ID NO: 11. In some embodiments, the IL-2 peptide consists of the sequence of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises the sequence of SEQ ID NO: 12. In some embodiments, the IL-2 peptide consists of the sequence of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises the sequence of SEQ ID NO: 13. In some embodiments, the IL-2 peptide consists of the sequence of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 14. In some embodiments, the IL-2 peptide comprises the sequence of SEQ ID NO: 14. In some embodiments, the IL-2 peptide consists of the sequence of SEQ ID NO: 14.

[0179] In some embodiments, the IL-2 peptide is a truncated variant, such as SEQ ID NO: 15 or 16, relative to wild-type IL-2. In some embodiments, truncation is an N-terminal deletion. In some embodiments, truncation is a C-terminal deletion. In some embodiments, truncation comprises both N-terminal and C-terminal deletions. For example, truncation may be the deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the N-terminus or C-terminus or both ends. In some embodiments, the IL-2 peptide 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 peptide 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 peptide comprises an N-terminal deletion of at least or about 2 residues. In some embodiments, the IL-2 peptide comprises an N-terminal deletion of at least or about 3 residues. In some embodiments, the IL-2 peptide comprises an N-terminal deletion of at least or about 4 residues. In some embodiments, the IL-2 peptide comprises an N-terminal deletion of at least or about 5 residues. In some embodiments, the IL-2 peptide comprises an N-terminal deletion of at least or about 6 residues. In some embodiments, the IL-2 peptide comprises an N-terminal deletion of at least or about 7 residues. In some embodiments, the IL-2 peptide comprises an N-terminal deletion of at least or about 8 residues. In some embodiments, the IL-2 peptide comprises an N-terminal deletion of at least or about 9 residues. In some embodiments, the IL-2 peptide 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 that references, for example, SEQ ID NO: 1 or has at least 80% sequence identity with 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 N-terminal addition of either alanine or methionine to the first amino acid of a sequence having at least 80% sequence identity with 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 regions 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 referenced 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 referenced to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 region 20-133, wherein the residue positions are referenced 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 referenced 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 referenced to the positions in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 regions 20-125, wherein the residue positions are referenced to those in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 regions 1-130, wherein the residue positions are referenced to those in SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises IL-2 regions 1-125, wherein the residue positions are referenced to those in SEQ ID NO: 1.

[0182] In some embodiments, this document describes an IL-2 conjugate comprising an isolated and purified IL-2 conjugate and a conjugated moiety. In some embodiments, the IL-2 conjugate has a reduced affinity for the IL-2 receptor βγ (IL-2Rβγ) subunit relative to the wild-type IL-2 peptide. In some embodiments, the conjugated moiety binds to an amino acid residue that interacts with IL-2Rβγ (e.g., at the IL-2 / IL-2Rβγ interface). In some embodiments, the conjugated moiety binds to an amino acid residue near the IL-2 / IL-2Rβγ interface (e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å from the IL-2 / IL-2Rβγ interface). As used herein, the residues involved at 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 conjugation portion is bound to amino acid positions selected from the sequence of reference SEQ ID NO: 1: 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, P8 1. Amino acid residues of 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 some embodiments, the conjugation portion binds to amino acid residues selected from amino acid positions K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108 of the sequence referenced SEQ ID NO: 1. In some embodiments, the conjugation portion binds to an amino acid residue selected from amino acid position L18 of the sequence of reference SEQ ID NO: 1. In some embodiments, the conjugation portion binds to an amino acid residue selected from amino acid position H15 of the sequence of reference SEQ ID NO: 1. In some embodiments, the conjugation portion binds to an amino acid residue selected from amino acid position D19 of the sequence of reference SEQ ID NO: 1.

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

[0185] In some embodiments, receptor signaling efficacy is measured by an ED50 value. In some embodiments, the IL-2 conjugate provides a first ED50 value for activating the IL-2βγ signaling complex and a second ED50 value for activating the IL-2αβγ signaling complex, wherein the difference between the first ED50 and the second ED50 values ​​is less than 10-fold. In some embodiments, the IL-2 conjugate provides a first ED50 value for activating the IL-2βγ signaling complex and a second ED50 value for activating the IL-2αβγ signaling complex, wherein the difference between the first ED50 and the second ED50 values ​​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 conjugation portion is directly or indirectly linked to the N-terminus or C-terminus of the IL-2 polypeptide, either via a linker peptide. In some embodiments, the conjugation portion (e.g., a polymer, protein, or peptide) is fused to the IL-2 gene at the N-terminus or C-terminus of IL-2, either directly or indirectly via a linker peptide. In some embodiments, the conjugation portion is linked to an N-terminal or C-terminal amino acid residue. In some embodiments, the conjugation portion is linked to a reactive group that binds to an N-terminal or C-terminal amino acid residue.

[0187] In some embodiments, IL-2 conjugates with reduced binding affinity for IL-2Rβγ can amplify the CD4+ T regulatory cell population. In some embodiments, IL-2 conjugates with reduced binding affinity for IL-2Rβγ can inhibit CD8+ T cell populations, including CD8+ T effector memory cells, vector-specific IFNγ-secreting CD8+ T cells, and transgene-specific IFNγ-secreting CD8+ T cells. In some embodiments, IL-2 conjugates with reduced binding affinity for IL-2Rβγ can inhibit the production of antibodies against transgenes and the production of IgG1 antibodies against transgenes. In some embodiments, the conjugate partially impairs or blocks the binding of IL-2 to IL-2Rβγ.

[0188] In some embodiments, the IL-2 conjugate retains a significant potency in activating the CD4+ T regulatory cell population via the IL-2Rαβγ complex, relative to the wild-type IL-2 peptide. In some embodiments, the activation of the IL-2 conjugate is equivalent to the activation of the wild-type IL-2 peptide. In other embodiments, the activation of the IL-2 conjugate is superior to the activation of the wild-type IL-2 peptide. In some embodiments, the receptor signaling efficacy of the IL-2 conjugate to the IL-2Rαβγ complex is superior to that of the wild-type IL-2 peptide. In some embodiments, the receptor signaling efficacy of the IL-2 conjugate is at least 50% higher than the corresponding efficacy of the wild-type IL-2 peptide. In some embodiments, the receptor signaling potency of the IL-2 conjugate is about 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 that of the wild-type IL-2 peptide. In such cases, the dose or concentration of the IL-2 conjugate used to achieve a level of CD4+ T regulatory cell population activation similar to that of the wild-type IL-2 peptide is lower than the dose or concentration used for the wild-type IL-2 peptide.

[0189] In some embodiments, the IL-2 conjugate retains the significant efficacy of the wild-type IL-2 peptide in activating the CD4+ T regulatory cell population via the IL-2Rαβγ complex. In some embodiments, the receptor signaling efficacy of the IL-2 conjugate to the IL-2Rβγ complex is lower than that of the wild-type IL-2 peptide to the IL-2Rβγ complex. In some embodiments, the receptor signaling efficacy of the IL-2 conjugate is about 25%, 2, 3, 4, 5, 10, 20, or 50 times lower than that of the corresponding wild-type IL-2 peptide.

[0190] In some embodiments, the IL-2 conjugate exhibits both first receptor signaling potency and second receptor signaling potency for IL-2Rαβγ. In some embodiments, the first receptor signaling potency for IL-2Rαβγ is an improved potency relative to the wild-type IL-2 peptide. In some embodiments, the second receptor signaling potency for IL-2Rβγ is an impaired potency relative to the wild-type IL-2 peptide. In some embodiments, the IL-2 conjugate exhibits both first receptor signaling potency and second receptor signaling potency for IL-2Rαβγ, wherein the first receptor signaling potency is at least 50%, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, 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 efficacy is at least 2 times higher than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 5 times higher than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 10 times higher than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 20 times higher than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 50 times higher than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 100 times higher than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 500 times higher than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 1000 times higher than the second receptor signaling efficacy. In some embodiments, the modified IL-2 peptide exhibits higher first receptor signaling efficacy than the wild-type IL-2 peptide for IL-2Rαβγ, and lower second receptor signaling efficacy than the wild-type IL-2 peptide for IL-2Rβγ. In some embodiments, the signaling efficiencies of both receptors are lower than their corresponding efficiencies in the wild-type IL-2 peptide. In other embodiments, the signaling efficiencies of both receptors are higher than their corresponding efficiencies in the wild-type IL-2 peptide.

[0191] In some embodiments, the IL-2 conjugate reduces toxic adverse events in subjects receiving the IL-2 conjugate. Exemplary toxic adverse events include eosinophilia, capillary leakage, and vascular leakage syndrome (VLS). In some embodiments, the IL-2 conjugate reduces the incidence of toxic adverse events in subjects receiving wild-type IL-2 or aldehyde interleukin by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to subjects receiving wild-type IL-2 or aldehyde interleukin. In some embodiments, the IL-2 conjugate reduces the severity of toxic adverse events in subjects receiving wild-type IL-2 or aldehyde interleukin by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to subjects receiving wild-type IL-2 or aldehyde interleukin.

[0192] In some embodiments, the adverse toxicity event is eosinophilia. In some embodiments, the IL-2 conjugate reduces the incidence of eosinophilia in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject receiving wild-type IL-2 or aldehyde. In some embodiments, the IL-2 conjugate reduces the severity of eosinophilia in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject receiving wild-type IL-2 or aldehyde.

[0193] In some embodiments, the adverse toxicity event is capillary leakage. In some embodiments, the IL-2 conjugate reduced the incidence of capillary leakage in subjects by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldehyde. In some embodiments, the IL-2 conjugate reduced the severity of capillary leakage in subjects by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldehyde.

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

[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 longer.

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

[0197] In some embodiments, the IL-2 conjugate has a plasma half-life of about 1 hour to about 18 hours, about 1 hour to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 4 hours to about 18 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 6 hours to about 18 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 18 hours, about 8 hours to about 12 hours, or about 8 hours to about 10 hours.

[0198] In some embodiments, the IL-2 conjugate has a plasma half-life that enables the proliferation and / or amplification of CD4+ T regulatory cells without exerting harmful effects such as apoptosis.

[0199] In some embodiments, the IL-2 conjugate has a prolonged plasma half-life relative to wild-type IL-2, for example, a prolonged 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 longer. In some embodiments, the IL-2 conjugate has a prolonged plasma half-life relative to wild-type IL-2 or aldehyde interleukin, for example, a prolonged 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 longer.

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

[0201] In some embodiments, the IL-2 conjugate comprises a mutation at residue position L18 of the sequence referring to SEQ ID NO: 1, and comprises a conjugate portion comprising 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 PEG at least partially determines the in vivo plasma half-life of the IL-2 conjugate. In some embodiments, PEG is consistent with a longer in vivo plasma half-life of the IL-2 conjugate compared to a smaller in vivo plasma half-life. In some embodiments, PEG is consistent with a shorter in vivo plasma half-life of the IL-2 conjugate compared to a larger in vivo plasma half-life. In some embodiments, the molecular weight of PEG does not affect, nor has a minimal effect on, the receptor signaling efficacy of the IL-2 conjugate for the IL-2α or IL-2αβγ signaling complex. In some embodiments, the molecular weight of PEG does not affect, or has minimal effect on, the desired reduced binding of the IL-2 conjugate to IL-2Rαβγ or the maintenance of the binding to the IL-2Rαβγ signaling complex, wherein the reduced binding to IL-2Rβγ is compared to the binding between the wild-type IL-2 peptide and IL-2Rβγ. In some embodiments, the molecular weight of PEG does not affect the formation of the modified IL-2 peptide / IL-2Rαβγ complex, wherein the reduced binding to IL-2Rβγ is compared to the binding between the wild-type IL-2 peptide and IL-2Rβγ.

[0202] In some embodiments, the IL-2 conjugate comprises a mutation at residue position 15, H15, of the sequence referenced in SEQ ID NO: 1, comprising a conjugation portion comprising 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 conjugation portion is attached to an amino acid position 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. Amino acid residues of 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, wherein the amino acid residues are numbered with reference to the sequence of SEQ ID NO: 1. In some embodiments, the amino acid positions are 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 additional mutations. In such cases, the amino acid is conjugated with an additional conjugation moiety to increase serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a non-natural amino acid before binding with the additional conjugation moiety.

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

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

[0207] In some embodiments, the IL-2 conjugate has reduced recruitment of the IL-2Rβγ subunit to the IL-2 / IL-2Rβγ complex. In some embodiments, the reduced recruitment is compared to the recruitment of the IL-2Rβγ subunit in an equivalent IL-2 polypeptide that does not contain non-natural amino acids (e.g., a wild-type IL-2 polypeptide). In some embodiments, the reduction in IL-2Rβγ subunit recruitment relative to an equivalent IL-2 polypeptide without non-natural amino acid modifications (e.g., a wild-type IL-2 polypeptide) is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99%.

[0208] In some embodiments, the reduction in IL-2Rβγ subunit recruitment relative to an equivalent IL-2 peptide without non-natural amino acid modifications (e.g., wild-type IL-2 peptide) 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. In some embodiments, the IL-2 conjugate further exhibits an increase in IL-2Rαβγ recruitment.

[0209] In some embodiments, the IL-2 conjugate exhibits an increased recruitment of IL-2Rαβγ to the IL-2 peptide. In some embodiments, the reduced recruitment is compared to the recruitment of IL-2Rαβγ by an equivalent IL-2 peptide that does not contain non-natural amino acids (e.g., a wild-type IL-2 peptide). In some embodiments, the increase in IL-2Rαβγ recruitment relative to an equivalent IL-2 peptide without non-natural amino acid modifications is an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99%. In some embodiments, the IL-2 conjugate further exhibits a reduced recruitment of IL-2Rβγ.

[0210] In some embodiments, the recruitment of IL-2Rαβγ is increased by 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 peptide without non-natural amino acid modifications (e.g., a wild-type IL-2 peptide). In some embodiments, the IL-2 conjugate further has a reduced recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex.

[0211] In some embodiments, the IL-2 conjugates described herein exhibit reduced receptor signaling efficacy for IL-2Rβγ. In some embodiments, the reduction in receptor signaling efficacy for IL-2Rβγ relative to the wild-type IL-2 peptide is approximately 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.

[0212] In some embodiments, receptor signaling efficacy is measured by EC50 values. In some embodiments, a decrease in receptor signaling efficacy is represented by an increase in EC50. In some embodiments, the increase in EC50 relative to the wild-type IL-2 peptide is approximately 50%, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 100, 200, 300, 400, 500, 1000, or more.

[0213] In some embodiments, receptor signaling efficacy is measured by the ED50 value. In some embodiments, a decrease in receptor signaling efficacy is an increase in ED50. In some embodiments, the increase in ED50 relative to the wild-type IL-2 peptide is about 50%, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, 50, 100, 200, 300, 400, 500, 1000, or more.

[0214] In some embodiments, the IL-2 conjugates described herein have an expanded therapeutic window compared to the therapeutic window of the wild-type IL-2 peptide. In some embodiments, the expanded therapeutic window is due to reduced binding between the IL-2 conjugate and the interleukin 2 receptor βγ (IL-2Rβγ), decreased receptor signaling efficacy of IL-2Rβγ, reduced recruitment of IL-2Rβγ subunits to the IL-2 / IL-2Rαβγ complex, or increased recruitment of IL-2Rαβγ to the IL-2 peptide. In some embodiments, the IL-2 conjugates do not impair the activation of the interleukin 2 αβγ receptor (IL-2Rαβγ).

[0215] In some embodiments, the IL-2 conjugate exhibits a first receptor signaling efficacy against the IL-2βγ signaling complex and a second receptor signaling efficacy against the IL-2αβγ signaling complex, wherein the difference between the first receptor signaling efficacy and the second receptor signaling efficacy is at least 50%. In some embodiments, the difference is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, or more. In some embodiments, the first receptor signaling efficacy is less than the second receptor signaling efficacy. In some embodiments, the first receptor signaling efficacy is at least 25%, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 500, 1000, or less than the second receptor signaling efficacy. In some embodiments, the receptor signaling efficacy of the IL-2 conjugate to the IL-2βγ signaling complex is lower than that to the second receptor signaling efficacy to the IL-2αβγ signaling complex. In some embodiments, the first receptor signaling efficacy of the IL-2 conjugate is at least 25% lower than that of the wild-type IL-2 peptide. In some embodiments, the first receptor signaling efficacy of the IL-2 conjugate is at least 25%, 2, 3, 4, 5, 10, 20, 50, 100, 200, or 500 times lower than that of the wild-type IL-2 peptide. In some embodiments, both the first and second receptor signaling efficiencies are lower than the corresponding efficiencies of the wild-type IL-2 peptide, but the first receptor signaling efficacy is lower than the second receptor signaling efficacy. In some embodiments, the difference between the first and second receptor signaling efficiencies increases the therapeutic window of the IL-2 conjugate.

[0216] In some embodiments, the conjugate partially impairs or blocks the receptor signaling efficacy of IL-2 and IL-2Rβγ, or reduces the recruitment of IL-2Rβ and / or IL-2Rγ subunits to the IL-2 / IL-2Rβγ complex. In some embodiments, the IL-2 conjugate further has a reduced recruitment of IL-2Rγ subunits to the IL-2 / IL-2Rβ complex.

[0217] In some embodiments, IL-2 conjugates with reduced receptor signaling efficacy against IL-2Rβγ can amplify CD4+ T regulatory (Treg) cells.

