Immuno-oncology combination therapy using IL-2 conjugates

Abstract

Disclosed herein are compositions, kits, and methods comprising interleukin (IL) conjugates (e.g., IL-2 conjugates) in combination with other agents or methods useful for the treatment of one or more indications, e.g., the treatment of proliferative disorders. Also described herein are pharmaceutical compositions and kits comprising one or more of the interleukin conjugates (e.g., IL-2 conjugates).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 887,400, filed August 15, 2019, U.S. Provisional Patent Application No. 62 / 903,187, filed September 20, 2019, and U.S. Provisional Patent Application No. 62 / 962,668, filed January 17, 2020, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on August 12, 2020, is named 2020-08-12_01183-0073-00PCT_seq_listing.txt, and is 128,000 bytes in size. [Background technology]

[0003] Different populations of T cells regulate the immune system to maintain immune homeostasis and immune tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological autoreactivity, while cytotoxic T cells target and destroy infected cells and / or cancerous cells. In some cases, the regulation of different populations of T cells provides options for the treatment of diseases or symptoms. In some cases, this is benefited by the presence of additional drugs or methods in combination therapy.

[0004] Thus, in one aspect, provided herein is a method of treating cancer in a subject, the method comprising administering to the subject an IL-2 conjugate in combination with one or more immune checkpoint inhibitors. Summary of the Invention [Means for solving the problem]

[0005] In certain embodiments, methods of treating cancer are described herein. The following embodiments are included:

[0006] Embodiment A1 is a method of treating cancer in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more immune checkpoint inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate has the structure of Formula (I): [ka] (In the formula: Z is CH2 and Y is [ka] and; Y is CH2 and Z is [ka] and; Z is CH2 and Y is [ka] is; or Y is CH2 and Z is [ka] and; 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, and 60 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) has been replaced by; The method, wherein the position of the structure of formula (I) in SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71.

[0007] Embodiment A2 is an IL-2 conjugate wherein Z is CH2 and Y is [ka] The method of embodiment A1, wherein

[0008] Embodiment A3 is an IL-2 conjugate wherein Y is CH2 and Z is [ka] The method of embodiment A1, wherein

[0009] Embodiment A4 is an IL-2 conjugate wherein Z is CH2 and Y is [ka] The method of embodiment A1, wherein

[0010] Embodiment A5 is an IL-2 conjugate wherein Y is CH2 and Z is [ka] The method of embodiment A1, wherein

[0011] Embodiment A6 is the method of any one of embodiments A1 to A5, wherein in the IL-2 conjugate, the PEG groups have an average molecular weight of 25 kDa, 30 kDa, or 35 kDa.

[0012] Embodiment A7 is the method of embodiment A6, wherein in the IL-2 conjugate, the PEG group has an average molecular weight of 30 kDa.

[0013] Embodiment A8 is the method of any one of embodiments A1 to A7, wherein in the IL-2 conjugate, the position of the structure of Formula (I) in SEQ ID NO: 3 is P64.

[0014] Embodiment A9 is an embodiment in which the structure of formula (I) has the structure of formula (X) or formula (XI), or a mixture of formula (X) and formula (XI): [ka] (In the formula: n is an integer ranging from about 2 to about 5000; The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 3 that is not replaced. The method of embodiment A1, wherein

[0015] Embodiment A10 is the method of embodiment A9, wherein in the IL-2 conjugate, the position of the structure of formula (X) or formula (XI) in SEQ ID NO: 3 is P64.

[0016] Embodiment A11 is an IL-2 conjugate wherein n is —(OCH2CH2) n The method of embodiment A9 or A10, wherein —OCH 3 is an integer such that the compound has a molecular weight of about 25 kDa, 30 kDa, or 35 kDa.

[0017] Embodiment A12 is an IL-2 conjugate wherein n is —(OCH2CH2) n The method of embodiment A11, wherein —OCH 3 is an integer such that the compound has a molecular weight of about 30 kDa.

[0018] Embodiment A13 is an embodiment in which the structure of formula (I) has the structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII): [ka] (In the formula: n is an integer ranging from about 2 to about 5000; The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 3 that is not replaced. The method of embodiment A1, wherein

[0019] Embodiment A14 is the method of embodiment A13, wherein in the IL-2 conjugate, the position of the structure of formula (XII) or formula (XIII) in SEQ ID NO: 3 is P64.

[0020] Embodiment A15 is an IL-2 conjugate wherein n is —(OCH2CH2) n The method of embodiment A13 or A14, wherein —OCH 3 is an integer such that the compound has a molecular weight of about 25 kDa, 30 kDa, or 35 kDa.

[0021] Embodiment A16 is an IL-2 conjugate wherein n is —(OCH2CH2) n The method of embodiment A15, wherein —OCH3 is an integer such that the compound has a molecular weight of about 30 kDa.

[0022] Embodiment A17 is a method of treating cancer in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more immune checkpoint inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 50, and [AzK_L1_PEG30kD] has the structure of Formula (IV) or Formula (V), or a mixture of the structures of Formula (IV) and Formula (V): [ka] (In the formula: 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, and 60 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) The method is as follows:

[0023] Embodiment A18 is the method of embodiment A17 wherein W is a PEG group having an average molecular weight selected from 25 kDa, 30 kDa, or 35 kDa.

[0024] Embodiment A19 is the method of embodiment A18, wherein W is a PEG group having an average molecular weight of 30 kDa.

[0025] Embodiment A20 is a method of treating cancer in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more immune checkpoint inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 50, and [AzK_L1_PEG30kD] has the structure of Formula (XII) or Formula (XIII), or a mixture of the structures of Formula (XII) and Formula (XIII): [ka] (In the formula: n is -(OCH2CH2) n -OCH3 is an integer such that it has a molecular weight of about 30 kDa; The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 50 that is not replaced. The method is as follows:

[0026] Embodiment A21 is the method of any one of embodiments A1 to A20, wherein the one or more immune checkpoint inhibitors are one or more PD-1 inhibitors.

[0027] Embodiment A22 is the method of embodiment A21, wherein the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, and cemiplimab.

[0028] Embodiment A23 is the method of embodiment A22, wherein the one or more PD-1 inhibitors is pembrolizumab.

[0029] Embodiment A24 is the method of embodiment A22, wherein the one or more PD-1 inhibitors is nivolumab.

[0030] Embodiment A25 is a method for treating a cancer comprising administering to a patient a therapeutically effective amount of ... The method of any one of embodiments A1-A24, wherein the cancer is selected from breast cancer, prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer or metastatic castration-resistant prostate cancer with a DNA damage response (DDR) deficiency, bladder cancer, ovarian cancer, tumors with moderate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), tumors with low to no PD-L1 expression, tumors that have disseminated systemically beyond their primary anatomic site of origin to the liver and CNS, and diffuse large B-cell lymphoma.

[0031] Embodiment A26 is the method of any one of embodiments A1 to A25, wherein the IL-2 conjugate is administered to the subject once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks.

[0032] Embodiment A27 is the method of any one of embodiments A1 to A26, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

[0033] Embodiment A28 is the method of any one of embodiments A1 to A27, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0034] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0035] [Figure 1] 1 shows a graph of the antitumor activity of Compound A administered IV on a QWx3 schedule from Study 1 in Example 11. Black arrows indicate the days on which Compound A was administered. [Figure 2] 1 shows a graph of tumor volume for Compound A administered IV on a QWx3 schedule from Study 1 in Example 11. [Figure 3] Figure 1 shows the tumor volume at day 15 post-treatment for each animal treated with Compound A QW x 3 dosing from Study 1 in Example 11. Black arrows indicate the days on which Compound A was administered. [Figure 4] 1 shows the tumor volume at 15 days post-treatment in each animal dosed Q2W×2 with Compound A from Study 1 of Example 11. [Figure 5] Figure 1 shows the mean tumor growth curves from treatment of mice with vehicle, 6 mg / kg Compound A (as a single agent), anti-PD-1 antibody (as a single agent), and the combination of 6 mg / kg Compound A and anti-PD-1 antibody from Study 2 in Example 11. Black arrows indicate the days on which Compound A was administered. [Figure 6]Figure 1 shows a graph of TGI% data at day 15 post-treatment in groups treated with a combination of Compound A and anti-PD-1 antibody, compared to groups treated with vehicle, Compound A alone, or anti-PD-1 antibody alone, from Study 2 in Example 11. *p<0.05, **p<0.01, and ***p<0.01 vs. vehicle control. ┴p<0.05 vs. anti-PD-1 antibody. #p<0.05 vs. Compound A. Data represent mean tumor volume ± SEM (14 mice / group). [Figure 7] Figure 1 shows a graph of Kaplan-Meier survival curves for treatment groups from Study 2 in Example 11. *p<0.05 vs. vehicle control. ┴p<0.05 vs. anti-PD-1 antibody. #p<0.05 vs. Compound A. [Figure 8] Figure 1 shows the mean tumor growth curves when Compound A was administered as a single agent at 1 mg / kg, 3 mg / kg, 6 mg / kg, and 9 mg / kg in Study 3 of Example 11. Data represent mean tumor volume ± SEM (14 mice / group, except for Compound A at 9 mg / kg, which had 12 mice / group). Black arrows indicate the days of Compound A administration. [Figure 9] Figure 1 shows individual tumor volumes at 15 days post-treatment from Study 3 in Example 11. Data represent individual tumor volumes; mean ± SEM and % TGI compared to vehicle control are also shown. ***p<0.01 vs. vehicle control. [Figure 10] Figure 1 shows a graph of Kaplan-Meier survival curves for treatment groups treated with vehicle (control), anti-PD-1 antibody alone, Compound A alone, and the combination of Compound A and anti-PD-1 antibody. *p<0.05 vs. vehicle control from Study 3 in Example 11. ┴p<0.05 vs. anti-PD-1 antibody. #p<0.05 vs. Compound A. [Figure 11A]Figure 11A shows representative cytokine level graphs for IL-2 and IL-2_P65[AzK_L1_PEG30kD]-1 alone and in combination with nivolumab (Nivo) or pembrolizumab (Pem) for a single donor in Example 12. Figure 11A shows graphs of IFN-gamma, IL-8, IL-6, TNF-alpha, IL-4, and IL-5 levels. [Figure 11B] Figure 11B shows representative cytokine level graphs for IL-2 and IL-2_P65[AzK_L1_PEG30kD]-1 alone and in combination with nivolumab (Nivo) or pembrolizumab (Pem) for a single donor in Example 12. Figure 11B shows graphs of IL-6, TNF-alpha, and IL-5 levels. [Figure 12] 1 shows the release of interferon gamma in a mixed lymphocyte reaction (MLR) assay for the combination of Compound B (IL-2_P65[AzK_L1_PEG30kD]-1) and pembrolizumab according to Example 13. [Figure 13] 1 shows the release of interferon gamma in a mixed lymphocyte reaction (MLR) assay for the combination of Compound B (IL-2_P65[AzK_L1_PEG30kD]-1) and nivolumab according to Example 13. [Figure 14] 1 shows the release of interferon gamma in a mixed lymphocyte reaction (MLR) assay for the combination of Compound B (IL-2_P65[AzK_L1_PEG30kD]-1) and nivolumab according to Example 13. [Figure 15] 1 shows the pharmacokinetic properties of Compound B obtained from Example 14. [Figure 16A] 1 shows the amounts of pSTAT5+ cells in peripheral blood CD8+ T cells, CD8+ memory T cells, NK cells, and Treg cells, respectively, after administration of Compound B according to Example 14. [Figure 16B] 1 shows the amounts of pSTAT5+ cells in peripheral blood CD8+ T cells, CD8+ memory T cells, NK cells, and Treg cells, respectively, after administration of Compound B according to Example 14. [Figure 16C]1 shows the amounts of pSTAT5+ cells in peripheral blood CD8+ T cells, CD8+ memory T cells, NK cells, and Treg cells, respectively, after administration of Compound B according to Example 14. [Figure 16D] 1 shows the amounts of pSTAT5+ cells in peripheral blood CD8+ T cells, CD8+ memory T cells, NK cells, and Treg cells, respectively, after administration of Compound B according to Example 14. [Figure 17-1] 17A-17G show activation of Ki67 in CD8+ T, NK, and Treg cell populations by Compound B, according to Example 14. [Figure 17-2] Continuation of Figure 17-1. [Figure 17-3] Continued from Figure 17-2. [Figure 17-4] Continued from Figure 17-3. [Figure 18-1] 18A-18D show the analysis of tumor samples (CD8+ T cell, NK cell, and Treg cell levels, and CD8+ / Treg ratio) after treatment with Compound B according to Example 14. [Figure 18-2] Continuation of Figure 18-1. [Figure 19] 1 shows TCR diversity after treatment with Compound B and a murine anti-PD-1 antibody according to Example 15. [Figure 20] Figure 1 shows TIL clonality on T cell compartments following the indicated treatments (e.g., Compound B and / or murine anti-PD-1 antibody) according to Example 15. [Figure 21] FIG. 10 shows T cell clonality after treatment with Compound B compared to vehicle control according to Example 15. [Figure 22] Figure 1 shows expression heatmaps from day 8 CT26 tumor samples after treatment with control (vehicle), Compound B (6 mg / kg), murine anti-PD-1 (10 mg / kg), or a combination of Compound B and murine anti-PD-1 according to Example 16 (N=10 mice per group). [Figure 23]Figures 23A-23C show infiltration analysis of key expression reporters of tumor microenvironment status: activated CD8+ effector and effector memory T cells, and cytolytic NK cells after treatment with Compound B according to Example 16. CTL = control (vehicle); Cmpd B = Compound B; aPD1 = murine anti-PD-1 antibody; Cmpd B aPD1 = combination of Compound B and murine anti-PD-1 antibody. [Figure 24A] Figure 1 shows the profile analysis of interferon-gamma gene expression signature levels in response to treatment according to Example 16. CTL = control (vehicle); Cmpd B = compound B; aPD1 = mouse anti-PD-1 antibody; Cmpd B aPD1 = combination of compound B and mouse anti-PD-1 antibody. [Figure 24B] Figure 1 shows the profile analysis of interferon-gamma gene expression signature levels in response to treatment according to Example 16. CTL = control (vehicle); Cmpd B = compound B; aPD1 = mouse anti-PD-1 antibody; Cmpd B aPD1 = combination of compound B and mouse anti-PD-1 antibody. [Figure 25] 10 shows survival and tumor growth assessment in tumor-free animals re-challenged according to Example 17. [Figure 26] 10 shows survival and tumor growth assessment in tumor-free animals re-challenged according to Example 17. [Figure 27] 27A and 27B show that Compound B promotes a global increase in peripheral blood memory T cells (CD3+), including memory CD8+ T cells, in mice re-challenged with Example 17. DETAILED DESCRIPTION OF THE INVENTION