[0218] In some embodiments, the proliferation of CD4+ Treg cells with the modified IL-2 / IL-2Rαβγ complex is equal to or greater than the proliferation of CD4+ Treg cells with the wild-type IL-2 peptide.

[0219] In some embodiments, the IL-2 / IL-2Rαβγ complex induces CD4+ Treg cell proliferation to a population sufficient to regulate disease progression in animal models. 1. Natural and non-natural amino acids

[0220] In some embodiments, non-natural amino acids are not conjugated to the conjugation site. In some embodiments, the cytokines described herein comprise non-natural amino acids, wherein the cytokines are conjugated to proteins, and the attachment site is not a non-natural amino acid.

[0221] In some embodiments, the amino acid residues described herein (e.g., within cytokines such as IL-2) are mutated to non-natural amino acids before binding to the conjugation moiety. In some embodiments, the mutation to a non-natural amino acid prevents or minimizes the immune system's self-antigen response. As used herein, the terms "non-natural amino acid" or "non-standard amino acid" refer to amino acids other than the 20 amino acids naturally present in proteins. Non-limiting examples of non-natural amino acids include: p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propynyloxyphenylalanine, p-propynyl-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, and m-azido-L-phenylalanine. Non-natural analogs of the amino acids p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronylphenylalanine, O-propytyrosine, L-phosphoserine, phosphonylserine, phosphonyltyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK), and tyrosine amino acids; non-natural analogs of glutamine amino acids; non-natural analogs of phenylalanine amino acids; non-natural analogs of serine amino acids; threonine. Non-natural analogues of amino acids; non-natural analogues of lysine amino acids; alkyl, aryl, acyl, azide, cyano, halogenated, hydrazine, acylhydrazine, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, selenized, esterified, thioacid, borate, boronate, phosphoric acid, phosphonyl, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, ketone, or amino-substituted amino acids or combinations thereof; amino acids with photoactivated crosslinking agents; spin-labeled amino acids; fluorescent amino acids; metal-bound amino acids; metal-containing amino acids; radioactive amino acids; light-shielded amino acids. And / or photoisomerizable amino acids; amino acids containing biotin or biotin analogs; ketone-containing amino acids; amino acids containing polyethylene glycol or polyether; heavily atom-substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with extended side chains; amino acids containing toxic groups; sugar-substituted amino acids; carbon-linked sugar-containing amino acids; redox-active amino acids; acids containing α-hydroxy groups; aminothio acids; α,α-disubstituted amino acids; β-amino acids; cyclic amino acids other than proline or histidine; and aromatic amino acids other than phenylalanine, tyrosine, or tryptophan. In some embodiments, the non-natural amino acid is azido-substituted lysine.

[0222] In some embodiments, the non-natural amino acid contains a selectively reactive group, or a reactive group for site-selective labeling of the target peptide. In some embodiments, the chemical reaction is a bioorthogonal reaction (e.g., a biocompatibility and selectivity reaction). In some embodiments, the chemical reaction is a Cu(I)-catalyzed or “copper-free” acetylene-azide triazole formation reaction, Staudinger ligation, anti-electron demand Diels-Alder (IEDDA) reaction, “photoclick” chemical reaction, or a metal-mediated process such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.

[0223] In some embodiments, the non-natural amino acids contain photoreactive groups that crosslink when irradiated with, for example, UV light.

[0224] In some embodiments, non-natural amino acids include light-masking amino acids.

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

[0226] In some embodiments, non-natural amino acids include 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-propynyloxyphenylalanine, o-propynyl-phenylalanine, m-propynyl-phenylalanine, p-propynyl-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 non-natural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, or 3-iodotyrosine.

[0228] In some embodiments, the non-natural amino acid is phenylselenocysteine.

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

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

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

[0232] In some embodiments, non-natural amino acids do not contain aromatic side chains.

[0233] In some embodiments, the non-natural amino acid contains an azide group.

[0234] In some embodiments, the non-natural amino acid comprises a Michael-acceptor group. In some embodiments, the Michael-acceptor group comprises an unsaturated portion capable of forming a covalent bond via a 1,2-addition reaction. In some embodiments, the Michael-acceptor group comprises an electron-deficient alkene or alkyne. In some embodiments, the Michael-acceptor group includes, but is not limited to, α,β-unsaturated ketones, aldehydes, sulfoxides, sulfones, nitriles, imines, or aromatic compounds.

[0235] In some embodiments, the non-natural amino acid is dehydroalanine.

[0236] In some embodiments, the non-natural amino acid contains an aldehyde or ketone group.

[0237] In some embodiments, the non-natural amino acid is a lysine derivative containing an aldehyde or ketone group.

[0238] In some embodiments, the non-natural amino acid is a lysine derivative containing one or more O, N, Se, or S atoms at the β, γ, or δ position. In some embodiments, the non-natural amino acid is a lysine derivative containing O, N, Se, or S atoms at the γ position.

[0239] In some embodiments, the non-natural amino acid is a lysine derivative, wherein the εN atom is replaced by an oxygen atom.

[0240] In some embodiments, the non-natural amino acid is a lysine derivative, which is a post-translational modified lysine that is not naturally occurring.

[0241] In some embodiments, the non-natural amino acid is an amino acid containing a side chain, wherein the sixth atom from the α position comprises a carbonyl group. In some embodiments, the non-natural amino acid is an amino acid containing a side chain, wherein the sixth atom from the α position comprises a carbonyl group, and the fifth atom from the α position is nitrogen. In some embodiments, the non-natural amino acid is an amino acid containing a side chain, wherein the seventh atom from the α position is an oxygen atom.

[0242] In some embodiments, the non-natural amino acid is a serine derivative containing selenium. In some embodiments, the non-natural amino acid is selenoserine (2-amino-3-hydroselenopropionic acid). In some embodiments, the non-natural amino acid is 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selenopropionic acid. In some embodiments, the non-natural amino acid is 2-amino-3-(phenylselenopropionic acid). In some embodiments, the non-natural amino acid contains selenium, wherein the oxidation of selenium results in the formation of a non-natural amino acid containing an olefin.

[0243] In some embodiments, the non-natural amino acid contains a cyclooctyne group.

[0244] In some embodiments, the non-natural amino acid comprises a trans-cyclooctene group.

[0245] In some embodiments, the non-natural amino acid comprises norbornene.

[0246] In some embodiments, the non-natural amino acid contains a cyclopropene group.

[0247] In some embodiments, the non-natural amino acid contains a diacylpyridinium group.

[0248] In some embodiments, the non-natural amino acid contains a tetrazine group.

[0249] In some embodiments, the non-natural amino acid is a lysine derivative wherein the side chain nitrogen is carbamylated. In some embodiments, the non-natural amino acid is a lysine derivative wherein the side chain nitrogen is acylated. In some embodiments, the non-natural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is N6-Boc-N6-methyllysine. In some embodiments, the non-natural amino acid is N6-acetyllysine. In some embodiments, the non-natural amino acid is pyrrolyllysine. In some embodiments, the non-natural amino acid is N6-trifluoroacetyllysine. In some embodiments, the non-natural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is 2-amino-6-{[(p-iodobenzyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is N6-prolyl-lysine. In some embodiments, the non-natural amino acid is 2-amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is N6-(cyclopentanecarbonyl)lysine. In some embodiments, the non-natural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is 2-amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is N6-(2-azidoethoxy)-carbonyl-lysine. In some embodiments, the non-natural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is 2-amino-6-{[(2-cyclooctyynyloxy)carbonyl]amino}hexanoic acid. In some embodiments, the non-natural amino acid is N6-(2-aminobut-3-ynyl)lysine. In some embodiments, the non-natural amino acid is 2-amino-6-((2-aminobut-3-ynyl)oxy)hexanoic acid. In some embodiments, the non-natural amino acid is N6-(allyloxycarbonyl)lysine. In some embodiments, the non-natural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In some embodiments, the non-natural amino acid is N6-(pentenyl-5-oxycarbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((butenyl-3-ynyloxy)carbonyl)-lysine. In some embodiments, the non-natural amino acid is N6-((pentenyl-4-ynyloxy)carbonyl)-lysine.In some embodiments, the non-natural amino acid is N6-(thiazolidin-4-carbonyl)lysine. In some embodiments, the non-natural amino acid is 2-amino-8-oxononanoic acid. In some embodiments, the non-natural amino acid is 2-amino-8-oxooctanoic acid. In some embodiments, the non-natural amino acid is N6-(2-oxoacetyl)lysine.

[0250] In some embodiments, the non-natural amino acid is N6-propionyllysine. In some embodiments, the non-natural amino acid is N6-butyryllysine. In some embodiments, the non-natural amino acid is N6-(but-2-enoyl)lysine. In some embodiments, the non-natural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is cysteyllysine. In some embodiments, the non-natural amino acid is N6-((1-(6-nitrobenzo[d][1,3]dioxane-5-yl)ethoxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((2-(3-methyl-3H-bisacrididin-3-yl)ethoxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((3-(3-methyl-3H-bisacrididin-3-yl)propoxy)carbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((m-nitrobenzyloxy)N6-methylcarbonyl)lysine. In some embodiments, the non-natural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)-lysine. In some embodiments, the non-natural amino acid is N6-((cycloheptane-3-en-1-yloxy)carbonyl)-L-lysine.

[0251] In some embodiments, the non-natural amino acid is 2-amino-3-(((((benzyloxy)carbonyl)amino)methyl)seleno)propionic acid.

[0252] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via repurposed amber, opal, or ochre stop codons.

[0253] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via 4-base codons.

[0254] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) using recycled rare sense codons.

[0255] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via synthetic codons containing non-natural nucleic acids.

[0256] In some embodiments, non-natural amino acids are incorporated into cytokines via orthogonal, modified synthase / tRNA pairs. Such orthogonal pairs contain non-natural synthases capable of providing non-natural amino acids to non-natural tRNA while minimizing: a) the incorporation of other endogenous amino acids into non-natural tRNA, and b) the incorporation of non-natural amino acids into other endogenous tRNA. Such orthogonal pairs contain tRNAs that can be provided by non-natural synthases while avoiding the incorporation of a) other endogenous amino acids by endogenous synthases. In some embodiments, such pairs have been identified from various organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, orthogonal synthase / tRNA pairs contain components from a single organism. In some embodiments, orthogonal synthase / tRNA pairs contain components from two different organisms. In some embodiments, orthogonal synthase / tRNA pairs contain components that promote the translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthase is a modified alanine synthase. In some embodiments, the orthogonal synthase is a modified arginine synthase. In some embodiments, the orthogonal synthase is a modified asparagine synthase. In some embodiments, the orthogonal synthase is a modified aspartate synthase. In some embodiments, the orthogonal synthase is a modified cysteine ​​synthase. In some embodiments, the orthogonal synthase is a modified glutamine synthase. In some embodiments, the orthogonal synthase is a modified glutamate synthase. In some embodiments, the orthogonal synthase is a modified alanine-glycine synthase. In some embodiments, the orthogonal synthase is a modified histidine synthase. In some embodiments, the orthogonal synthase is a modified leucine synthase. In some embodiments, the orthogonal synthase is a modified isoleucine synthase. In some embodiments, the orthogonal synthase is a modified lysine synthase. In some embodiments, the orthogonal synthase is a modified methionine synthase. In some embodiments, the orthogonal synthase is a modified phenylalanine synthase. In some embodiments, the orthogonal synthase is a modified proline synthase. In some embodiments, the orthogonal synthase is a modified serine synthase. In some embodiments, the orthogonal synthase is a modified threonine synthase. In some embodiments, the orthogonal synthase is a modified tryptophan synthase. In some embodiments, the orthogonal synthase is a modified tyrosine synthase. In some embodiments, the orthogonal synthase is a modified valine synthase. In some embodiments, the orthogonal synthase is a modified phosphoserine synthase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartic acid tRNA.In some embodiments, the orthogonal tRNA is a modified cysteine ​​tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine-glycine tRNA. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

[0257] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pairs. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair, the Escherichia coli TyrRS (Ec-Tyr) / Bacillus stearothermophilus tRNACUA pair, the Escherichia coli LeuRS (Ec-Leu) / Bacillus stearothermophilus tRNACUA pair, and the pyrrolidone-lysyl-tRNA pair. In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via Mj-TyrRS / tRNA pairs. Exemplary UAAs that can be incorporated via Mj-TyrRS / tRNA pairs include, but are not limited to, para-substituted phenylalanine derivatives, such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives, such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronphenylalanine; and o-nitrobenzyltyrosine.

[0258] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via Ec-Tyr / tRNACUA or Ec-Leu / tRNACUA pairs. Exemplary UAAs that can be incorporated via Ec-Tyr / tRNACUA or Ec-Leu / tRNACUA pairs include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; o-propargyltyrosine; α-aminooctanoic acid, o-methyltyrosine, o-nitrobenzylcysteine; and 3-(naphthyl-2-ylamino)-2-aminopropionic acid.

[0259] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via pyrrolidone-lysyl-tRNA pairs. In some embodiments, PylRS are obtained from archaea, for example, from methanogenic archaea. In some embodiments, PylRS are obtained from *Methanosarcina barkeri*, *Methanosarcina mazei*, or *Methanosarcina acetivorans*. Exemplary UAAs that can be incorporated via pyrrololysyl-tRNA pairs include, but are not limited to, amide and carbamate-substituted lysines, such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamide)hexanoic acid, N-ε-D-prolyl-L-lysine, and N-ε-cyclopentyloxycarbonyl-L-lysine; N-ε-acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzene[d][1,3]dioxacyclopenten-5-yl)ethoxy)carbonyl]-L-lysine; and N-ε-(1-methylcyclopropen-2-encarboxamide)lysine. In some embodiments, the IL-2 conjugates disclosed herein can be prepared using *Methanococcus martensii* Pyl tRNA, which is selectively supplied with non-natural amino acids, such as N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK), by *Methanococcus pasteurella* pyrrololysyl-tRNA synthetase (MbPylRS). Other methods are known to those skilled in the art, such as those described in: Zhang et al., *Nature* 2017, 551(7682): 644-647.

[0260] In some embodiments, non-natural amino acids are incorporated into the cytokines (e.g., IL peptides) described herein using synthases disclosed in US 9,988,619 and US 9,938,516. Exemplary UAAs that can be incorporated by such synthases include p-methylazido-L-phenylalanine, aralkyl, heterocyclic, heteroaryl non-natural amino acids, etc. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophene, or other heterocycles. In some embodiments, such amino acids include azides, tetrazines, or other chemical groups capable of conjugating with a coupling partner (such as a water-soluble portion). In some embodiments, such synthases are expressed and used to incorporate UAAs into cytokines in vivo. In some embodiments, such synthases are used to incorporate UAAs into cytokines using a cell-free translation system.

[0261] In some embodiments, non-natural amino acids are incorporated into the cytokines (e.g., IL peptides) described herein via naturally occurring synthetic enzymes. In some embodiments, non-natural amino acids are incorporated into the cytokines by organisms that are auxotrophic for one or more amino acids. In some embodiments, synthetic enzymes corresponding to auxotrophic amino acids are capable of loading the corresponding tRNA with the non-natural amino acid. In some embodiments, the non-natural amino acid is selenocysteine ​​or a derivative thereof. In some embodiments, the non-natural amino acid is selenomethionine or a derivative thereof. In some embodiments, the non-natural amino acid is an aromatic amino acid, wherein the aromatic amino acid contains an aryl halide, such as an iodide. In embodiments, the non-natural amino acid is structurally similar to an auxotrophic amino acid.

[0262] In some embodiments, non-natural amino acids include those described and illustrated in, for example, International Publication No. WO 2021 / 050554 A1, which is incorporated herein by reference in its entirety.