[0036] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter. To the extent that any of the material incorporated herein by reference conflicts with the statements of this disclosure, the statements will control, to that extent.

[0037] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "include," "includes," and "included," is not limiting.

[0038] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the invention, but not necessarily in all embodiments.

[0039] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 5 μL" means "about 5 μL" and also "5 μL." Typically, the term "about" includes amounts that would be expected to be within experimental error, e.g., within 15%, 10%, or 5%.

[0040] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0041] The term "or" is used in its inclusive equivalent of "and / or" unless the context clearly dictates otherwise.

[0042] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of the terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent) of a health care worker (e.g., a physician, registered nurse, nurse practitioner, physician's assistant, hospital worker, or hospice worker).

[0043] As used herein, the terms "substantial" or "substantial" with respect to binding affinity refer to a change in the binding affinity of a cytokine (e.g., an IL-2 polypeptide) sufficient to affect the binding of the cytokine (e.g., an IL-2 polypeptide) to a target receptor. In some examples, the term refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some examples, the term refers to a change of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more.

[0044] In some instances, the term "substantial" or "substantial," in reference to the activation of one or more cell populations via a cytokine signaling complex, refers to a change sufficient to activate the cell population. In some instances, the change that activates the cell population is measured as receptor signaling potency. In such instances, an EC50 value is provided. In other instances, an ED50 value is provided. In additional instances, a concentration or dosage of the cytokine is provided.

[0045] As used herein, the term "potency" refers to the amount of cytokine (e.g., IL-2 polypeptide) required to produce a target effect. In some instances, the term "potency" refers to the amount of cytokine (e.g., IL-2 polypeptide) required to activate a target cytokine receptor (e.g., IL-2 receptor). In other instances, the term "potency" refers to the amount of cytokine (e.g., IL-2 polypeptide) required to activate a target cell population. In some instances, potency is measured as the ED50 (50% effective dose), or the dose required to produce 50% of the maximum effect. In other cases, potency is measured as the EC50 (50% effective concentration), or the dose required to produce a target effect in 50% of the population.

[0046] As used herein, the term "unnatural amino acid" refers to an amino acid other than the 20 naturally occurring amino acids. Exemplary unnatural amino acids are described in Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry 285(15):11039-11044 (2010), the disclosure of which is incorporated herein by reference.

[0047] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen is EGFR.

[0048] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope. This does not include possible variant antibodies, e.g., containing naturally occurring mutations or mutations that arise during the production of a monoclonal antibody preparation, such as variants typically present in minor amounts. In contrast to polyclonal antibody preparations, which generally include different antibodies targeting different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation targets a single determinant on an antigen. Thus, the modifier "monoclonal" describes the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0049] As used herein, "nucleotide" refers to a compound comprising a nucleoside moiety and a phosphate moiety. Exemplary naturally occurring nucleotides include, but are not limited to, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), and guanosine monophosphate (GMP), deoxyadenosine triphosphate ( Deoxyribonucleotides include dATP, deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine diphosphate (dADP), thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP), and deoxyguanosine monophosphate (dGMP). Exemplary naturally occurring deoxyribonucleotides containing deoxyribose as the sugar moiety include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary naturally occurring ribonucleotides that contain ribose as the sugar moiety include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.

[0050] As used herein, "base" or "nucleobase" refers to at least the nucleobase portion of a nucleoside or nucleotide (nucleosides and nucleotides, including ribo- or deoxyribo-variants), and in some cases contains further modifications to the sugar portion of the nucleoside or nucleotide. In some cases, "base" is also used to refer to the entire nucleoside or nucleotide (e.g., a "base" can be incorporated into DNA by DNA polymerase or into RNA by RNA polymerase). However, the term "base" should not be interpreted as necessarily representing the entire nucleoside or nucleotide, unless required by context. In the chemical structures of bases or nucleobases provided herein, only the base of the nucleoside or nucleotide is shown, and for clarity, the sugar portion and, optionally, any phosphate residues are omitted. When used in the chemical structures of bases or nucleobases provided herein, a wavy line represents a nucleoside or nucleotide linkage in which the sugar portion of the nucleoside or nucleotide may be further modified. In some embodiments, the wavy line represents the linkage of a base or nucleobase to a sugar moiety of a nucleoside or nucleotide, such as a pentose. In some embodiments, the pentose is a ribose or deoxyribose.

[0051] In some embodiments, a nucleobase is generally the heterocyclic base moiety of a nucleoside. Nucleobases can be naturally occurring, but can also be modified, may not bear any similarity to natural bases, and / or may be synthesized, for example, by organic synthesis. In certain embodiments, a nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide, which atom or group of atoms can interact with a base of another nucleic acid with or without hydrogen bonding. In certain embodiments, a non-natural nucleobase is not derived from a natural nucleobase. It should be noted that non-natural nucleobases do not necessarily have basic properties, but are still referred to as nucleobases for brevity. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase can be linked to deoxyribose or ribose, while "d" without parentheses indicates that the nucleobase is linked to deoxyribose.

[0052] As used herein, a "nucleoside" is a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides with mimetic bases and / or sugars. Nucleosides include nucleosides containing any of a variety of substituents. A nucleoside may be a glycosidic compound formed through a glycosidic bond between a nucleobase and a reducing group of a sugar.

[0053] An "analog" of a chemical structure, as the term is used herein, refers to a chemical structure that may not be readily synthetically derived from the parent structure, but that maintains substantial similarity to the parent structure. In some embodiments, a nucleotide analog is a non-natural nucleotide. In some embodiments, a nucleoside analog is a non-natural nucleoside. Related chemical structures that are readily synthetically derived from the parent chemical structure are referred to as "derivatives."

[0054] While various features of the invention may be described in the context of a single embodiment, the features may also be provided individually or in any suitable combination. Conversely, although the invention may be described herein for clarity in the context of several separate embodiments, the invention may also be practiced in a single embodiment.

[0055] IL-2 conjugates Cytokines include a family of cell signaling proteins, such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors that play a role in innate and adaptive immune cell homeostasis. Cytokines are produced by immune cells, such as macrophages, B lymphocytes, T lymphocytes, and mast cells, endothelial cells, fibroblasts, and various stromal cells. In some cases, cytokines regulate the balance between humoral and cellular immune responses.

[0056] Interleukins are signaling proteins that regulate the development and differentiation of T and B lymphocytes, cells of the monocyte lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4 T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue residents.

[0057] Interleukin-2 (IL-2) is a pleiotropic type-1 cytokine whose structure contains a four-α-helical bundle of 15.5 kDa. The precursor of IL-2 is 153 amino acid residues in length, with the first 20 amino acids forming a signal peptide and the remaining 21–153 amino acids forming the mature form. IL-2 is produced primarily by CD4+ T cells after antigen stimulation, and to a lesser extent by CD8+ cells, natural killer (NK) cells, and natural killer T (NKT) cells, activated dendritic cells (DCs), and mast cells. IL-2 signaling occurs through interaction with a specific combination of IL-2 receptor (IL-2R) subunits: IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132). The interaction of IL-2 with IL-2Rα is approximately 10 -8 K of M d The interaction of IL-2 with IL-2Rβ and IL-2Rγ occurs at approximately 10 -9 K of M d The interaction of IL-2 with all three subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, occurs at approximately >10 -11 K of M d form a "high affinity" IL-2 receptor complex.