[0263] In some embodiments, the non-natural amino acid comprises lysine or a phenylalanine derivative or analogue. In some embodiments, the non-natural amino acid comprises a lysine derivative or a lysine analogue. In some embodiments, the non-natural amino acid comprises pyrrolysine (Pyl). In some embodiments, the non-natural amino acid comprises a phenylalanine derivative or a phenylalanine analogue. In some embodiments, the non-natural amino acid is the non-natural amino acid described in the following literature: 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, non-natural amino acids incorporated into the cytokines described herein (e.g., IL peptides) are 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 synthases include p-methylazido-L-phenylalanine, aralkyl, heterocyclic, and heteroaryl, as well as lysine derivative non-natural amino acids. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophene, or other heterocycles. In some embodiments, such amino acids include azides, tetrazine, or other chemical groups capable of conjugating to a coupling partner (such as a water-soluble portion). In some embodiments, the UAA comprises an azide attached to an aromatic portion via an alkyl linker. In some embodiments, the alkyl linker is a C1-C10 linker. In some embodiments, the UAA comprises a tetrazine attached to an aromatic moiety via an alkyl linker. In some embodiments, the UAA comprises a tetrazine attached to an aromatic moiety via an amino group. In some embodiments, the UAA comprises a tetrazine attached to an aromatic moiety via an alkylamino group. In some embodiments, the UAA comprises an azide attached to the terminal nitrogen of an amino acid side chain via an alkyl chain (e.g., N6 of a lysine derivative, or N5, N4, or N3 of a derivative containing a shorter alkyl side chain). In some embodiments, the UAA comprises a tetrazine attached to the terminal nitrogen of an amino acid side chain via an alkyl chain. In some embodiments, the UAA comprises an azide or tetrazine attached to an amide via an alkyl linker. In some embodiments, the UAA is a carbamate or amide containing an azide or tetrazine of 3-aminoalanine, serine, lysine, or a derivative thereof. In some embodiments, such a UAA is incorporated into cytokines in vivo. In some embodiments, such a UAA is incorporated into cytokines in a cell-free system. 2. Joined parts

[0265] In some embodiments, this document discloses conjugated moieties that bind to one or more cytokines described above (e.g., interleukins, IFN, or TNF). In some embodiments, the conjugated moieties are molecules that disrupt the interaction between the cytokine and its receptor. In some embodiments, the conjugated moieties are any molecules that, when bound to a cytokine, enable the cytokine conjugate to modulate an immune response. In some embodiments, the conjugated moieties bind to the cytokine via covalent bonds. In some embodiments, the cytokines described herein are attached to a conjugated moiety having a triazole group. In some embodiments, the cytokines described herein are attached to a conjugated moiety having a dihydropyridazine or pyridazine group. In some embodiments, the conjugated moieties comprise a water-soluble polymer. In other cases, the conjugated moieties comprise a protein or a fragment thereof. In still other cases, the conjugated moieties comprise a peptide. In still other cases, the conjugated moieties comprise a nucleic acid. In still other cases, the conjugated moieties comprise a small molecule. In other cases, the conjugated portion comprises a biological conjugate (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 conjugated portion increases serum half-life and / or improves stability. In some embodiments, the conjugated portion reduces the interaction of cytokines with one or more cytokine receptor domains or subunits. In other cases, the conjugated portion blocks the interaction of cytokines with one or more cytokine domains or subunits and their homologous receptors. In some embodiments, the cytokine conjugates described herein comprise multiple conjugated portions. In some embodiments, the conjugated portion is attached to a non-natural amino acid in a cytokine peptide. In some embodiments, the cytokine conjugate is attached to a non-natural amino acid in a cytokine peptide. In some embodiments, the conjugation portion is attached to an N- or C-terminal amino acid of the cytokine peptide. Various combinations of sites are disclosed herein, such as a first conjugation portion attaching to a non-natural amino acid in the cytokine peptide, and a second conjugation portion attaching to an N- or C-terminal amino acid of the cytokine peptide. In some embodiments, a single conjugation portion is attached to multiple residues of a cytokine peptide (e.g., a staple). In some embodiments, the conjugation portion is attached to both an N-terminal and a C-terminal amino acid of the cytokine peptide.

[0266] In some embodiments, the conjugation portion described herein is a water-soluble polymer. In some embodiments, the water-soluble polymer is non-peptide, non-toxic, and biocompatible. As used herein, a substance is considered biocompatible if, when used alone or in combination with another substance (e.g., an active agent, such as a cytokine portion), its beneficial effects on living tissue (e.g., when administered to a patient) outweigh any harmful effects, as evaluated by a clinician (e.g., a physician, toxicologist, or clinical development specialist). In some embodiments, the water-soluble polymer is further non-immunogenic. In some embodiments, a substance is considered non-immunogenic if, when evaluated by a clinician (e.g., a physician, toxicologist, or clinical development specialist), its intended use in vivo does not produce an undesirable immune response (e.g., antibody formation), or if an immune response is produced but not considered clinically significant or important.

[0267] In some embodiments, the water-soluble polymer is characterized by having about 2 to about 300 ends. 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, etc.; poly(oxyethylated polyols); poly(enols); poly(vinylpyrrolidone); poly(hydroxyalkylmethylacrylamide); poly(hydroxyalkyl methacrylate); poly(sugars); poly(α-hydroxy acids); poly(vinyl alcohol) (PVA); polyacrylamide (PAAm); poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA); polydimethylacrylamide (PDAAm); polyphosphazene; polyoxazoline (“POZ”) (described in WO 2008 / 106186); poly(N-acryloylmorpholine); and any combination of the foregoing substances.

[0268] In some embodiments, the water-soluble polymer is not limited to a specific structure. In some embodiments, the water-soluble polymer is a linear (e.g., end-capped, such as alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), dendritic (or star-shaped) architecture, each having or not having one or more degradable bonds. Furthermore, the internal structure of the water-soluble polymer can be organized in any number of different repeating patterns and can be selected from the group consisting of: homopolymers, alternating copolymers, random copolymers, block copolymers, alternating trimers, random trimers, and block trimers.

[0269] In some embodiments, the water-soluble polymer is represented by the length of the repeating polymer units (e.g., the number n of polyethylene glycol units). In some embodiments, the water-soluble polymer has the following structure:

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

[0271] The wavy line indicates attachment to a connector, reactive group, or non-natural amino acid, "Cap" indicates a capping group (e.g., 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 does not exceed 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some embodiments, n represents the 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 ranges of, greater than 5 kDa to about 100 kDa, about 6 kDa to about 90 kDa, about 10 kDa to about 85 kDa, greater than 10 kDa to about 85 kDa, about 20 kDa to about 85 kDa, about 53 kDa to about 85 kDa, about 25 kDa to about 120 kDa, about 29 kDa to about 120 kDa, about 35 kDa to about 120 kDa, and about 40 kDa to about 120 kDa.

[0273] Exemplary weight-average molecular weights of water-soluble polymers include approximately 100 Daltons, approximately 200 Daltons, approximately 300 Daltons, approximately 400 Daltons, approximately 500 Daltons, approximately 600 Daltons, approximately 700 Daltons, approximately 750 Daltons, approximately 800 Daltons, approximately 900 Daltons, approximately 1 kDa, approximately 1.5 kDa, approximately 2 kDa, approximately 2.2 kDa, approximately 2.5 kDa, approximately 3 kDa, approximately 4 kDa, approximately 4.4 kDa, approximately 4.5 kDa, approximately 5 kDa, approximately 5.5 kDa, approximately 6 kDa, approximately 7 kDa, approximately 7.5 kDa, approximately 8 kDa, approximately 9 kDa, approximately 10 kDa, approximately 11 kDa, approximately 12 kDa, approximately 13 kDa, approximately 14 kDa, approximately 15 kDa, approximately 20 kDa, approximately 22.5 kDa, approximately 25 kDa, approximately 30 kDa, approximately 35 kDa, and approximately 40 kDa. kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, and about 75 kDa. Branched forms of water-soluble polymers with a total molecular weight of any of the foregoing can also be used (e.g., a branched 40 kDa water-soluble polymer comprising two 20 kDa polymers). In one or more embodiments, the conjugate will not have any PEG portion directly or indirectly attached to PEG with a weight-average molecular weight of less than about 6 kDa.

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

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

[0276] In some embodiments, the PEG group comprising the IL-2 conjugate disclosed herein is a straight-chain or branched PEG group. In some embodiments, the PEG group is a straight-chain 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 straight-chain or branched methoxy PEG group. In some embodiments, the PEG group is a straight-chain methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. In some embodiments, the PEG group is a straight-chain or branched PEG group with an average molecular weight of about 100 Daltons to about 150 kDa. Exemplary ranges include, for example, weight-average molecular weights in the ranges of greater than 5 kDa to about 100 kDa, about 6 kDa to about 90 kDa, about 10 kDa to about 85 kDa, greater than 10 kDa to about 85 kDa, about 20 kDa to about 85 kDa, about 53 kDa to about 85 kDa, about 25 kDa to about 120 kDa, about 29 kDa to about 120 kDa, about 35 kDa to about 120 kDa, and about 40 kDa to about 120 kDa. Exemplary weight-average molecular weights of PEG groups include approximately 100 Daltons, approximately 200 Daltons, approximately 300 Daltons, approximately 400 Daltons, approximately 500 Daltons, approximately 600 Daltons, approximately 700 Daltons, approximately 750 Daltons, approximately 800 Daltons, approximately 900 Daltons, approximately 1 kDa, approximately 1.5 kDa, approximately 2 kDa, approximately 2.2 kDa, approximately 2.5 kDa, approximately 3 kDa, approximately 4 kDa, approximately 4.4 kDa, approximately 4.5 kDa, approximately 5 kDa, approximately 5.5 kDa, approximately 6 kDa, approximately 7 kDa, approximately 7.5 kDa, approximately 8 kDa, approximately 9 kDa, approximately 10 kDa, approximately 11 kDa, approximately 12 kDa, approximately 13 kDa, approximately 14 kDa, approximately 15 kDa, approximately 20 kDa, approximately 22.5 kDa, approximately 25 kDa, approximately 30 kDa, approximately 35 kDa, and approximately 40 kDa. 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 straight-chain PEG group having the average molecular weight disclosed above. In some embodiments, the PEG group is a branched PEG group having the average molecular weight disclosed above. In some embodiments, the PEG group comprising the IL-2 conjugate disclosed herein is a straight-chain or branched PEG group having a defined molecular weight ± 10%, 15%, 20%, or 25%.For example, within the scope of this disclosure are IL-2 conjugates that contain PEG groups having a molecular weight of 30,000 Da ± 3,000 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 conjugate disclosed herein is a straight-chain or branched PEG group with an average molecular weight of about 5 kDa to about 60 kDa. In some embodiments, the PEG group is a straight-chain or branched PEG group with 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 straight-chain or branched PEG group with 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 straight-chain or branched PEG group with 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 straight-chain PEG group with 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 PEG group with 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 conjugate disclosed herein is a straight-chain methoxy PEG group with an average molecular weight of about 5 kDa to about 60 kDa. In some embodiments, the PEG group is a straight-chain methoxy PEG group with 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 straight-chain methoxy PEG group with 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 straight-chain methoxy PEG group with 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 straight-chain methoxy PEG group with 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 straight-chain methoxy PEG group with an average molecular weight of about 30 kDa. In some embodiments, the PEG group is a straight-chain methoxy PEG group with an average molecular weight of about 50 kDa. In some embodiments, the PEG group is a straight-chain methoxy PEG group with an average molecular weight of about 60 kDa. In some embodiments, the PEG group comprising the IL-2 conjugate disclosed herein is a straight-chain methoxy PEG group having a defined molecular weight ± 10%, 15%, 20%, or 25%. For example, within the scope of this disclosure are IL-2 conjugates that contain a straight-chain methoxy PEG group having a molecular weight of 30,000 Da ± 3,000 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 conjugate disclosed herein is a branched methoxy PEG group with an average molecular weight of about 5 kDa to about 60 kDa. In some embodiments, the PEG group is a branched methoxy PEG group with 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 branched methoxy PEG group with 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 with 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 with 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 comprising the IL-2 conjugate disclosed herein is a branched methoxy PEG group having a defined molecular weight ± 10%, 15%, 20%, or 25%. For example, within the scope of this disclosure are IL-2 conjugates that contain branched methoxy PEG groups having a molecular weight of 30,000 Da ± 3,000 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 PEG from Nektar Therapeutics; and Y-shaped PEG derivatives from JenKem Technology.

[0281] In some embodiments, the IL-2 peptide described herein is conjugated with a water-soluble polymer selected from: poly(alkylene glycols), such as polyethylene glycol (“PEG”), poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol, etc.; poly(oxyethylated polyols); poly(enols); poly(vinylpyrrolidone); poly(hydroxyalkylmethylacrylamide); poly(hydroxyalkyl methacrylate); poly(sugars); poly(α-hydroxy acids); poly(vinyl alcohol) (PVA); polyacrylamide (PAAm); polydimethylacrylamide (PDAAm); poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA); polyphosphazene; polyoxazoline (“POZ”); poly(N-acryloylmorpholine); and combinations thereof. In some embodiments, the IL-2 peptide is conjugated with PEG (e.g., PEGylation). In some embodiments, the IL-2 peptide is conjugated with PPG. In some embodiments, the IL-2 peptide is conjugated with POZ. In some embodiments, the IL-2 peptide is conjugated with PVP.

[0282] In some embodiments, the water-soluble polymer comprises polyglycerol (PG). In some embodiments, the polyglycerol is 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 embodiments, the polyglycerol is linear PG (LPG). In other cases, polyglycerol is an intermediate-functional PG, a linear-block-hyperbranched PG (e.g., as described by Wurm et al., "Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates" Biomacromolecules 13:1161-1171 (2012)) or a side-chain 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, the cytokine (e.g., interleukin, IFN, or TNF) peptides described herein are conjugated to PGs (e.g., HPG, LPG, intermediate-functional PGs, linear-block-hyperbranched PGs, or side-chain functional PGs). In some embodiments, the cytokine is an IL-2 peptide. In some embodiments, the IL-2 peptide is conjugated to PGs, intermediate-functional PGs, or linear-block-hyperbranched PGs.

[0284] In some embodiments, the water-soluble polymer is a biodegradable synthetic PEG substitute. Exemplary biodegradable synthetic PEG substitutes include, but are not limited to, poly[oligomeric (ethylene glycol)methyl methacrylate] (POEGMA); backbone-modified PEG derivatives generated by polymerization of telechelic or end-functionalized PEG-type macromonomers; PEG derivatives containing comonomers with biodegradable bonds, such as poly[(ethylene oxide)-co-(methylene oxide)][P(EO-co-MEO)]; cyclic enone acetals, such as 5,6-benzo-2-methylene-1,3-dioxane (BMDO), 2-methylene-1,3-dioxane (MDO), and 2-methylene-4-phenyl-1,3-dioxane (MPDL) copolymerized with OEGMA; or poly-(ε-caprolactone)-grafted-poly(ethylene oxide) (PCL-g-PEO).

[0285] In some embodiments, the cytokine (e.g., interleukin, IFN, or TNF) peptides described herein are conjugated to a degradable synthetic PEG substitute, such as, for example, POEGM; a main-chain modified PEG derivative generated by polymerization of telechelic or end-functionalized PEG-like macromonomers; P(EO-co-MEO); cyclic enone acetals, such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO. In some embodiments, the cytokine is an IL-2 peptide. In some embodiments, the IL-2 peptide is conjugated to a degradable synthetic PEG substitute, such as, for example, POEGM; a main-chain modified PEG derivative generated by polymerization of telechelic or end-functionalized PEG-like macromonomers; P(EO-co-MEO); cyclic enone acetals, such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.

[0286] In some embodiments, the water-soluble polymer comprises a zwitterion. Exemplary zwitterions include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methacryloyloxyethylphosphorylcholine) (PMPC). In some embodiments, cytokine peptides described herein (e.g., interleukins, IFN, or TNF) are conjugated to zwitterions such as PSBMA, PCBMA, or PMPC. In some embodiments, the cytokine is an IL-2 peptide. In some embodiments, an IL-2 peptide is conjugated to a zwitterion such as PSBMA, PCBMA, or PMPC.

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

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

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

[0290] In some embodiments, the water-soluble polymer comprises a polysaccharide. Exemplary classes of polysaccharides include N-linked polysaccharides, O-linked polysaccharides, glycolipids, O-GlcNAc, and glycosaminoglycans. In some embodiments, a cytokine (e.g., interleukin, IFN, or TNF) peptide is conjugated to the polysaccharide. In some embodiments, an IL-2 peptide is conjugated to an N-linked polysaccharide. In some embodiments, an IL-2 peptide is conjugated to an O-linked polysaccharide. In some embodiments, an IL-2 peptide is conjugated to a glycolipid. In some embodiments, an IL-2 peptide is conjugated to an O-GlcNAc. In some embodiments, an IL-2 peptide is conjugated to a glycosaminoglycan.

[0291] In some embodiments, the water-soluble polymer comprises a polyoxazoline polymer. The polyoxazoline polymer is a linearly synthesized polymer and, similar to PEG, exhibits low polydispersity. In some embodiments, the polyoxazoline polymer is a polydisperse polyoxazoline polymer characterized by an average molecular weight. In some embodiments, the average molecular weight of the polyoxazoline polymer includes, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 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, the polyoxazoline polymer includes poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), or poly(2-propyl-2-oxazoline) (PPOZ). In some embodiments, a cytokine (e.g., interleukin, IFN, or TNF) peptide is conjugated to the polyoxazoline polymer. In some embodiments, an IL-2 peptide is conjugated to the polyoxazoline polymer. In some embodiments, an IL-2 peptide is conjugated to PMOZ. In some embodiments, an IL-2 peptide is conjugated to PEOZ. In some embodiments, an IL-2 peptide is conjugated to PPOZ.

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

[0293] In some embodiments, the water-soluble polymer comprises a polyamine. A polyamine is an organic polymer containing two or more primary amino groups. In some embodiments, the polyamine includes branched polyamines, linear polyamines, or cyclic polyamines. In some embodiments, the polyamine is a low molecular weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine. In some embodiments, a cytokine (e.g., interleukin, IFN, or TNF) peptide is conjugated to a polyamine. In some embodiments, an IL-2 peptide is conjugated to a polyamine. In some embodiments, the IL-2 peptide is conjugated to putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, or piperazine.

[0294] In some embodiments, the water-soluble polymer is described in U.S. Patent Nos. 7,744,861, 8,273,833, and 7,803,777. In some embodiments, the cytokine (e.g., interleukin, IFN, or TNF) peptide is conjugated to a linker as described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777. In some embodiments, the IL-2 peptide is conjugated to a linker as described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777.