[0058] In some instances, IL-2 signaling through the "high affinity" IL-2Rαβγ complex regulates the activation and proliferation of regulatory T cells, or CD4 + CD25 + Foxp3 + Regulatory T (Treg) cells are effector cells, e.g., CD4 + T cells, CD8 +They mediate the maintenance of immune homeostasis by suppressing T cells, B cells, NK cells, and NKT cells. In some instances, Treg cells arise from the thymus (tTreg cells) or are derived from naive T cells in the periphery (pTreg cells). In some cases, Treg cells are considered mediators of peripheral tolerance. Indeed, one study showed that CD25-depleted peripheral CD4 + T cell transplantation induces various autoimmune diseases in nude mice, while CD4 + CD25 + Cotransplantation of T cells suppressed the development of autoimmunity (Sakaguchi, et al., "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)," J. Immunol. 155(3):1151-1164 (1995), the disclosure of which is incorporated herein by reference). Expanding the Treg cell population downregulates effector T cell proliferation and suppresses autoimmunity and T cell antitumor responses.

[0059] IL-2 signaling through the "intermediate affinity" IL-2Rβγ complex mediates CD8 + Regulates the activation and proliferation of effector T (Teff) cells, NK cells, and NKT cells. CD8 + Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, Tcon, or killer T cells) are T lymphocytes that recognize and kill damaged, cancerous, and pathogen-infected cells. NK and NKT cells are CD8 + Similar to Teff cells, they are several types of lymphocytes that target cancerous and pathogen-infected cells.

[0060] In some cases, IL-2 signaling regulates T cell responses and is subsequently utilized in cancer treatment. For example, IL-2 is administered at high doses to induce the expansion of Teff cell populations for cancer treatment. However, high-dose IL-2 also leads to the concomitant stimulation of Treg cells, which blunts the anti-tumor immune response. High-dose IL-2 also induces toxic adverse events mediated by the involvement of IL-2R alpha chain-expressing cells in the vasculature, including type 2 innate immune cells (ILC-2), eosinophils, and endothelial cells. This leads to eosinophilia, capillary leakage, and vascular leak syndrome (VLS).

[0061] Adoptive cell therapy allows physicians to effectively harness a patient's own immune cells to fight disease, such as proliferative diseases (e.g., cancer) and infectious diseases. Disclosed herein, in some embodiments, are methods of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the one or more additional agents can include one or more immune checkpoint inhibitors.

[0062] In some embodiments, described herein are interleukin 2 (IL-2) conjugates. In some embodiments, described herein are exemplary polypeptides shown in Table 1. In some embodiments, the IL-2 conjugates described herein are exemplified in Table 1.

[0063] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0064] [AzK] = N6-((2-azidoethoxy)-carbonyl)-L-lysine (Chemical Abstracts Registry No. 1167421-25-1). [AzK_PEG]=N6-((2-azidoethoxy)-carbonyl)-L-lysine is stably conjugated to PEG via DBCO-mediated click chemistry to form a compound comprising the structure of formula (II) or formula (III). For example, PEG5kD is specified to represent a linear polyethylene glycol chain having an average molecular weight of 5 kilodaltons and capped with a methoxy group. The ratio of positional isomers produced from the click reaction is about 1:1 or greater. The term "DBCO" refers to a chemical moiety comprising a dibenzocyclooctyne group, for example, the mPEG-DBCO compound shown in Scheme 1 of Example 2. [AzK_L1_PEG]=N6-((2-azidoethoxy-)carbonyl)-L-lysine is stably conjugated to PEG via DBCO-mediated click chemistry to form a compound comprising the structure of formula (IV) or formula (V). For example, PEG5kD is specified to represent a linear polyethylene glycol chain having an average molecular weight of 5 kilodaltons and capped with a methoxy group. The ratio of positional isomers produced from the click reaction is about 1:1 or greater. The term "DBCO" refers to a chemical moiety comprising a dibenzocyclooctyne group, for example, the mPEG-DBCO compound shown in Scheme 1 of Example 2.

[0065] 1. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein at least one amino acid residue in the IL-2 conjugate has the structure of Formula (I): [ka] (In the formula: Z is CH2 and Y is [ka] and; Y is CH2 and Z is [ka] and; Z is CH2 and Y is [ka] is; or Y is CH2 and Z is [ka] and; 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, and 60 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) The method is described herein, where

[0066] Here and throughout, the term "IL-2 conjugate" encompasses pharmaceutically acceptable salts, solvates, and hydrates of the structure shown.

[0067] Herein and throughout, the structure of Formula (I) encompasses its pharmaceutically acceptable salts, solvates, or hydrates. In some embodiments, the structure of Formula (I), or any embodiment or variation thereof, is provided as its pharmaceutically acceptable salt. In some embodiments, the structure of Formula (I), or any embodiment or variation thereof, is provided as its solvate. In some embodiments, the structure of Formula (I), or any embodiment or variation thereof, is provided as its hydrate. In some embodiments, the structure of Formula (I), or any embodiment or variation thereof, is provided as the free base.

[0068] In some embodiments of the methods described herein, in the IL-2 conjugate, Z is CH2 and Y is [ka] In some embodiments of the methods described herein, in the IL-2 conjugate, Y is CH2 and Z is [ka] In some embodiments of the methods described herein, Z is CH2 and Y is [ka] In some embodiments of the methods described herein, in the IL-2 conjugate, Z is CH2 and Y is [ka] In some embodiments of the methods described herein, in the IL-2 conjugate, Y is CH2 and Z is [ka] Here and throughout, the Z and Y embodiments also include pharmaceutically acceptable salts, solvates, or hydrates thereof.

[0069] In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa, and 30 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 5 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 10 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 15 kDa. In some embodiments, the methods use an IL-2 conjugate in which the PEG groups in the IL-2 conjugate have an average molecular weight of 20 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 25 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 30 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 35 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 40 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 45 kDa. In some embodiments of the methods described herein, the PEG groups in the IL-2 conjugate have an average molecular weight of 50 kDa. In some embodiments, the methods use an IL-2 conjugate in which the PEG groups in the IL-2 conjugate have an average molecular weight of 60 kDa.

[0070] In some embodiments of the methods described herein, in the IL-2 conjugate, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is relative to the position of SEQ ID NO: 3. In some embodiments of the methods described herein, in the IL-2 conjugate, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from F41, E61, and P64, and the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is relative to the position of SEQ ID NO: 3.

[0071] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 15-19, and [AzK_PEG] has the structure of Formula (II) or Formula (III), or a mixture of Formulas (II) and (III): [ka] (In the formula: 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, and 60 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0072] Herein and throughout, the structure of formula (II) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (III) includes pharmaceutically acceptable salts, solvates, or hydrates thereof.

[0073] In some embodiments, [AzK_PEG] is a mixture of Formula (II) and Formula (III).

[0074] In some embodiments, [AzK_PEG] has the structure of formula (II): [ka] It has.

[0075] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15. In some embodiments, W in the structure of Formula (II) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, W in the structure of Formula (II) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of Formula (II) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of Formula (II) is a PEG group having an average molecular weight of 30 kDa.

[0076] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method uses an IL-2 conjugate, wherein W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 30 kDa.

[0077] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method uses an IL-2 conjugate, wherein W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 30 kDa.

[0078] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 30 kDa.

[0079] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (II) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (II) is a PEG group having an average molecular weight of 30 kDa.

[0080] In some embodiments, [AzK_PEG] has the structure of formula (III) [ka] It has.

[0081] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight of 30 kDa.

[0082] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight of 30 kDa.

[0083] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method uses an IL-2 conjugate, wherein W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight of 30 kDa.

[0084] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18. In some embodiments, W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of formula (III) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of formula (III) is a PEG group having an average molecular weight of 30 kDa.

[0085] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (III) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (III) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (III) is a PEG group having an average molecular weight of 30 kDa.

[0086] In some embodiments of the methods disclosed herein, an IL-2 conjugate is used, having an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] contains 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, and 60 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] contains a PEG group having an average molecular weight of 5 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] contains a PEG group having an average molecular weight of 10 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 15 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 20 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 25 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 30 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 35 kDa.In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 40 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 45 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 50 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains a PEG group having an average molecular weight of 60 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 15, 16, 17, 18, and 19, and [AzK_PEG] contains 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, and 60 kDa, and the PEG group is a methoxy PEG group, a linear methoxy PEG group, or a branched methoxy PEG group.

[0087] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 20-24, and [AzK_PEG5kD] has the structure of Formula (II) or Formula (III), or a mixture of Formulas (II) and (III): [ka] (In the formula: W is a PEG group having an average molecular weight of 5 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue); or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0088] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24.

[0089] In some embodiments, the methods use an IL-2 conjugate, wherein [AzK_PEG5kD] has the structure of formula (II): [ka] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24.

[0090] In some embodiments, the method comprises the step of: [ka] or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24.

[0091] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 25-29, and [AzK_PEG30kD] has a structure of Formula (II) or Formula (III), or a mixture of the structures of Formulas (II) and (III): [ka] (In the formula: W is a PEG group having an average molecular weight of 30 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0092] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29.

[0093] In some embodiments, the methods disclosed herein provide for the preparation of [AzK_PEG30kD] having the structure of formula (II): [ka] or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29.

[0094] In some embodiments, the method further comprises the step of: [ka] In some embodiments, an IL-2 conjugate having the amino acid sequence of SEQ ID NO: 25 is used. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29.

[0095] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 15-19, and [AzK_PEG] is a mixture of structures of formula (II) and formula (III): [ka] (In the formula: 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, and 60 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0096] In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III), including the total amount of [AzK_PEG] in the IL-2 conjugate, is about 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III), including the total amount of [AzK_PEG] in the IL-2 conjugate, is greater than 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II) to the amount of the structure of Formula (III), including the total amount of [AzK_PEG] in the IL-2 conjugate, is less than 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein W is a linear or branched PEG group. In some embodiments, the method employs an IL-2 conjugate, wherein W is a linear PEG group. In some embodiments, the method employs an IL-2 conjugate, wherein W is a branched PEG group. In some embodiments, the method employs an IL-2 conjugate wherein W is a methoxy PEG group. In some embodiments, the method employs an IL-2 conjugate wherein the methoxy PEG group is linear or branched. In some embodiments, the method employs an IL-2 conjugate wherein the methoxy PEG group is linear. In some embodiments, the method employs an IL-2 conjugate wherein the methoxy PEG group is branched.