[0295] In some embodiments, the conjugation portion 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 embodiments, the fatty acid is an unsaturated fatty acid. Exemplary fatty acids include, but are not limited to, fatty acids comprising about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the 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 with IL-2. In some embodiments, the lipid is a fatty acid, such as a saturated fatty acid or an unsaturated fatty acid. In some embodiments, the fatty acid has about 6 to about 26 carbon atoms, about 6 to about 24 carbon atoms, about 6 to about 22 carbon atoms, about 6 to about 20 carbon atoms, about 6 to about 18 carbon atoms, about 20 to about 26 carbon atoms, about 12 to about 26 carbon atoms, about 12 to about 24 carbon atoms, about 12 to about 22 carbon atoms, about 12 to about 20 carbon atoms, or about 12 to about 18 carbon atoms. In some embodiments, the length of 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 some embodiments, fatty acids include caproic acid, hexanoic acid, enanthic acid, heptanoic acid, caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, hexadecanoic acid, margaricacid, stearic acid, nonadecanoic acid, arachidic acid, heneicosylic acid, heneicosanoic acid, behenic acid, dodecanoic acid, and tridecanoic acid. The following are listed: trichosanoic acid, ligustic acid, pentacosyl acid, or ceric acid.

[0297] In some embodiments, IL-2 lipid conjugates enhance serum stability and / or serum half-life.

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

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

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

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

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

[0303] In some embodiments, a cytokine peptide (e.g., interleukin, IFN, or TNF) is conjugated to transferrin. In some embodiments, the cytokine peptide is conjugated to human transferrin. In other embodiments, the cytokine peptide is conjugated to a functional fragment of transferrin.

[0304] In some embodiments, the IL-2 peptide is conjugated to transferrin. In some embodiments, the IL-2 peptide is conjugated to human transferrin. In other embodiments, the IL-2 peptide is conjugated to a functional fragment of transferrin.

[0305] In some embodiments, the conjugated portion is transthyretin (TTR). Transthyretin is a transporter located in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine (T4) and retinol-binding protein that binds to retinol.

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

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

[0308] In some embodiments, the conjugated portion is an antibody or a binding fragment thereof. In some embodiments, the antibody or its binding fragment thereof includes a humanized antibody or its binding fragment thereof, a mouse antibody or its binding fragment thereof, a chimeric antibody or its binding fragment thereof, a monoclonal antibody or its binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a biscFv, (scFv)2, a biantibody, a microantibody, a nanobody, a triantibody, a tetraantibody, a human antibody (humabody), a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its binding fragment thereof, a bispecific antibody or its binding fragment thereof, or a chemically modified derivative thereof.

[0309] In some embodiments, the concatenation portion includes scFv, double scFv, (scFv)2, dsFv, or sdAb. In some embodiments, the concatenation portion includes scFv. In some embodiments, the concatenation portion includes double scFv. In some embodiments, the concatenation portion includes (scFv)2. In some embodiments, the concatenation portion includes dsFv. In some embodiments, the concatenation portion includes sdAb.

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

[0311] In some embodiments, cytokine peptides (e.g., interleukins, IFN, or TNF) are conjugated to antibodies or their binding fragments. In some embodiments, cytokine peptides are conjugated to humanized antibodies or their binding fragments, mouse antibodies or their binding fragments, chimeric antibodies or their binding fragments, monoclonal antibodies or their binding fragments, monovalent Fab', bivalent Fab2, F(ab)'3 fragments, single-chain variable fragments (scFv), biscFv, (scFv)2, biantibodies, microantibodies, nanoantibodies, triantibodies, tetraantibodies, human antibodies, disulfide-stabilized Fv proteins (dsFv), single-domain antibodies (sdAb), Ig NAR, camel antibodies or their binding fragments, bispecific antibodies or their binding fragments, or chemically modified derivatives thereof. In other cases, cytokine peptides are conjugated to the Fc portion of antibodies. In other cases, cytokine peptides are conjugated to the Fc portion of IgG (e.g., IgG1, IgG3, or IgG4).

[0312] In some embodiments, the IL-2 peptide is conjugated to an antibody or a binding fragment thereof. In some embodiments, the IL-2 peptide is conjugated to a humanized antibody or a binding fragment thereof, a mouse antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a biscFv, (scFv)2, a biantibody, a microantibody, a nanobody, a triantibody, a tetraantibody, a human antibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In other embodiments, the IL-2 peptide is conjugated to the Fc portion of an antibody. In other embodiments, the IL-2 peptide is conjugated to the Fc portion of IgG (e.g., IgG1, IgG3, or IgG4).

[0313] In some embodiments, the IL-2 peptide is conjugated with a water-soluble polymer (e.g., PEG) and an antibody or a binding fragment thereof. In some embodiments, the antibody or its binding fragment includes a humanized antibody or its binding fragment, a mouse antibody or its binding fragment, a chimeric antibody or its binding fragment, a monoclonal antibody or its binding fragment, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a biscFv, (scFv)2, a biantibody, a microantibody, a nanobody, a triantibody, a tetraantibody, a human antibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its binding fragment, a bispecific antibody or its binding fragment, or a chemically modified derivative thereof. In some embodiments, the antibody or its binding fragment includes scFv, a biscFv, (scFv)2, dsFv, or sdAb. In some embodiments, the antibody or its binding fragment includes scFv. In some embodiments, the antibody or its binding fragment directs the IL-2 conjugate to target cells, and the water-soluble polymer enhances stability and / or serum half-life.

[0314] In some embodiments, one or more IL-2 peptide-water-soluble polymer (e.g., PEG) conjugates are further bound to an antibody or a binding fragment 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 about 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 still other cases, the ratio of the IL-2 conjugate to the antibody is about 6:1 or higher.

[0315] In some embodiments, one or more IL-2 peptide-water-soluble polymer (e.g., PEG) conjugates bind directly to antibodies or their binding fragments. In other embodiments, IL-2 conjugates bind indirectly to antibodies or their binding fragments via linkers. Exemplary linkers include homobifunctional linkers, heterobifunctional linkers, maleimide-based linkers, zero-trace linkers, self-disintegrating linkers, spacers, etc.

[0316] In some embodiments, the antibody or its binding fragment binds directly or indirectly to the IL-2 polypeptide portion of an 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 located at a site that will not prevent the binding of the IL-2 polypeptide to IL-2Rβγ. In other cases, the conjugation site of the antibody to the IL-2 polypeptide is located at a site that partially blocks the binding of the IL-2 polypeptide to IL-2Rβγ. In still other cases, the conjugation site of the antibody to the IL-2 polypeptide is located at a site that will prevent or further prevent the binding of the IL-2 polypeptide to IL-2Rα. In other embodiments, the antibody or its binding fragment binds directly or indirectly to the water-soluble polymer portion of an IL-2 polypeptide-water-soluble polymer (e.g., PEG) conjugate.

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

[0318] In some embodiments, the conjugated portion is an XTEN™ peptide (Amunix Operating Corporation), and the modification is referred to as XTENation. XTENation is the fusion of a nucleic acid encoding a target polypeptide with a nucleic acid encoding an XTEN™ peptide (Amunix Operating Corporation), which is a long, unstructured hydrophilic peptide containing varying percentages of six amino acids: Ala, Glu, Gly, Ser, and Thr. In some embodiments, the 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 when combined with the target polypeptide. In some embodiments, cytokine peptides (e.g., interleukins, IFN, or TNF) are conjugated to the XTEN peptide. In some embodiments, an IL-2 peptide is conjugated to the XTEN peptide.

[0319] In some embodiments, the conjugated portion is a glycine-rich high-amino acid polymer (HAP), and the modification is referred to as HAP-ization. HAP-ization is the fusion of a nucleic acid encoding a target polypeptide with a nucleic acid encoding a glycine-rich high-amino acid polymer (HAP). In some embodiments, the HAP polymer contains a (Gly4Ser)n repeat motif and sometimes has a length of about 50, 100, 150, 200, 250, 300, or more residues. In some embodiments, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the HAP. In some embodiments, an IL-2 polypeptide is conjugated to the HAP.

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

[0321] In some embodiments, the conjugated portion is an elastin-like polypeptide (ELP), and the modification is referred to as ELP-ization. ELP-ization is the fusion of a nucleic acid encoding the target polypeptide with a nucleic acid encoding the elastin-like polypeptide (ELP). The ELP contains a VPGxG repeat motif, where x is any amino acid except proline. In some embodiments, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to the ELP. In some embodiments, an IL-2 polypeptide is conjugated to the ELP.

[0322] In some embodiments, the conjugated portion is a CTP peptide. A CTP peptide is a peptide comprising 30 or 31 amino acid residues (FQSSSS). KAPPPS LPSPS RLPGPS DTPILPQ (SEQ ID NO: 17) or FQDSSSS KAPPPS LPSPS RLPGPS DTPILPQ (SEQ ID NO: 18)), where S This represents the O-glycosylation site (OPKO). In some embodiments, the CTP peptide is fused to a cytokine peptide (e.g., IL-2 peptide) gene. In some embodiments, the cytokine peptide (e.g., IL-2 peptide) is conjugated to the CTP peptide.

[0323] In some embodiments, cytokines (e.g., IL-2 peptides) are modified via glutamylation. Glutamylation (or polyglutamylation) is a reversible post-translational modification of glutamate in which the γ-carboxyl group of glutamate forms a peptide-like bond with the amino group of free glutamate, wherein the α-carboxyl group extends into the polyglutamate chain.

[0324] In some embodiments, cytokines (e.g., IL-2 peptides) are modified with gelatin-like protein (GLK) polymers. 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 the amino acid residues Pro, Gly, Glu, Qln, Asn, Ser, and Lys. In some embodiments, the GLK polymer has a length of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150 residues or longer.

[0325] In some embodiments, the conjugation portion comprises an extracellular biomarker. In some embodiments, the extracellular biomarker is a tumor antigen. In some embodiments, exemplary extracellular biomarkers include CD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8, and B220. In some embodiments, the conjugation portion is bonded to or conjugated to a cytokine (e.g., IL-2). In some embodiments, the conjugation portion is a gene fusion, for example, at the N-terminus or C-terminus of a cytokine (e.g., IL-2).

[0326] In some embodiments, the conjugation portion comprises a molecule derived from a post-translational modification. Examples of post-translational modifications in some embodiments include myristylation, palmitoylation, isopreneylation (or pentenylation) (e.g., farnesylation or geraniylgeraniylation), glycosylphosphatidylinositylation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., addition of an alkyl group, such as methyl or ethyl), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycosylation, carbamylation, glutamylation, or deamidation. In some embodiments, cytokines (e.g., IL-2) are modified by post-translational modifications such as myristylation, palmitoylation, isopreneylation (or pentenylation) (e.g., farnesylation or geraniylgeraniylation), glycosylphosphatidylinositylation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., addition of an alkyl group, such as methyl or ethyl), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycosylation, carbamylation, glutamylation, or deamidation. 3. Joining

[0327] In some embodiments, useful functional reactive groups for conjugating or binding the conjugation portion to the cytokine peptide (e.g., IL-2 peptide) described herein include, for example, zero-order or higher-order linkers. In some embodiments, non-natural amino acids incorporated into the interleukin described herein contain functional reactive groups. In some embodiments, linkers contain functional reactive groups that react with non-natural amino acids incorporated into the interleukin described herein. In some embodiments, the conjugation portion contains functional reactive groups that react with non-natural amino acids incorporated into the interleukin described herein. In some embodiments, the conjugation portion contains functional reactive groups that react with the linker described herein (optionally pre-attached to the cytokine peptide). In some embodiments, the linker contains reactive groups that react with non-natural amino acids in the cytokine peptide described herein. In some embodiments, higher-order linkers comprise bifunctional linkers, such as homo-bifunctional linkers or hetero-bifunctional linkers. Exemplary bifunctional connectors include, but are not limited to, Lomant's reagents: dithiobis(succinimide propionate) DSP, 3',3'-dithiobis(sulfosuccinimide propionate) (DTSSP), succinimide dioctanoate (DSS), bis(sulfosuccinimide) succinimide (BS), succinimide tartrate (DST), and disulfosuccinimide tartrate (sulfo DST), ethylene glycol bis(succinimide) succinate (EGS), glutarate disuccinimide (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl adipamide (DMA), dimethyl heptamethimide (DMP), dimethyl octanoimide (DMS), dimethyl-3,3'-dithiobismalonimide (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionylamino)butane (DPDPB), bismaleimide hexane (BMH); compounds containing aryl halides (DFDNB), such as... Examples include 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylic acid)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N'-vinyl-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0328] In some embodiments, the bifunctional connector comprises a heterobifunctional connector. Exemplary heterobifunctional connectors include, but are not limited to, amine-reactive and thiol-crosslinking agents, such as succinimide N-3-(2-pyridyldithio)propionate (sPDP), long-chain succinimide N-3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain succinimide N-3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimide-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), and 6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoic acid sulfosuccinimide. Amino esters (sulfo-LC-sMPT), 4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid succinimide ester (sMCC), 4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester (sulfo-sMCC), m-maleiminobenzoyl-N-hydroxysuccinimide ester (MB), m-maleiminobenzoyl-N-hydroxysuccinimide ester (sulfo-MB), N-(4-iodoacetyl)aminobenzoic acid succinimide ester (sIAB), (4-iodoacetyl)aminobenzoic acid sulfosuccinimide ester Amino esters (sulfo-sIAB), 4-(p-maleiminophenyl)butyrate succinimide ester (sMPB), 4-(p-maleiminophenyl)butyrate sulfosuccinimide ester (sulfo-sMPB), N-(γ-maleiminobutyryloxy)succinimide ester (GMB), N-(γ-maleiminobutyryloxy)sulfosuccinimide ester (sulfo-GMB), 6-((iodoacetyl)amino)hexanoate succinimide ester (sIAX), 6-[6-(((iodoacetyl)amino)hexanoate)amino]hexanoate succinimide ester ( sIAXX), 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylic acid succinimide (sIAC), 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoic acid succinimide (sIACX), p-nitrophenyliodoacetic acid (NPIA); carbonyl reactive and thiol reactive crosslinking agents, such as 4-(4-N-maleiminophenyl)butyric acid hydrazide (MPBH), 4-(N-maleiminomethyl)cyclohexane-1-carboxy-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH);Amine-reactive and photoreactive crosslinking agents, such as N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfo-NHs-AsA), (4-azidosalicylamido)hexanoic acid sulfosuccinimide ester (sulfo-NHs-LC-AsA), 2-(ρ-azidosalicylamido)ethyl-1,3'-dithiopropionic acid sulfosuccinimide ester (sAsD), N-4-azidobenzoic acid hydroxysuccinimide ester (HsAB), N-4-azidobenzoic acid hydroxysulfosuccinimide ester (sulfo-H sAB), N-6-(4'-azido-2'-nitrophenylamino)hexanoic acid succinimide (sANPAH), 6-(4'-azido-2'-nitrophenylamino)hexanoic acid sulfosuccinimide (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), 2-(m-azido-o-nitrobenzoylamino)-ethyl-1,3'-dithiopropionic acid sulfosuccinimide (sAND), N-4-(4-azidophenyl)1,3'-dithiopropionic acid succinimide (sADP), N-(4-azidophenyl)-1 3'-Dithiopropionic acid sulfosuccinimide (sulfo-sADP), 4-(ρ-azidophenyl)butyric acid sulfosuccinimide (sulfo-sAPB), 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionic acid sulfosuccinimide (sAED), 7-azido-4-methylcoumarin-3-acetic acid sulfosuccinimide (sulfo-sAMCA), ρ-nitrophenyldiazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP); thiol reactivity and light Reactive crosslinking agents, such as 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionic acid amide (APDP), benzophenone-4-iodoacetamide; benzophenone-4-maleimide carbonyl reactive and photoreactive crosslinking agents, such as ρ-azidobenzoylhydrazine (ABH); carboxylic acid reactive and photoreactive crosslinking agents, such as 4-(ρ-azidosalicylamido)butylamine (AsBA); and arginine reactive and photoreactive crosslinking agents, such as ρ-azidophenylglyoxal (APG).

[0329] In some embodiments, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on the binding moiety (e.g., on the conjugation moiety or on IL-2). Exemplary electrophilic groups include carbonyl groups—such as aldehydes, ketones, carboxylic acids, esters, amides, ketenes, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include acylhydrazides, oximes, amino groups, hydrazides, thioureas, carboxylic acid hydrazides, and aryl acylhydrazides. In some embodiments, non-natural amino acids incorporated into the interleukins described herein comprise electrophilic groups.

[0330] In some embodiments, the connector is a cuttable connector. In some embodiments, the connector is a non-cuttable connector. In some embodiments, the non-cuttable connector is a dipeptide connector. In some embodiments, the cuttable connector is a dipeptide connector. In some embodiments, the dipeptide connector is valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), and valine-lysine (Val-Lys). In some embodiments, the dipeptide connector is valine-citrulline.

[0331] In some embodiments, the linker is a peptide linker that contains, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids. In some embodiments, the peptide linker contains at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or fewer amino acids. In other embodiments, the peptide linker contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.

[0332] In some embodiments, the connector includes a self-decomposing connector portion. In some embodiments, the self-decomposing connector portion includes p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the connector includes a dipeptide connector portion and a self-decomposing connector portion. In some embodiments, the self-decomposing connector portion is as described in U.S. Patent No. 9,089,614 and WIPO Application No. WO 2015038426.

[0333] In some embodiments, the cuttable connector is glucuronic acid. In some embodiments, the cuttable connector is an acid-cuttable connector. In some embodiments, the acid-cuttable connector is hydrazine. In some embodiments, the cuttable connector is a reducible connector.

[0334] In some embodiments, the connector 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 hexanoic acid, thereby forming a maleimide-hexanoyl (mc). In some embodiments, the connector comprises maleimino-hexanoyl (mc). In some embodiments, the connector is maleimino-hexanoyl (mc). In other cases, the maleimide group comprises maleimide-methyl, such as 4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid succinimide ester (sMCC) or 4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester (sulfo-sMCC) as described above.

[0335] In some embodiments, the maleimide group is a self-stabilized maleimide. In some embodiments, the self-stabilized maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis for the ring hydrolysis of thiosuccinimide, thereby eliminating the maleimide via an elimination reaction through a reverse Michael reaction. In some embodiments, the self-stabilized maleimide is the maleimide group described in Lyon et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some embodiments, the linker comprises a self-stabilized maleimide. In some embodiments, the linker is a self-stabilized maleimide.

[0336] This paper describes IL-2 conjugates having the structure of formula (I): Formula (I); in: Z is CH2 and Y is ;or Y is CH2 and Z is ; W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, and 50 kDa; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and

[0337] X+1 indicates the attachment site to the following amino acid residue. In some embodiments, X is an amino acid position of recombinant human IL-2, wherein the amino acid position refers to the position in SEQ ID NO: 1; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments of the IL-2 conjugate of formula (I), Z is CH2 and Y is In some embodiments of the IL-2 conjugate of formula (I), Y is CH2 and Z is This paper further provides IL-2 conjugates, where Z is CH2 and Y is... , or its pharmaceutically acceptable salt, solvate, or hydrate. This article further provides IL-2 conjugates, wherein Z is CH2 and Y is . And W is a PEG group having an average molecular weight selected from 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, and 60 kDa. This paper further provides IL-2 conjugates, wherein Y is CH2 and Z is... In some embodiments of the IL-2 conjugates of formula (I), the PEG group has an average molecular weight selected from 5 kDa, 10 kDa, 30 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, and 60 kDa. Examples of Z and Y herein and throughout also cover their pharmaceutically acceptable salts, solvates, or hydrates. This document further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 30 kDa. This document further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 35 kDa. This document further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 40 kDa. This document further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 45 kDa. This document further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 50 kDa. This document further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 55 kDa. This document further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 60 kDa. This document further provides IL-2 conjugates wherein formula (I) The positions of the structures in the amino acid sequences of the IL-2 conjugates are 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, H 78, 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. This article further describes IL-2 conjugates, wherein the structure of formula (VI) or (VII) or a mixture of (VI) and (VII) is selected from the amino acid sequence of the IL-2 conjugate at the position of K8, L11, E14, H15, L18, D19, M22, N87, E99 or D108.In some embodiments of the 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 the IL-2 conjugate of formula (I), X is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, and D108. In some embodiments, the IL-2 conjugate of formula (I) comprises a sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the IL-2 conjugate of formula (I) comprises the sequence of SEQ ID NO: 1.

[0338] This paper describes IL-2 conjugates having the structure of formula (II): Equation (II); Wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, and 60 kDa; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the following amino acid residue. In some embodiments, X is an amino acid position of recombinant human IL-2 having the following structure: The amino acid is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99 or D108, wherein the position of the amino acid is referenced to the position of the sequence in SEQ ID NO: 1.

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

[0340] This document describes IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein [AzK_PEG50kDa] has a structure of formula (II) or formula (III) or a mixture of formulas (II) and (III). This document further describes IL-2 conjugates wherein the ratio of the amount of formula (II) structure constituting the total amount of [AzK_PEG50kDa] in the IL-2 conjugate to the amount of formula (III) structure is approximately 1:1. This document further describes IL-2 conjugates wherein the ratio of the amount of formula (II) structure constituting the total amount of [AzK_PEG50kDa] in the IL-2 conjugate to the amount of formula (III) structure is greater than 1:1. This document further describes IL-2 conjugates wherein the ratio of the amount of formula (II) structure constituting the total amount of [AzK_PEG50kDa] in the IL-2 conjugate to the amount of formula (III) structure is less than 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (II) constituting the total [AzK_PEG30kDa] to the amount of structure (III) is approximately 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (II) constituting the total [AzK_PEG30kDa] to the amount of structure (III) is greater than 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (II) constituting the total [AzK_PEG30kDa] to the amount of structure (III) is less than 1:1.

[0341] In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. Examples of formula (II) and / or (III) herein and throughout the text also cover pharmaceutically acceptable salts, solvates, or hydrates thereof. IL-2 conjugates are further described herein, wherein [AzK_PEG] is a mixture of formulas (II) and (III). In formulas (II) and / or (III) In some examples of the IL-2 conjugates, 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, and F77 of recombinant human IL-2. 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 the IL-2 conjugates 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 the IL-2 conjugates 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, or 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] This document further describes IL-2 conjugates, wherein the IL-2 conjugates have an amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. This document further describes IL-2 conjugates, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, or 60 kDa. This document further describes IL-2 conjugates, wherein W is a PEG group having an average molecular weight selected from 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, or 60 kDa. This document further describes IL-2 conjugates, wherein W is a PEG group having an average molecular weight of 30 kDa. This document further provides IL-2 conjugates, wherein the PEG group has an average molecular weight of 35 kDa. This article further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 40 kDa. This article further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 45 kDa. This article further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 50 kDa. This article further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 55 kDa. This article further provides IL-2 conjugates wherein the PEG group has an average molecular weight of 60 kDa. This article further describes IL-2 conjugates wherein the IL-2 conjugate has an amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. This article further describes IL-2 conjugates wherein the IL-2 conjugate has an amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. This article further describes IL-2 conjugates, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, or 60 kDa. This article further describes IL-2 conjugates, wherein W is a PEG group having an average molecular weight selected from 50 kDa and 30 kDa.

[0343] This article describes IL-2 conjugates with the structure of formula (IV): Formula (IV); Wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, and 60 kDa; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the following amino acid residue. In some embodiments, X is an amino acid position of recombinant human IL-2 having the following structure: The amino acid is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99 or D108, wherein the position of the amino acid is referenced to the position of the sequence in SEQ ID NO: 1. This paper describes IL-2 conjugates with the structure of formula (V): Formula (V); Wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, and 60 kDa; and X is the amino acid position of recombinant human IL-2 with the following structure:

[0344] The amino acid is selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108, wherein the amino acid refers to the position of the sequence in SEQ ID NO: 1. Examples of formula (IV) and / or (V) herein and throughout the text also cover pharmaceutically acceptable salts, solvates, or hydrates thereof. IL-2 conjugates are further described herein, wherein [AzK_L1_PEG] is a mixture of formulas (IV) and (V). IL-2 conjugates are further described herein, wherein [AzK_L1_PEG] has the structure of formula (IV): Formula (IV)

[0345] Herein and throughout the text, the structure of formula (IV) encompasses its pharmaceutically acceptable salt, solvate, or hydrate. This document further describes IL-2 conjugates having an amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. This document further describes IL-2 conjugates where W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, or 60 kDa. This document further describes IL-2 conjugates where W is a PEG group having an average molecular weight selected from 50 kDa and 30 kDa. This document further describes IL-2 conjugates where W is a PEG group having an average molecular weight of 5 kDa. This document further describes IL-2 conjugates where W is a PEG group having an average molecular weight of 30 kDa. This article further describes IL-2 conjugates having an amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. This article further describes IL-2 conjugates where W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, or 60 kDa. This article further describes IL-2 conjugates where W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. This article further describes IL-2 conjugates where W is a PEG group having an average molecular weight of 30 kDa. This article further provides IL-2 conjugates where the PEG group has an average molecular weight of 35 kDa. This article further provides IL-2 conjugates where the PEG group has an average molecular weight of 40 kDa. This article further provides IL-2 conjugates where the PEG group has an average molecular weight of 45 kDa. This paper further provides IL-2 conjugates in which the PEG group has an average molecular weight of 50 kDa. This paper further provides IL-2 conjugates in which the PEG group has an average molecular weight of 55 kDa. This paper further provides IL-2 conjugates in which the PEG group has an average molecular weight of 60 kDa. This paper further describes IL-2 conjugates in which [AzK_L1_PEG] has the structure of formula (V): Formula (V)

[0346] Herein and throughout the text, the structure of formula (V) encompasses its pharmaceutically acceptable salt, solvate, or hydrate. This document further describes IL-2 conjugates having an amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. This document further describes IL-2 conjugates where W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, or 60 kDa. This document further describes IL-2 conjugates where W is a PEG group having an average molecular weight selected from 50 kDa and 30 kDa. This document further describes IL-2 conjugates where W is a PEG group having an average molecular weight of 30 kDa. This document further provides IL-2 conjugates where the PEG group has an average molecular weight of 35 kDa. This article further provides IL-2 conjugates in which the PEG group has an average molecular weight of 40 kDa. This article further provides IL-2 conjugates in which the PEG group has an average molecular weight of 45 kDa. This article further provides IL-2 conjugates in which the PEG group has an average molecular weight of 50 kDa. This article further provides IL-2 conjugates in which the PEG group has an average molecular weight of 55 kDa. This article further provides IL-2 conjugates in which the PEG group has an average molecular weight of 60 kDa. This article further describes IL-2 conjugates in which the IL-2 conjugates have the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0347] This paper further describes IL-2 conjugates in which the ratio of the amount of formula (IV) structure constituting the total [AzK_L1_PEG] to the amount of formula (V) structure is approximately 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of formula (IV) structure constituting the total [AzK_L1_PEG] to the amount of formula (V) structure is greater than 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of formula (IV) structure constituting the total [AzK_L1_PEG] to the amount of formula (V) structure is less than 1:1. This paper further describes IL-2 conjugates in which W is a straight-chain or branched PEG group. This paper further describes IL-2 conjugates in which W is a straight-chain PEG group. This paper further describes IL-2 conjugates in which W is a branched PEG group. This paper further describes IL-2 conjugates in which W is a methoxy PEG group. This article further describes IL-2 conjugates in which the methoxyPEG group is linear or branched. This article further describes IL-2 conjugates in which the methoxyPEG group is linear. This article further describes IL-2 conjugates in which the methoxyPEG group is branched. This article describes IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein [AzK_L1_PEG50kDa] has the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V): Formula (IV); Formula (V); in: W is a PEG group with an average molecular weight of 50 kDa; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the next amino acid residue. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (IV) constituting the total [AzK_L1_PEG50kDa] to the amount of structure (V) is approximately 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (IV) constituting the total [AzK_L1_PEG50kDa] to the amount of structure (V) is greater than 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (IV) constituting the total [AzK_L1_PEG50kDa] to the amount of structure (V) is less than 1:1. This article describes IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1, [AzK_L1_PEG30kDa] having the structure of formula (IV) or formula (V), or a mixture of the structures of formula (IV) and formula (V): Formula (IV); Formula (V); in: W is a PEG group with an average molecular weight of 30 kDa; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the next amino acid residue.

[0348] This paper further describes IL-2 conjugates in which the ratio of the amount of structure (IV) constituting the total [AzK_L1_PEG30kDa] to the amount of structure (V) is approximately 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (IV) constituting the total [AzK_L1_PEG30kDa] to the amount of structure (V) is greater than 1:1. This paper further describes IL-2 conjugates in which the ratio of the amount of structure (IV) constituting the total [AzK_L1_PEG30kDa] to the amount of structure (V) is less than 1:1. This article describes IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1, where [Azk_L1_PEG] is a mixture of structures of formula (IV) and formula (V): Formula (IV); Formula (V); in: W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, and 60 kDa; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the next amino acid residue.

[0349] In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. IL-2 conjugates are further described herein where the ratio of the amount of formula (IV) structure constituting the total [AzK_L1_PEG] in the IL-2 conjugate to the amount of formula (V) structure is about 1:1. IL-2 conjugates are further described herein where the ratio of the amount of formula (IV) structure constituting the total [AzK_L1_PEG] in the IL-2 conjugate to the amount of formula (V) structure is greater than 1:1. IL-2 conjugates are further described herein where the ratio of the amount of formula (IV) structure constituting the total [AzK_L1_PEG] in the IL-2 conjugate to the amount of formula (V) structure is less than 1:1. IL-2 conjugates are further described herein where W is a linear or branched PEG group. IL-2 conjugates are further described herein where W is a linear PEG group. IL-2 conjugates are further described herein where W is a branched PEG group. This paper further describes IL-2 conjugates, wherein W is a methoxy PEG group. This paper further describes IL-2 conjugates, wherein the methoxy PEG group is linear or branched. This paper further describes IL-2 conjugates, wherein the methoxy PEG group is linear. This paper further describes IL-2 conjugates, wherein the methoxy PEG group is branched.

[0350] In some embodiments of the IL-2 conjugates 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. In some embodiments of the IL-2 conjugates 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 the IL-2 conjugates 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, or 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, the IL-2 conjugate of formula (IV) or formula (V) comprises a sequence of any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1.

[0352] This article describes IL-2 conjugates containing the amino acid sequence SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII): Formula (VI); Equation (VII); in: n is an integer in the range of approximately 2 to approximately 5000; and X has the following structure: ; X-1 indicates the attachment site to the preceding amino acid residue; and

[0353] X+1 indicates the attachment site to the following amino acid residue. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. Examples of formula (VI) and / or (VII) herein and throughout the text also cover pharmaceutically acceptable salts, solvates, or hydrates thereof. IL-2 conjugates of formula (VI) or (VII), or (VI) and (VII) are further described herein. The positions of the mixture's structure in the amino acid sequence of the IL-2 conjugate are 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. This paper further describes IL-2 conjugates in which the structure of formula (VI) or (VII) or a mixture of (VI) and (VII) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. This paper further describes IL-2 conjugates in which the structure of formula (VI) or (VII) or a mixture of (VI) and (VII) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from H15 and L18.

[0354] This article describes IL-2 conjugates containing the amino acid sequence SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX): Formula (VIII); Formula (IX); in: n is an integer in the range of approximately 2 to approximately 5000; and X has the following structure: X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the following amino acid residue. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate thereof. Examples of formula (VIII) and / or (IX) herein and throughout also cover pharmaceutically acceptable salts, solvates, or hydrates thereof. IL-2 conjugates are further described herein, wherein formula (VIII) or (IX) or (VIII) and (IX) are... The positions of the mixture's structure in the amino acid sequence of the IL-2 conjugate are 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. This paper further describes IL-2 conjugates in which the structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. This paper further describes IL-2 conjugates in which the structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from H15 and L18.

[0355] This article describes IL-2 conjugates containing the amino acid sequence SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (X) or (XI) or a mixture of (X) and (XI): Formula (X); Formula (XI); in: n is an integer in the range of approximately 2 to approximately 5000; and The wavy line indicates a covalent bond with an amino acid residue that has not been substituted in SEQ ID NO: 1. Examples of formula (X) and / or (XI) herein and throughout the text also cover their pharmaceutically acceptable salts, solvates, or hydrates. IL-2 conjugates of formula (X) or (XI), or (X) and (XI), are further described herein. The positions of the mixture's structure in the amino acid sequence of the IL-2 conjugate are 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. This paper further describes IL-2 conjugates in which the structure of formula (X) or (XI) or a mixture of (X) and (XI) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. This paper further describes IL-2 conjugates in which the structure of formula (X) or (XI) or a mixture of (X) and (XI) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from H15 and L18.

[0356] This article describes an IL-2 conjugate containing the amino acid sequence SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII): Formula (XII); Formula (XIII); in: n is an integer in the range of approximately 2 to approximately 5000; and

[0357] The wavy line indicates a covalent bond with an amino acid residue that has not been substituted in SEQ ID NO: 1. Examples of formula (XII) and / or (XIII) herein and throughout the text also cover their pharmaceutically acceptable salts, solvates, or hydrates. IL-2 conjugates of formula (XII) or (XIII), or (XII) and (XIII), are further described herein. The positions of the mixture's structure in the amino acid sequence of the IL-2 conjugate are 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. This paper further describes IL-2 conjugates in which the structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108. This paper further describes IL-2 conjugates in which the structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII) is selected from the amino acid sequence of the IL-2 conjugate at positions selected from H15 and L18.

[0358] This document describes pharmaceutical compositions of formula (I), formula (IV), or formula (V). In some embodiments, the pharmaceutical composition of formula (I), formula (IV), or formula (V) comprises a sequence containing any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the pharmaceutical composition of formula (I), formula (IV), or formula (V) comprises a sequence containing any one of SEQ ID NO: 4. This document describes pharmaceutical compositions of formula (I), formula (VI), or formula (VII). In some embodiments, the pharmaceutical composition of formula (I), formula (VI), or formula (VII) comprises a sequence containing any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the pharmaceutical composition of formula (I), formula (VI), or formula (VII) comprises a sequence containing any one of SEQ ID NO: 4. This document describes pharmaceutical compositions of formula (I), formula (VIII), or formula (IX). In some embodiments, the pharmaceutical composition of formula (I), formula (VIII), or formula (IX) comprises a sequence containing any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the pharmaceutical composition of formula (I), formula (VIII), or formula (IX) comprises a sequence containing any one of SEQ ID NO: 4. Pharmaceutical compositions of formula (I), formula (X), or formula (XI) are described herein. In some embodiments, the pharmaceutical composition of formula (I), formula (X), or formula (XI) comprises a sequence containing any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the pharmaceutical composition of formula (I), formula (X), or formula (XI) comprises a sequence containing any one of SEQ ID NO: 4. Pharmaceutical compositions of formula (XII), formula (XIII), or formula (V) are described herein. In some embodiments, the pharmaceutical composition of formula (I), formula (XII), or formula (XIII) comprises a sequence containing any one of SEQ ID NO: 2-14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the pharmaceutical composition of formula (I), formula (XII), or formula (XIII) comprises a sequence containing any one of SEQ ID NO: 4.