[0097] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 20-24, and [AzK_PEG5kD] is a mixture of structures of formula (II) and formula (III): [ka] (In the formula: W is a PEG group having an average molecular weight of 5 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue); or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0098] In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II), including the total amount of [AzK_PEG5kD] in the IL-2 conjugate, to the amount of the structure of Formula (III) is about 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II), including the total amount of [AzK_PEG5kD] in the IL-2 conjugate, to the amount of the structure of Formula (III) is greater than 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II), including the total amount of [AzK_PEG5kD] in the IL-2 conjugate, to the amount of the structure of Formula (III) is less than 1:1.

[0099] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 25-29, and [AzK_PEG30kD] is a mixture of structures of Formula (II) and Formula (III): [ka] (In the formula: W is a PEG group having an average molecular weight of 30 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue); or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0100] In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II), including the total amount of [AzK_PEG30kD] in the IL-2 conjugate, to the amount of the structure of Formula (III) is about 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II), including the total amount of [AzK_PEG30kD] in the IL-2 conjugate, to the amount of the structure of Formula (III) is greater than 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (II), including the total amount of [AzK_PEG30kD] in the IL-2 conjugate, to the amount of the structure of Formula (III) is less than 1:1.

[0101] In some embodiments, the method employs an IL-2 conjugate described herein comprising a structure of Formula (II) or Formula (III), or a mixture of Formulas (II) and (III), wherein W is a linear or branched PEG group. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of Formula (II) or Formula (III) is a linear PEG group. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of Formula (II) or Formula (III) is a branched PEG group. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of Formula (II) or Formula (III) is a methoxy PEG group. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of Formula (II) or Formula (III) is a linear or branched methoxy PEG group. In some embodiments, the method employs an IL-2 conjugate wherein the methoxy PEG group in the structure of Formula (II) or Formula (III) is linear. In some embodiments, the methods use an IL-2 conjugate in which the methoxy PEG group in the structure of formula (II) or formula (III) is branched.

[0102] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 40-44, and [AzK_L1_PEG] has the structure of Formula (IV) or Formula (V), or a mixture of Formulas (IV) and (V): [ka] (In the formula: 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, and 60 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue); or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0103] Herein and throughout, the structure of formula (IV) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (V) includes pharmaceutically acceptable salts, solvates, or hydrates thereof.

[0104] In some embodiments, the method uses an IL-2 conjugate wherein [AzK_L1_PEG] is a mixture of Formula (IV) and Formula (V).

[0105] In some embodiments, the method comprises the step of: [ka] An IL-2 conjugate having the formula:

[0106] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 30 kDa.

[0107] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 30 kDa.

[0108] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 30 kDa.

[0109] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 30 kDa.

[0110] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 44. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (IV) is a PEG group having an average molecular weight of 30 kDa.

[0111] In some embodiments, the method further comprises the step of: [ka] An IL-2 conjugate having the formula:

[0112] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 30 kDa.

[0113] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 30 kDa.

[0114] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 30 kDa.

[0115] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate wherein W in the structure of formula (V) is a PEG group having an average molecular weight of 30 kDa.

[0116] In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, and [AzK_L1_PEG] contains 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, and 60 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, and [AzK_L1_PEG] contains a PEG group having an average molecular weight of 5 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, and [AzK_L1_PEG] contains a PEG group having an average molecular weight of 10 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains a PEG group having an average molecular weight of 15 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains a PEG group having an average molecular weight of 20 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains a PEG group having an average molecular weight of 25 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains a PEG group having an average molecular weight of 30 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains a PEG group having an average molecular weight of 35 kDa.In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, and [AzK_L1_PEG] contains a PEG group having an average molecular weight of 40 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, and [AzK_L1_PEG] contains a PEG group having an average molecular weight of 45 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, and [AzK_L1_PEG] contains a PEG group having an average molecular weight of 50 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, and [AzK_L1_PEG] contains a PEG group having an average molecular weight of 60 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains 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, and 60 kDa, and the PEG group is a methoxy PEG group, a linear methoxy PEG group, or a branched methoxy PEG group.

[0117] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 45-49, and [AzK_L1_PEG5kD] has the structure of Formula (IV) or Formula (V), or a mixture of Formulas (IV) and (V): [ka] (In the formula: W is a PEG group having an average molecular weight of 5 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0118] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49.

[0119] In some embodiments, the method comprises the step of: [ka] or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49.

[0120] In some embodiments, the method comprises the step of: [ka] In some embodiments, an IL-2 conjugate having the amino acid sequence of SEQ ID NO: 45 is used. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49.

[0121] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 50-54, and [AzK_L1_PEG30kD] has a structure of Formula (IV) or Formula (V), or a mixture of the structures of Formulas (IV) and (V): [ka] (In the formula: W is a PEG group having an average molecular weight of 30 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0122] In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54.

[0123] In some embodiments, the method comprises: wherein [AzK_L1_PEG30kD] has the structure of formula (IV): [ka] or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54.

[0124] In some embodiments, the method further comprises the step of: [ka] In some embodiments, an IL-2 conjugate having the amino acid sequence of SEQ ID NO: 50 is used. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54.

[0125] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 40-44, and [Azk_L1_PEG] is a mixture of structures of formula (IV) and formula (V): [ka] (In the formula: 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, and 60 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue); or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0126] In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG] in the IL-2 conjugate, to the amount of the structure of Formula (V) is about 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG] in the IL-2 conjugate, to the amount of the structure of Formula (V) is greater than 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG] in the IL-2 conjugate, to the amount of the structure of Formula (V) is less than 1:1.