[0359] In some embodiments described herein, the conjugation reaction comprises an anti-electron-demanding cycloaddition reaction involving a diene and a dienophile. In some embodiments, the diene comprises a tetrazine. In some embodiments, the dienophile comprises an alkene. In some embodiments, the dienophile comprises an alkyne. In some embodiments, the alkyne is a strained alkyne. In some embodiments, the alkene is a strained diene. In some embodiments, the alkyne is a trans-cyclooctyne. In some embodiments, the alkyne is a cyclooctene. In some embodiments, the alkene is a cyclopropene. In some embodiments, the alkene is a fluorocyclopropene. In some embodiments, the conjugation reaction described herein results in the formation of a cytokine peptide by attachment of a 6-membered heterocyclic ring containing two nitrogen atoms to a linker or conjugation portion.

[0360] In some embodiments described herein, the conjugation reactions include olefin metathesis reactions. In some embodiments, the conjugation reactions described herein include the reaction of an olefin and an alkyne with a ruthenium catalyst. In some embodiments, the conjugation reactions described herein include the reaction of two olefins with a ruthenium catalyst. In some embodiments, the conjugation reactions described herein include the reaction of two alkynes with a ruthenium catalyst. In some embodiments, the conjugation reactions described herein include the reaction of an olefin or alkyne with a ruthenium catalyst and an amino acid containing an allyl group. In some embodiments, the conjugation reactions described herein include the reaction of an olefin or alkyne with a ruthenium catalyst and an amino acid containing an allyl sulfide or selenide. In some embodiments, the ruthenium catalyst is a Hoveda-Grubbs second-generation catalyst. In some embodiments, the olefin metathesis reaction includes the reaction of one or more strained olefins or alkynes.

[0361] In some embodiments described herein, the conjugation reactions include cross-coupling reactions. In some embodiments, the cross-coupling reaction comprises a transition metal catalyst, such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or other transition metal catalysts, and one or more ligands. In some embodiments, the transition metal catalyst is water-soluble. In some embodiments described herein, the conjugation reactions include the Suzuki-Miyaura cross-coupling reaction. In some embodiments described herein, the conjugation reactions include a reaction of an aryl halide (or trifluoromethanesulfonate or toluenesulfonate), an aryl or alkenylboronic acid, and a palladium catalyst. In some embodiments described herein, the conjugation reactions include a sage cross-coupling reaction. In some embodiments described herein, the conjugation reactions include a reaction of an aryl halide (or trifluoromethanesulfonate or toluenesulfonate), an alkyne, and a palladium catalyst. In some embodiments, the cross-coupling reaction results in the attachment of a linker or conjugated portion to a cytokine peptide via a carbon-carbon bond.

[0362] In some embodiments described herein, the conjugation reaction includes a deprotection or "uncageing" reaction of the reactive group prior to conjugation. In some embodiments, the conjugation reaction described herein includes uncageing the reactive group with light before the conjugation reaction. In some embodiments, the reactive group is protected with an aralkyl moiety comprising one or more nitro groups. In some embodiments, uncageing of the reactive group yields a free amine, sulfide, or other reactive group. In some embodiments, the conjugation reaction described herein includes uncageing the reactive group with a transition metal catalyst before the conjugation reaction. In some embodiments, the transition metal catalyst comprises palladium and one or more ligands. In some embodiments, the reactive group is protected with an allyl moiety. In some embodiments, the reactive group is protected with an allyl carbamate. In some embodiments, the reactive group is protected with an acetylacetic moiety. In some embodiments, the reactive group is protected with an acetylacetic carbamate. In some embodiments, the 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, the conjugation reactions include ligand-directed reactions, wherein (optionally) a ligand attached to a reactive group facilitates a conjugation site between the reactive group and a cytokine peptide. In some embodiments, the ligand is cleaved during or after the reaction of the cytokine peptide with the reactive group. In some embodiments, the conjugation site of the cytokine peptide is a non-natural amino acid as described herein. In some embodiments, the reactive group comprises a leaving group, such as an electron-deficient aryl or heteroaryl group. In some embodiments, the reactive group comprises a leaving group, such as an electron-deficient alkyl group replaced by a cytokine peptide. In some embodiments, the conjugation reactions described herein include a reaction of a radical scavenger with a radical substance. In some embodiments, the conjugation reactions described herein include an oxidative radical addition reaction. In some embodiments, the radical scavenger is an arylamine. In some embodiments, the radical substance is a tyrosine amide. In some embodiments, the radical substance is generated via a ruthenium catalyst (such as [Ru(bpy)3]) and light.

[0364] Enzymatic reactions may optionally be used in the conjugation reactions described herein. Exemplary enzymatic conjugations include SortA-mediated conjugation, TG-mediated conjugation, or FGE-mediated conjugation. In some embodiments, the conjugation reactions described herein involve the native protein linkage (NPL) of a terminal 1-amino-2-thio group to a thioester to form an amide bond.

[0365] This document describes various conjugation reactions for reacting a linker or conjugated portion with a cytokine peptide, wherein the reaction occurs together with a non-natural amino acid in the cytokine peptide. In some embodiments, the conjugation reaction includes forming a disulfide bond at a non-natural amino acid residue. In some embodiments, the conjugation reaction includes a 1,4-Michael addition reaction of a non-natural amino acid. In some embodiments, the conjugation reaction includes a cyanobenzothiazole linker to a non-natural amino acid. In some embodiments, the conjugation reaction includes crosslinking with an acetone moiety such as 1,3-dichloro-2-propanone. In some embodiments, the conjugation reaction includes a 1,4-Michael addition reaction with dehydroalanine, which is formed by the reaction of a non-natural amino acid with O-tris(methyl)sulfonylhydroxylamine. In some embodiments, the conjugation reaction includes the reaction of a non-natural amino acid with a triazolinide (TAD) or a TAD derivative. In some embodiments, the conjugation reaction includes the reaction of a non-natural amino acid with rhodium carbenoid.

[0366] Various conjugation reactions are used to conjugate linkers, conjugated moieties, and non-natural amino acids incorporated into the cytokine peptides described herein. Such conjugation reactions are generally compatible with aqueous conditions, such as “bioorthogonal” reactions. In some embodiments, the conjugation reaction is mediated by chemical reagents, such as catalysts, light, or reactive chemical groups found on the linker, conjugated moieties, or non-natural amino acids. In some embodiments, the conjugation reaction is enzyme-mediated. In some embodiments, the conjugation reactions used herein are described in Gong, Y., Pan, L. Tett. Lett. [Tetrahedral Communications] 2015, 56, 2123. In some embodiments, the conjugation reactions used herein are described in Chen, X.; Wu, YW. Org. Biomol. Chem. [Organic and Biomolecular Chemistry] 2016, 14, 5417.

[0367] In some embodiments described herein, the conjugation reactions include 1,3-dipolar cycloaddition reactions. In some embodiments, the 1,3-dipolar cycloaddition reaction includes a reaction of an azide with a phosphine (“click” reaction). In some embodiments, the conjugation reaction is catalyzed by copper. In some embodiments, the conjugation reactions described herein produce cytokine peptides comprising a linker or conjugation moiety attached by a triazole. In some embodiments, the conjugation reactions described herein include a reaction of an azide with a strained olefin. In some embodiments, the conjugation reactions described herein include a reaction of an azide with a strained alkyne. In some embodiments, the conjugation reactions described herein include a reaction of an azide with a cycloalkyne (e.g., DBCO).

[0368] In some embodiments described herein, the conjugation reactions include: , where X is the position of the non-natural amino acid contained in the IL-2 conjugate, such as in any one of SEQ ID NO: 2 to 14 or a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments, the conjugation portion comprises a water-soluble polymer. In some embodiments, the reactive group comprises an alkyne or an azide. In some embodiments described herein, the conjugation reaction described herein includes: , where X is the position of the non-natural amino acid contained in the IL-2 conjugate, such as in any one of SEQ ID NO: 2 to 14 or in a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments described herein, the conjugation reaction described herein includes: , where X is the position of the non-natural amino acid contained in the IL-2 conjugate, such as in any one of SEQ ID NO: 2 to 14 or in a sequence having at least 80% sequence identity with SEQ ID NO: 1. In some embodiments described herein, the conjugation reaction described herein includes: X is the position of a non-natural amino acid contained in the IL-2 conjugate, such as in any one of SEQ ID NO: 2 to 14 or in a sequence having at least 80% sequence identity with SEQ ID NO: 1.

[0369] In some embodiments described herein, the conjugation reactions comprise cycloaddition reactions between azide moieties (such as those contained in proteins comprising amino acid residues derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK)) and strained cycloalkynes (such as strained cycloalkynes derived from DBCO as a chemical moieties comprising a dibenzocyclooctynyl group). The PEG group comprising the DBCO moieties is commercially available or can be prepared by methods known to those skilled in the art.

[0370] Conjugation reactions, such as the click reactions described herein, can produce a single regioisomer or a mixture of regioisomers. In some embodiments, the regioisomer ratio is about 1:1. In some embodiments, the regioisomer ratio is about 2:1. In some embodiments, the regioisomer ratio is about 1.5:1. In some embodiments, the regioisomer ratio is about 1.2:1. In some embodiments, the regioisomer ratio is about 1.1:1. In some embodiments, the regioisomer ratio is greater than 1:1. 4. Cytokine peptide production

[0371] In some embodiments, the IL-2 conjugates described herein containing non-natural amino acid mutations are recombinantly generated or chemically synthesized. In some embodiments, the IL-2 conjugates described herein are recombinantly generated, for example, through a host cell system or in a cell-free system.

[0372] In some embodiments, the IL-2 conjugate is generated through recombinant generation via a host cell system. In some embodiments, the host cell is a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell, or plant cell) or a prokaryotic cell (e.g., Gram-positive or Gram-negative bacteria). In some embodiments, the eukaryotic host cell is a mammalian host cell. In some embodiments, the mammalian host cell is a stable cell line, or a cell line that has incorporated the genetic material of interest into its own genome and has the ability to express the product of the genetic material after many generations of cell division. In other cases, the mammalian host cell is a transient cell line, or a cell line that has not incorporated the genetic material of interest into its own genome and cannot express the product of the genetic material after many generations of cell division.

[0373] Exemplary mammalian host cells include, but are not limited to, the following cell lines: 293T, 293A, 293FT, 293F, 293H, A549, MDCK, CHO DG44, CHO-S, CHO-K1, Expi293F™, Flp-In™ T-REx™ 293, Flp-In™-293, Flp-In™-3T3, Flp-In™-BHK, Flp-In™-CHO, Flp-In™-CV-1, Flp-In™-Jurkat, FreeStyle™ 293-F, FreeStyle™ CHO-S, GripTite™ 293 MSR, GS-CHO, HepaRG™, and T-REx™. Jurkat cell line, Per.C6 cell line, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

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

[0375] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H and X-33, and Saccharomyces cerevisiae yeast strains such as INVSC1.

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

[0377] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0378] In some embodiments, suitable polynucleotide molecules or vectors for generating the IL-2 polypeptide described herein include any suitable vectors derived from eukaryotic or prokaryotic sources. Exemplary polynucleotide molecules or vectors include vectors derived from bacteria (e.g., *Escherichia coli*), insects, yeast (e.g., *Pichia pastoris*), algae, or mammals. Bacterial vectors include, for example, pACYC177, pASK75, the pBAD vector series, the pBADM vector series, the pET vector series, the pETM vector series, the pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, the pQE vector series, pRSET A, pRSET B, pRSET C, the 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-MAT2, 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® target vector, pAO815 Pichia pastoris vector, pFLD1 Pichia pastoris vector, pGAPZA, B and C Pichia pastoris vector, pPIC3.5K Pichia pastoris vector, pPIC6 A, B and C Pichia pastoris vector, pPIC9K Pichia pastoris vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B and C yeast vector, or pYES3 / CT yeast vector.

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

[0382] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary transient mammalian expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV6a,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 stable mammalian expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0383] In some embodiments, the cell-free system is used to generate the cytokine (e.g., IL-2) peptides described herein. In some embodiments, the cell-free system comprises a mixture of cytoplasmic and / or nuclear components derived from cells and is suitable for in vitro nucleic acid synthesis. In some embodiments, the cell-free system utilizes prokaryotic cell components. In other cases, the cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis is achieved in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, archaea, or HeLa cells. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.

[0384] Cell-free translation systems vary and include components such as plasmids, mRNA, DNA, tRNA, synthases, releasing factors, ribosomes, chaperone proteins, translation initiation and elongation factors, native and / or non-native amino acids, and / or other components for protein expression. These components may optionally be modified to improve yield, increase synthesis rate, improve protein product fidelity, or incorporate non-native amino acids. In some embodiments, the cytokines described herein are synthesized using a cell-free translation system described in US 8,778,631, US 2017 / 0283469, US 2018 / 0051065, US 2014 / 0315245, or US 8,778,631. In some embodiments, the cell-free translation system includes modified releasing factors, or even removes one or more releasing factors from the system. In some embodiments, the cell-free translation system has a reduced protease concentration. In some embodiments, the cell-free translation system includes modified tRNA having redistributed codons for encoding non-native amino acids. In some embodiments, the synthetic enzymes described herein for incorporating non-natural amino acids are used in cell-free translation systems. In some embodiments, tRNA is preloaded with non-natural amino acids using enzymatic or chemical methods before being added to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organisms.

[0385] In some embodiments, cytokine (e.g., IL-2) peptides are generated in a circular arrangement via an expression host system or through a cell-free system.

[0386] In this disclosure, orthogonal or extended genetic codes may be used, wherein one or more specific codons present in the nucleic acid sequence of a cytokine (e.g., IL-2) polypeptide are assigned to encode non-natural amino acids, such that they can be genetically incorporated into the cytokine (e.g., IL-2) using orthogonal tRNA synthetase / tRNA pairs. Orthogonal tRNA synthetase / tRNA pairs are capable of providing non-natural amino acids to tRNA and are capable of incorporating non-natural amino acids into the polypeptide chain in response to codons.

[0387] In some embodiments, the codon is an amber, ochre, opal, or tetrad codon. In some embodiments, the codon corresponds to an orthogonal tRNA that will be used to carry non-natural amino acids. In some embodiments, the codon is an amber codon. In other cases, the codon is an orthogonal codon.

[0388] In some embodiments, the codon is a quadruplet codon, which can be decoded by orthogonal ribosomal ribosome ribo-Q1. In some embodiments, the quadruplet codon is as described 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, the codons used in this disclosure are re-encoded codons, such as synonymous codons or rare codons replaced by alternative codons. In some embodiments, the re-encoded codons are as described in: Napolitano et al., “Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli” PNAS, 113(38): E5588-5597 (2016). In some embodiments, the re-encoded codons are 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, non-natural nucleic acids are used to induce the incorporation of one or more non-natural amino acids into a cytokine (e.g., IL-2). Exemplary non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenated uracil and cytosine, and 5-propynyluracil and cytosine. 6-Azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated especially 5-bromine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, and 3-deadenine and 3-deadenine. Certain non-natural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2-substituted purines, N-6-substituted purines, O-6-substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that increase the stability of double-strand formation, universal nucleic acids, hydrophobic nucleic acids, hybrid nucleic acids, size-expanding nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl 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, 5-halocytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uridine Pyrimidines (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated, especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazoguanine, 7-deazoadenine, 3-deazoguanine, 3-deazoadenine, tricyclic pyrimidines, phenoxazinecytidine ([5,4-b][l,4] benzoxazine-2(3H)-one), phenothiazine-cytoside (1H-pyrimido[5,4-b][l,4]benzoxazine-2(3H)-one), G-cup, phenothiazine-cytoside (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazine-2(3H)-one), carbazole-cytoside (2H-pyrimido[4,5-b]indole-2-one), pyridine-indole-cytoside (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimido-2-one), purine or pyrimidine bases Those replaced by other heterocycles, 7-deadenine, 7-deadenine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, cyclocytosine, cytarabine, 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 Pyrimidines, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazoguanine, 7-deazo-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, 5,175,2 73, 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. [Bioorganic and Medicinal Chemistry] 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI (ed.), John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie,International Edition of Applied Chemistry, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, edited by Crooke and Lebleu, CRC Press, 1993, 273-288. Additional base modifications can be found, for example, in: US Patent No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition of Applied Chemistry, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pp. 289-302, edited by Crooke and Lebleu, CRC Press, 1993.

[0391] Non-natural nucleic acids comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and in some embodiments, the nucleic acid comprises one or more heterocyclic bases in addition to the five major base components of naturally occurring nucleic acids. For example, in some embodiments, the heterocyclic bases include uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4-aminopyrrolo[2,3-d]pyrimidine-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidine-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidine-3-yl, wherein the purine is attached to the sugar moieties of the nucleic acid via the 9-position, the pyrimidine via the 1-position, the pyrrolopyrimidine via the 7-position, and the pyrazolopyrimidine via the 1-position.

[0392] In some embodiments, the nucleotide analogue is also modified at the phosphate ester moiety. Modified phosphate ester moieties include, but are not limited to, those modified at the link between two nucleotides, and include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphonates, phosphamids including 3'-aminophosphamids and aminoalkylphosphamids, thiophosphamids, thioalkylphosphonates, thioalkyl phosphate triesters, and boran phosphates. It should be understood that these phosphate ester bonds or modified phosphate ester bonds between the two nucleotides are linked by a 3'-5' bond or a 2'-5' bond, and the bond contains opposite polarities, 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 US patents disclose how to prepare and use nucleotides containing modified phosphate esters, including but 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, and 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, non-natural nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleoside (PCT / US2002 / 006460), 5'-substituted DNA and RNA derivatives (PCT / US 2011 / 033961; Saha et al., J. OrgChem., 1995, 60, 788-789; Wang et al., Bioorganic & MedicinalChemistry 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 [Swiss Chimica Sinica], 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 of monophosphates prepared by modified bases (Wang et al., Nucleosides, Nucleotides & Nucleic Acids [Nucleosides, Nucleotides & Nucleic Acids], 2004, 23 (1 & 2), 317-337).

[0394] In some embodiments, the non-natural nucleic acid includes modifications at the 5' and 2' positions of the sugar ring (PCT / US94 / 02993), such as 5'-CH2-substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some embodiments, the non-natural nucleic acid includes amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, wherein the 3'-linked nucleoside in the dimer (5' to 3') comprises 2'-OCH3 and 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids can include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Non-natural nucleic acids can include 5'-methylenephosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett. [Tetrahedral Communications], 1993, 34, 2723-2726; Collingwood et al., Synlett [Synlett], 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta [Swiss Chimica Acta], 2002, 85, 2777-2806). Non-natural nucleic acids can include 2'-substituted 5'-phosphonate monomers (US 2006 / 0074035) and other modified 5'-phosphonate monomers (WO1997 / 35869). Non-natural nucleic acids may include 5'-modified methylene phosphonate monomers (EP 614907 and EP629633). Non-natural nucleic acids may include analogs of 5' or 6'-phosphonate ribonucleosides containing hydroxyl groups at the 5' and / or 6'-positions (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033).Non-natural nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers with 5'-phosphate groups (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids can include nucleosides with a 6'-phosphonate group, wherein the 5' or / and 6'-positions are unsubstituted or substituted with: thiotert-butyl (SC(CH3)3) (and its analogues); methylene amino (CH2NH2) (and its analogues) or cyano (CN) (and its analogues) (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, non-natural nucleic acids also include modifications to the sugar moiety. In some embodiments, the nucleic acid contains one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides can confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological properties. In some embodiments, the nucleic acid comprises a chemically modified furanose ring moiety. Examples of chemically modified furanose rings include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents; bridging two ring atoms to form a bicyclic nucleic acid (BNA); and using S, N(R) or C(R1)(R2) (R = H, C1-C) 12 Alkyl or protecting group) replacing the ribosyl epoxy atom; and combinations thereof. Examples of chemically modified sugars can be found in WO 2008 / 101157, US 2005 / 0130923 and WO 2007 / 134181.

[0396] In some embodiments, the modified nucleic acid comprises a modified sugar or sugar analogue. Therefore, in addition to ribose and deoxyribose, the sugar moiety can also be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lythose, or a sugar “analyte” cyclopentyl. The sugar can be in the form of pyranosyl or furanosyl. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be attached to the corresponding heterocyclic base in an [α] or [β] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogues, 2'-amino-RNA analogues, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs and the corresponding "nucleosides" is known, wherein such sugars or analogs are attached to heterocyclic bases (nucleic acid bases). Sugar modifications and combinations with other modifications are also possible.

[0397] Modifications to the sugar moiety include both natural and non-natural modifications of ribose and deoxyribose. 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 groups can be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. 2' sugar modifications also include, but are not limited to, -O[(CH2)] n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2 and -O(CH2) n ON[(CH2)n CH3)]2, where n and m are 1 to approximately 10.

[0398] Other modifications at position 2' include, but are not limited to: C1 to C 10Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl alkyl groups, O-alkylaryl groups, O-aryl alkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2, CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalation groups, groups used to improve the pharmacokinetic properties of oligonucleotides, or groups used to improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions of the sugar, specifically at the 3' position of the sugar on the 3' terminal nucleotide, or in oligonucleotides linked at 2'-5', and at the 5' position of the 5' terminal nucleotide as a target cell. Modified sugars can also include those containing modified sugars at the bridging epoxy, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimics, such as a cyclobutyl moiety, instead of furanylpentose. Numerous US patents disclose the preparation of such modified sugar structures and describe in detail a series of base modifications, such as US patent numbers 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, and 5,650. 8,873, 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 these patents is incorporated herein by reference in its entirety.

[0399] Examples of nucleic acids having modified sugar moieties include, but are not limited to, nucleic acids comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position may also be selected from allyl, amino, azide, thio, O-allyl, O-(C1-C)... 1O Alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R) m (R) n) and O-CH2-C(=O)-N(R m (R) n ), where each R m and R n Independently, it is H or substituted or unsubstituted C1-C 10 alkyl.

[0400] In some embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In some such embodiments, the bicyclic nucleic acid comprises a bridge between a 4' ribosyl ring atom and a 2' ribosyl ring atom. In some embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge comprises 4' to 2' bicyclic nucleic acids. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, one of the following: 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 their analogues (see U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' and their analogues (see WO 2009 / 006478; WO 2008 / 150729; US 2004 / 0171570; U.S. Patent No. 7,427,672; Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134 and WO 2008 / 154401).See also, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahllestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007. 129(26)8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs [Modern Research Drug Opinions], 2001, 2, 558-561; Braasch et al., Chem. Biol [Biochemistry], 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther. [Modern Molecular Therapy Opinions], 2001, 3, 239-243; US 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 Publications WO 2004 / 106356, WO 1994 / 14226, WO2005 / 021570, WO 2007 / 090071, and WO 2007 / 134181; US ​​Patent Publications US 2004 / 0171570, US2007 / 0287831, and US 2008 / 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 Application Nos. PCT / US2008 / 064591, PCT US2008 / 066154, PCT US2008 / 068922 and PCT / DK98 / 00393.

[0401] In some embodiments, the nucleic acid includes linked nucleic acids. Nucleic acids can be linked together using any inter-nucleic acid bond. Two main categories of inter-nucleic acid linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing inter-nucleic acid bonds include, but are not limited to, phosphodiesters, triphosphates, methylphosphonates, phosphatases, and thiophosphates (P=S). Representative non-phosphophosphate linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thiocarbamate (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N -Dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In some embodiments, the internucleotide bonds having chiral atoms can be prepared as racemic mixtures, as individual enantiomers, such as alkylphosphonates and thiophosphates. Non-natural nucleic acids may contain a single modification. Non-natural nucleic acids may contain multiple modifications within one part or between different parts.

[0402] Phosphate modification of the main chain of nucleic acids includes, but is not limited to, methylphosphonates, thiophosphates, phosphatases (bridging or non-bridging), triphosphates, dithiophosphates, dithiophosphates, and borophosphates, and can be used in any combination. Other non-phosphate bonds can also be used.

[0403] In some embodiments, main chain modifications (e.g., methylphosphonates, thiophosphates, phosphatases, and dithiophosphate nucleotide bonds) can confer immunomodulatory activity and / or enhance the stability of modified nucleic acids in vivo.

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

[0405] In some embodiments, backbone modification includes replacing phosphodiester bonds with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include: anionic nucleoside internucleotide bonds; N3' to P5' phosphoamide modifications; boron phosphate DNA; pre-oligonucleotides; neutral nucleoside internucleotide bonds, such as methylphosphonates; amide-linked DNA; methylene (methylimino) bonds; methyl acetal and thiomethyl acetal bonds; backbones containing sulfonyl groups; morpholine oligomers; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic acid guanidine (DNG) oligomers (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). Modified nucleic acids may comprise chimeric or hybrid backbones containing one or more modifications, such as combinations of phosphodiester bonds, such as combinations of phosphodiester and thiophosphate bonds.

[0406] Substituents in phosphate esters include, for example, short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatom and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatom or heterocyclic nucleoside bonds. These include those having: morpholine bonds (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; aminosulfonic acid backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 components. Numerous U.S. patents disclose how to prepare and use these types of phosphate ester substitutes, and include, but are not limited to, U.S. patent numbers 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, and 5,479. 0,967, 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 should be understood that in nucleotide substituents, both the sugar and phosphate ester moieties of the nucleotide can be replaced, for example, by an amide bond (aminoethylglycine) (PNA). U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262 teach how to prepare and use PNA molecules, each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), bile acids (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), and thioethers such as hexyl-S-triphenylmethanethiol (Manoharan et al., Ann. KY. Acad. Sci.).[Annals of the New York Academy of Sciences], 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. [Bioorganic Chemistry and Medicinal Chemistry Communications], 1993, 3, 2765-2770), thioglycolic acid (Oberhauser et al., Nucl. Acids Res. [Nucleic Acid Research], 1992, 20, 533-538), fatty chains such as dodecyldiol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. [FEBS Communications], 1990, 259, 327-330; Svinarchuk et al., Biochimie [Biochemistry], 1993, 75, 49-54), phospholipids such as dihexadecyl-rac-glycerol or l-di-O-hexadecyl-rac-glycerol-SH-phosphonate triethylammonium (Manoharan et al., Tetrahedron Lett. [Tetrahedron Communications], 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res. [Nucleic Acid Research], 1990, 18, 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides [Nucleosides and Nucleotides], 1995, 14, 969-973), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett. [Tetrahedron Communications], 1995, 36, 3651-3654), palmitoyl moiety (Mishra et al., Biochem. Biophys. Acta [Chinese Journal of Biochemistry and Biophysics]), 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxocholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.).[Journal of Pharmacology and Experimental Therapeutics], 1996, 277, 923-937). Numerous US patents have taught the preparation of such conjugates, including but not limited to US patent numbers 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, and 5,11. 8,802, 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, 5,39 1,723, 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 non-natural nucleic acid further forms non-natural base pairs. Exemplary non-natural nucleotides capable of forming non-natural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and any combination thereof. Other examples of non-natural nucleotides capable of forming non-natural UBPs and used to prepare the IL-2 conjugates disclosed herein can 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. [Journal of the American Chemical Society] 2013, 135:5408-5419; and Malyshev et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 2012, 109:12005-12010. In some embodiments, non-natural nucleotides include:

[0408] In some embodiments, the non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be derived from compounds having the following formula. R2 is selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide; and The wavy line indicates a bond with a ribosyl or 2'-deoxyribosyl group, wherein the 5'-hydroxyl group of the ribosyl or 2'-deoxyribosyl moiety is in free form, or optionally bonded to a monophosphate, diphosphate, or triphosphate group.

[0409] In some embodiments, the non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be derived from , , , , and In some embodiments, the non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein include , , , , and Or its salt.

[0410] In some embodiments, non-natural base pairs generate non-natural amino acids, as described in Dumas et al., “Designing logical codon reassignment - Expanding the chemistry in biology”, Chemical Science, 6:50-69 (2015).

[0411] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via synthetic codons comprising non-natural nucleic acids. In some embodiments, non-natural amino acids are incorporated into cytokines via orthogonal, modified synthase / tRNA pairs. Such orthogonal pairs comprise non-natural synthases capable of providing non-natural amino acids to non-natural tRNAs while minimizing: a) the incorporation of other endogenous amino acids into non-natural tRNAs, and b) the incorporation of non-natural amino acids into other endogenous tRNAs. Such orthogonal pairs comprise tRNAs capable of being provided by non-natural synthases while avoiding the incorporation of a) other endogenous amino acids by endogenous synthases. In some embodiments, such pairs have been identified from various organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, orthogonal synthase / tRNA pairs comprise components from a single organism. In some embodiments, orthogonal synthase / tRNA pairs comprise components from two different organisms. In some embodiments, orthogonal synthase / tRNA pairs comprise components that promote the translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthase is a modified alanine synthase. In some embodiments, the orthogonal synthase is a modified arginine synthase. In some embodiments, the orthogonal synthase is a modified asparagine synthase. In some embodiments, the orthogonal synthase is a modified aspartate synthase. In some embodiments, the orthogonal synthase is a modified cysteine ​​synthase. In some embodiments, the orthogonal synthase is a modified glutamine synthase. In some embodiments, the orthogonal synthase is a modified glutamate synthase. In some embodiments, the orthogonal synthase is a modified alanine-glycine synthase. In some embodiments, the orthogonal synthase is a modified histidine synthase. In some embodiments, the orthogonal synthase is a modified leucine synthase. In some embodiments, the orthogonal synthase is a modified isoleucine synthase. In some embodiments, the orthogonal synthase is a modified lysine synthase. In some embodiments, the orthogonal synthase is a modified methionine synthase. In some embodiments, the orthogonal synthase is a modified phenylalanine synthase. In some embodiments, the orthogonal synthase is a modified proline synthase. In some embodiments, the orthogonal synthase is a modified serine synthase. In some embodiments, the orthogonal synthase is a modified threonine synthase. In some embodiments, the orthogonal synthase is a modified tryptophan synthase. In some embodiments, the orthogonal synthase is a modified tyrosine synthase. In some embodiments, the orthogonal synthase is a modified valine synthase. In some embodiments, the orthogonal synthase is a modified phosphoserine synthase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA.In some embodiments, the orthogonal tRNA is a modified aspartic tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine ​​tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine-glycine tRNA. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

[0412] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanococcus japonicus (Mj-Tyr) aaRS / tRNA pair and the Escherichia coli TyrRS (Ec-Tyr) / Bacillus stearothermophilus tRNA pair. CUA P.E. coli LeuRS (Ec-Leu) / Bacillus stearothermophilus tRNA CUA Pyrrololysyl-tRNA pairs, and pyrrololysyl-tRNA pairs. In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via Mj-TyrRS / tRNA pairs. Exemplary UAAs that can be incorporated via Mj-TyrRS / tRNA pairs include, but are not limited to, para-substituted phenylalanine derivatives, such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives, such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronphenylalanine; and o-nitrobenzyltyrosine.

[0413] In some embodiments, non-natural amino acids are transmitted via Ec-Tyr / tRNA. CUA or Ec-Leu / tRNACUA It is incorporated into cytokines (e.g., IL peptides). This can be achieved via Ec-Tyr / tRNA. CUA or Ec-Leu / tRNA CUA The exemplary UAAs incorporated include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide or azide substituents; o-propargyltyrosine; α-aminooctanoic acid, o-methyltyrosine, o-nitrobenzylcysteine; and 3-(naphthyl-2-ylamino)-2-amino-propionic acid.

[0414] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL peptides) via pyrrololysyl-tRNA pairs. In some embodiments, PylRS is obtained from archaea, for example, from methanogenic archaea. In some embodiments, PylRS is obtained from *Methanococcus pastoris*, *Methanococcus martensii*, or *Methanococcus acetate*. Exemplary UAAs that can be incorporated via pyrrololysyl-tRNA pairs include, but are not limited to, amide and carbamate-substituted lysines, such as 2-amino-6-((R)-tetrahydrofuran-2-carbamoyl)hexanoic acid, N-ε- D -prolyl- L -Lysine, and N-ε-cyclopentyloxycarbonyl- L -Lysine; N-ε-Acryloyl- L -Lysine; N-ε-[(1-(6-nitrobenzene[d][1,3]dioxacyclopenten-5-yl)ethoxy)carbonyl]- L -lysine; and N-ε-(1-methylcycloprop-2-enecarboxamide)lysine. In some embodiments, the IL-2 conjugates disclosed herein can be prepared by using *Methanococcus martensii* tRNA, which is selectively supplied with non-natural amino acids, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), by *Methanococcus pasteurella* pyrrololysyl-tRNA synthetase (Mb PylRS). Other methods are known to those skilled in the art, such as those described below: Zhang et al., Nature [Nature] 2017, 551(7682): 644-647.

[0415] In some embodiments, non-natural amino acids are incorporated into the cytokines (e.g., IL peptides) described herein using synthases disclosed in US 9,988,619 and US 9,938,516.

[0416] Host cells in which the constructs or vectors disclosed herein are introduced are cultured or maintained in a suitable culture medium to produce tRNA, tRNA synthetase, and the target protein. The culture medium also contains one or more non-natural amino acids, allowing the target protein to incorporate one or more non-natural amino acids. In some embodiments, nucleoside triphosphate transporters (NTTs) from bacteria, plants, or algae are also present in the host cells. In some embodiments, the IL-2 conjugates disclosed herein are prepared using host cells expressing NTTs. 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 (Pseudomicroalgae), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (Phaeodactylum tricornutum), GsNTT (sulfophilic primitive red algae), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis), PamNTT1, PamNTT2 (Chlamydia trachomatis), CcNTT (Caedibacter caryophilus), and RpNTT1 (Rickettsia prowleri). In some embodiments, 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 [Nature] 2017, 551(7682): 644-647; Malyshev et al., Nature [Nature] 2014 (509(7500), 385-388; and Zhang et al., Proc NatlAcad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 2017, 114:1317-1322.

[0417] Orthogonal tRNA synthetase / tRNA pairs provide non-natural amino acids to tRNA and incorporate these amino acids into the polypeptide chain in response to a codon. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair and the Escherichia coli TyrRS (Ec-Tyr) / Bacillus stearothermophilus tRNA pair.CUA P.E. coli LeuRS (Ec-Leu) / Bacillus stearothermophilus tRNA CUA The aaRS-tRNA pairs, and pyrrololysyl-tRNA pairs. Other aaRS-tRNA pairs that may be used according to this disclosure include those derived from *Methanococcus martensii*, described in: Feldman et al., *J Am Chem Soc.*, 2018 140:1447-1454; and Zhang et al., *Proceedings of the National Academy of Sciences of the United States of America*, 2017, 114:1317-1322.

[0418] In some embodiments, methods are provided for preparing the IL-2 conjugates disclosed herein in a cell system expressing NTT and tRNA synthetase. In some embodiments described herein, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from *Methanococcus japonicus*, *Escherichia coli* TyrRS (Ec-Tyr) / *Bacillus stearothermophilus*, and *Methanococcus martensii*. In some embodiments, the NTT is PtNTT1, and the tRNA synthetase is derived from *Methanococcus japonicus*, *Escherichia coli* TyrRS (Ec-Tyr) / *Bacillus stearothermophilus*, or *Methanococcus martensii*. In some embodiments, the NTT is PtNTT2, and the tRNA synthetase is derived from *Methanococcus japonicus*, *Escherichia coli* TyrRS (Ec-Tyr) / *Bacillus stearothermophilus*, or *Methanococcus martensii*. In some embodiments, the NTT is PtNTT3, and the tRNA synthetase is derived from *Methanococcus japonicus*, *Escherichia coli TyrRS (Ec-Tyr)* / *Bacillus stearothermophilus*, or *Methanococcus martensii*. In some embodiments, the NTT is PtNTT3, and the tRNA synthetase is derived from *Methanococcus japonicus*, *Escherichia coli TyrRS (Ec-Tyr)* / *Bacillus stearothermophilus*, or *Methanococcus martensii*. In some embodiments, the NTT is PtNTT4, and the tRNA synthetase is derived from *Methanococcus japonicus*, *Escherichia coli TyrRS (Ec-Tyr)* / *Bacillus stearothermophilus*, or *Methanococcus martensii*. In some embodiments, the NTT is PtNTT5, and the tRNA synthetase is derived from *Methanococcus japonicus*, *Escherichia coli TyrRS (Ec-Tyr)* / *Bacillus stearothermophilus*, or *Methanococcus martensii*. In some embodiments, NTT is PtNTT6, and the tRNA synthetase is derived from Methanococcus jannulatae, Escherichia coli TyrRS (Ec-Tyr) / Bacillus stearothermophilus, or Methanococcus martensii.

[0419] In some embodiments, the IL-2 conjugates disclosed herein can be prepared in cells, such as *Escherichia coli*, comprising (a) the nucleoside triphosphate transporter PtNTT2 (including truncated variants in which the first 65 amino acid residues of the full-length protein are missing), (b) a plasmid containing a double-stranded oligonucleotide encoding an IL-2 variant having a desired amino acid sequence and containing a non-natural base pair comprising a first and a second non-natural nucleotide to provide a codon at the desired position where a non-natural amino acid, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), (c) a plasmid encoding tRNA derived from *Methanococcus martensii* and containing a non-natural nucleotide to provide a recognized anticodon (the codon for the IL-2 variant) in place of its native sequence, and (d) a plasmid encoding pyrrolidone-lysyl-tRNA synthetase (Mb) derived from *Methanococcus martensii*. The plasmid (PylRS) can be the same plasmid encoding tRNA or a different plasmid. In some embodiments, the cell is further supplemented with a deoxyribonucleotide triphosphate containing one or more non-natural bases. In some embodiments, the cell is further supplemented with a ribonucleotide triphosphate containing one or more non-natural bases. In some embodiments, the cell is further supplemented with one or more non-natural amino acids, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the double-stranded oligonucleotide encoding the amino acid sequence of the desired IL-2 variant contains the codon AXC at positions 8, 11, 14, 15, 18, 19, 22, 87, 99, or 108 of the sequence encoding, for example, the protein having SEQ ID NO: 1 (aldeleukin), where X is a non-natural nucleotide. In some embodiments, the cell further comprises a plasmid, which may be a protein expression plasmid or another plasmid encoding an orthogonal tRNA gene from *Methanococcus masculinii*, containing an AXC-matched anticodon GYT in place of its native sequence, where Y is a complementary non-natural nucleotide and may be the same as or different from the non-natural nucleotide in the codon. In some embodiments, the non-natural nucleotide in the codon is different from and complementary to the non-natural nucleotide in the anticodon. In some embodiments, the non-natural nucleotide in the codon is the same as the non-natural nucleotide in the anticodon. In some embodiments, the first and second non-natural nucleotides of the non-natural base pairs in the double-stranded oligonucleotide may be derived from... , and In some embodiments, the first and second non-natural nucleotides of the non-natural base pairs in the double-stranded oligonucleotide may be derived from... and In some embodiments, the triphosphates of the first and second non-natural nucleotides include , and Or its salts. In some embodiments, the triphosphates of the first and second non-natural nucleotides include and Or its salts. In some embodiments, the mRNA-derived double-stranded oligonucleotide comprising the first and second non-natural nucleotides may contain a codon containing a derivative derived from... , and Non-natural nucleotides. In some embodiments, *Methanococcus martensii* tRNA may contain an anticodon that includes a non-natural nucleotide that recognizes a codon containing a non-natural nucleotide of mRNA. The anticodon in *Methanococcus martensii* tRNA may contain non-natural nucleotides derived from... , and Non-natural nucleotides. In some embodiments, the mRNA contains non-natural nucleotides derived from... Non-natural nucleotides. In some embodiments, the mRNA contains non-natural nucleotides derived from... Non-natural nucleotides. In some embodiments, the mRNA contains non-natural nucleotides derived from... Non-natural nucleotides. In some embodiments, the tRNA contains non-natural nucleotides derived from... Non-natural nucleotides. In some embodiments, the tRNA contains non-natural nucleotides derived from... Non-natural nucleotides. In some embodiments, the tRNA contains non-natural nucleotides derived from... Non-natural nucleotides. In some embodiments, the mRNA contains non-natural nucleotides derived from... The tRNA contains non-natural nucleotides, and the tRNA contains nucleotides derived from Non-natural nucleotides. In some embodiments, the mRNA contains non-natural nucleotides derived from... The tRNA contains non-natural nucleotides, and the tRNA contains nucleotides derived from The host cells were cultured in a medium containing appropriate nutrients and supplemented with (a) triphosphates of deoxyribonucleosides containing one or more non-natural bases, which are necessary for the replication of one or more plasmids encoding codon-carrying cytokine genes; (b) triphosphates of ribonucleosides containing one or more non-natural bases, which are necessary for the transcription of (i) mRNA corresponding to the coding sequence of the cytokine and containing codons contain...

Claims

1. A method of delivering a gene therapy agent to the cells of a subject, the method comprising administering an IL-2 conjugate to the subject, wherein the gene therapy agent is administered to the subject before, simultaneously with, or after the IL-2 conjugate, wherein the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate moiety, and the non-natural amino acid linked to the conjugate moiety is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or located in reference SEQ ID NO:

1. The positions of the sequence 1 are: 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, P 81, 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.

2. A method of treating an individual in need with a gene therapy agent, the method comprising administering an IL-2 conjugate to the subject, wherein the gene therapy agent is administered to the subject before, simultaneously with, or after the IL-2 conjugate, and the IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate moiety, and the non-natural amino acid linked to the conjugate moiety is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or located in reference SEQ ID NO:

1. The positions of the sequence 1 are: 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, P 81, 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.

3. A method for increasing the expression of a gene therapy agent, the method comprising: IL-2 conjugates were administered to the subjects; The gene therapy agent was administered to the subject before, simultaneously with, or after the IL-2 conjugate, and The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

4. A method for reducing the immune response to a gene therapy agent, the method comprising: IL-2 conjugates were administered to the subjects; The gene therapy agent was administered to the subject before, simultaneously with, or after the IL-2 conjugate, and The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

5. A method for preventing immune-related adverse events in subjects, the method comprising: IL-2 conjugates were administered to the subjects; The gene therapy agent was administered to the subject before, simultaneously with, or after the IL-2 conjugate, and The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

6. The method of any of the preceding claims, wherein the method further comprises, prior to administering 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 expression of one or more activated biomarkers or an increase in the expression of one or more activated biomarkers in these immune cells, wherein the expression or increase in the expression of the one or more activated biomarkers after incubation with the gene therapy agent identifies the subject as requiring the IL-2 conjugate.

7. A method for selecting a subject to be treated 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 expression of one or more activated biomarkers or an increase in the expression of one or more activated biomarkers in the immune cells, wherein the expression or increase in the expression of the one or more activated biomarkers after incubation with the gene therapy agent identifies the subject to be treated with the gene therapy agent and the IL-2 conjugate; and c) selecting the subject to be treated with the gene therapy agent and the IL-2 conjugate identified in step b). The IL-2 conjugate comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a non-natural amino acid linked to the conjugate, and the non-natural amino acid linked to the conjugate is located in the amino acid sequence to preferentially reduce the binding of the IL-2 conjugate to IL-2Rβγ relative to IL-2Rαβγ, or is located in the reference SEQ ID NO: The positions of the sequence 1 are: 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, P 81, 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.

8. The method of claim 1, further comprising 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 to 8, wherein the immune cell is a lymphocyte, T cell, CD8+ T cell, effector T cell, cytotoxic T cell or NK cell.

10. The method as described in any of the preceding claims, wherein the non-natural amino acid is connected to the conjugate via a linker.

11. The method of claim 1, wherein the connector comprises a homobifunctional connector, a heterobifunctional connector, a cleavable or uncleavable dipeptide connector, a maleimide group, a spacer, or a combination thereof.

12. The method of any of the preceding claims, wherein the non-natural amino acid is a substituted lysine, a substituted phenylalanine, a substituted histidine, a substituted cysteine, contains an azide group, contains an alkynyl group, contains an aldehyde group, contains an aromatic side chain, or contains a ketone group.

13. The method of any one of the preceding claims, wherein the at least one non-natural amino acid comprises N6-azidoethoxy-L-lysine, N6-((2-azidoethoxy)-carbonyl)-L-lysine, N6-propynylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetraazine lysine, allyloxycarbonyl lysine, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, p-propynyloxyphenylalanine, p-propynyl-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)seleno)propionic acid or 2-amino-3-(phenylseleno)propionic acid.

14. The method of any of the preceding claims, wherein the non-natural amino acid is azide-substituted lysine.

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

16. The method of any of the preceding claims, wherein the conjugated portion comprises a water-soluble polymer.

17. The method of the 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(enol), poly(vinylpyrrolidone), poly(hydroxyalkyl methylacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or combinations thereof.

18. The method of claim 1, wherein the conjugation portion comprises PEG.

19. The method of the preceding claim, wherein the conjugated portion is PEG with a molecular weight of about 10-85 kDa or selected from about 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa and 85 kDa.

20. The method as claimed in any of the preceding claims, wherein the conjugated portion is PEG with a molecular weight of about 20-70 kDa or selected from about 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa and 70 kDa.

21. The method as claimed in any of the preceding claims, wherein the conjugated portion is PEG with a molecular weight of about 30-60 kDa or selected from about 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa and 60 kDa.

22. The method as claimed in any of the preceding claims, wherein the amino acid linked to the conjugation portion has the structure of formula (I): Formula (I); in: Z is CH2 and Y is ; Y is CH2 and Z is ; Z is CH2 and Y is ;or Y is CH2 and Z is ; W is a PEG group; and X has the following structure: ； X-1 indicates the attachment site to the preceding amino acid residue; and X+1 indicates the attachment site to the next amino acid residue.

23. The method as claimed in the preceding claim, wherein Z is CH2 and Y is Or Y is CH2 and Z is .

24. The method as claimed in the preceding claim, wherein the structure of formula (I) has the structure of formula (IV) or formula (V): Formula (IV); Formula (V); in: W is the PEG group, with a molecular weight of approximately 5-60 kDa or approximately 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa or 60 kDa.

25. The method of any of the preceding claims, wherein the IL-2 conjugate further comprises an alanine or methionine at the N-terminus of the first amino acid of the sequence having at least 80% sequence identity with SEQ ID NO:

1.

26. The method of any 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 with SEQ ID NO:

1.

27. The method of any of the preceding claims, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the positions 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 are replaced by this non-natural amino acid.

28. The method as described in any of the preceding claims, wherein positions K8, L11, E14, H15, L18, D19, M22, N87, E99, or D108 of the sequence referenced to SEQ ID NO: 1 are replaced by the non-natural amino acid.

29. The method of the preceding claim, wherein position L18 of the sequence referring to SEQ ID NO: 1 is replaced by the non-natural amino acid.

30. The method of claim 28, wherein position H15 of the sequence referring to SEQ ID NO: 1 is replaced by the non-natural amino acid.

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

32. The method of any of the preceding claims, wherein the non-natural amino acid and / or the conjugated portion impairs or blocks the receptor signaling efficacy of the IL-2 conjugate for IL-2Rβγ, or reduces the recruitment of the IL-2Rγ subunit to the IL-2 / IL-2Rβ complex.

33. The method as described in any of the preceding claims, wherein the IL-2 conjugate has lower receptor signaling efficacy for IL-2Rβγ than wild-type IL-2 has.

34. The method as claimed in any of the preceding claims, wherein the receptor signaling efficacy of the IL-2 conjugate to IL-2Rαβγ is greater than or equal to the receptor signaling efficacy of wild-type IL-2 to IL-2Rαβγ.

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

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

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

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

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

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

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

42. The method of any of the preceding claims, wherein the gene therapy agent encodes a transgenic product, and the IL-2 conjugate prolongs the expression of the transgenic product in a subject who is given the gene therapy agent but not the IL-2 conjugate.

43. The method of the preceding claim, wherein the extended expression of the transgenic product is for 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 of the preceding claims, wherein the gene therapy agent comprises a viral vector.

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

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

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

48. The method of the 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 AAV2N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, and an AAV... V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid rAAV2 / HBoV1 (chimeric AAV / human bocavirus type 1), AAV2HBKO capsid, AAVPHP.B capsid or AAVPHP.eB capsid or functional variants thereof.

49. The method of the preceding claim, wherein the AAV capsid comprises a tyrosine mutation, a heparin-binding mutation, or an HBKO mutation.

50. The method of any one of claims 47 to 49, wherein the AAV viral particle comprises an AAV genome containing one or more inverted terminal repeats (ITRs), wherein the one or more ITRs are AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR, AAVrh10 ITR, AAV11 ITR, or AAV12 ITR.

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

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

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

54. The method of the preceding claim, wherein the adenovirus particle comprises a capsid from adenovirus serotypes 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, AdHu3, AdHu4, AdHu24, AdHu26, AdHu34, AdHu35, AdHu36, AdHu37, AdHu41, AdHu48, AdHu49, AdHu50, AdC6, AdC7, AdC69, bovine Ad 3, canine Ad 2, sheep Ad, or swine Ad 3, or functional variants thereof.

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

56. The method of the preceding claim, wherein the lentiviral particle is pseudotyped with vesicular stomatitis virus (VSV), lymphocytic choriomeningovirus (LCMV), Ross River virus (RRV), Ebola virus, Marburg virus, Mokola virus, rabies virus, RD114, or a functional variant thereof.

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

58. The method of the 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 to 41, wherein the gene therapy agent comprises lipid nanoparticles.

60. The method of any of the preceding claims, wherein the gene therapy agent comprises a nucleic acid encoding a heterologous transgene.

61. The method of claim 1, wherein the heterologous transgene is operatively linked to a promoter.

62. The method of the 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 to 62, wherein the nucleic acid comprises terminally closed DNA (ceDNA).

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

65. The method of any of the preceding claims, wherein the gene therapy agent and the IL-2 conjugate are administered simultaneously to the subject.

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

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

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

69. The method of the 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 to 64, wherein the IL-2 conjugate is administered before, simultaneously with, or after the administration of the gene therapy agent.

71. The method as described in any of the preceding claims, wherein the individual suffers from a disease or condition suitable for treatment by gene therapy.

72. The method of the preceding claim, wherein the disease or condition is a single-gene disease or condition.

73. The method of any of the preceding claims, wherein the gene therapy agent is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, intracranially, intracerebral sac, intradurally, intraocularly, intravenously, intraocularly, intracerebellomedullary cistern, or intrahepatically.

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

75. The method of any of the preceding claims, wherein the IL-2 conjugate is administered intravenously, intraperitoneally, intraarterially, intramuscularly, subcutaneously, intracranially, intracerebral sac, intradurally, intraocularly, intracerebellomedullary cistern, or intrahepatically.

76. The method as described in any of the preceding claims, wherein the subject is a mammal.

77. The method as described in any of the preceding claims, wherein the subject is a primate.

78. The method as described in any of the preceding claims, wherein the subject is a human.

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

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

82. The method of any 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.

83. The method of claim 1, 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 IL-2 conjugates in the preparation of a medicament for use in the method as described in any one of the preceding claims.

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

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