[0127] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 45-49, and [AzK_L1_PEG5kD] is a mixture of structures of formula (IV) and formula (V): [ka] (In the formula: W is a PEG group having an average molecular weight of 5 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue); or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0128] In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate, to the amount of the structure of Formula (V) is about 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate, to the amount of the structure of Formula (V) is greater than 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate, to the amount of the structure of Formula (V) is less than 1:1.

[0129] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 50-54, and [AzK_L1 PEG30kD] is a mixture of structures of Formula (IV) and Formula (V): [ka] (In the formula: W is a PEG group having an average molecular weight of 30 kDa; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue); or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0130] In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate, to the amount of the structure of Formula (V) is about 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate, to the amount of the structure of Formula (V) is greater than 1:1. In some embodiments, the method employs an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (IV), including the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate, to the amount of the structure of Formula (V) is less than 1:1.

[0131] In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 5 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 30 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 10 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 15 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 20 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 25 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 30 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 35 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 40 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a PEG group having an average molecular weight of 45 kDa. In some embodiments, the methods use an IL-2 conjugate in which W in the structure of formula (IV) or formula (V) is a PEG group having an average molecular weight of 50 kDa.In some embodiments, the method employs an IL-2 conjugate in which W in the structure of formula (IV) or formula (V) is a PEG group having an average molecular weight of 55 kDa. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of formula (IV) or formula (V) is a PEG group having an average molecular weight of 60 kDa.

[0132] In some embodiments, the method employs an IL-2 conjugate described herein comprising a structure of Formula (IV) or Formula (V), or a mixture of Formulas (IV) and (V), wherein W is a linear or branched PEG group. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a linear PEG group. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a branched PEG group. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a methoxy PEG group. In some embodiments, the method employs an IL-2 conjugate in which W in the structure of Formula (IV) or Formula (V) is a linear or branched methoxy PEG group. In some embodiments, the method employs an IL-2 conjugate in which the methoxy PEG group in the structure of Formula (IV) or Formula (V) is linear. In some embodiments, the methods use an IL-2 conjugate in which the methoxy PEG group in the structure of formula (IV) or formula (V) is branched.

[0133] For the IL-2 conjugates used in the methods described herein, an exemplary structure of a methoxy PEG group is depicted in the mPEG-DBCO structure in Scheme 1 of Example 2. An exemplary structure of a methoxy PEG group is depicted in the following mPEG-DBCO structure: [ka] As illustrated in

[0134] 1. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein at least one amino acid residue in the IL-2 conjugate has the structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII): [ka] (In the formula: n is an integer ranging from about 2 to about 5000; X is of the structure: [ka] and X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0135] Herein and throughout, the structure of formula (VI) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (VII) includes pharmaceutically acceptable salts, solvates, or hydrates thereof.

[0136] In some embodiments, n in compounds of Formula (VI) and Formula (VII) is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 27 5, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.In some embodiments, n in the compounds of Formula (VI) and Formula (VII) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1479, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1 is an integer selected from 478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0137] In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate is relative to the position of SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is K34. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is F41. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is F43. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is K42. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37.In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is T40. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is E67. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is Y44. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is V68. In some embodiments, the position of the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and Formula (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71.

[0138] In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (VI) to the amount of the structure of Formula (VII), including the total amount of the IL-2 conjugate, is about 1:1. In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (VI) to the amount of the structure of Formula (VII), including the total amount of the IL-2 conjugate, is greater than 1:1. In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (VI) to the amount of the structure of Formula (VII), including the total amount of the IL-2 conjugate, is less than 1:1.

[0139] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), and the replaced amino acid residues in SEQ ID NO: 3 are K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L7 1, where n is selected from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 12 50, or an integer from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575.In some embodiments, n in the compounds of Formula (VI) and Formula (VII) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1479, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1 is an integer selected from 478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0140] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from F41, F43, K42, E61, and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (VI) and Formula (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0141] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from E61 and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (VI) and Formula (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0142] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), where the replaced amino acid residue in SEQ ID NO: 3 is E61, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, the method uses an IL-2 conjugate in which n in the compounds of Formula (VI) and Formula (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0143] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), wherein the replaced amino acid residue in SEQ ID NO: 3 is P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (VI) and Formula (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0144] In some embodiments, n in the structures of Formula (VI) and Formula (VII) indicates that the molecular weight of the PEG moiety is from about 1,000 daltons to about 200,000 daltons, or from about 2,000 daltons to about 150,000 daltons, or from about 3,000 daltons to about 125,000 daltons, or from about 4,000 daltons to about 100,000 daltons, or from about 5,000 daltons to about 100,000 daltons, or from about 6,000 daltons to about 90,000 daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons. Daltons, or about 5,000 Daltons to about 70,000 Daltons, or about 5,000 Daltons to about 65,000 Daltons, or about 5,000 Daltons to about 60,000 Daltons, or about 5,000 Daltons to about 50,000 Daltons, or about 6,000 Daltons to about 50,000 Daltons, or about 7,000 Daltons to about 50,000 Daltons, or about 7,000 Daltons to about 45,000 Daltons, or about 7,000 Daltons to about 40,000 Daltons, or about 8,000 Daltons to about 40,000 Daltons, or about 8,500 Daltons daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 daltons to about 45,000 daltons, or about 9,000 daltons to about 40,000 daltons, or about 9,000 daltons to about 35,000 daltons, or about 9,000 daltons to about 30,000 daltons, or about 9,500 daltons to about 35,000 daltons, or about 9,500 daltons to about 30,000 daltons, or about 10,000 daltons to about 50,000 daltons, or about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,000 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 40,000 daltons, or about 15,000 daltons to about 35,000 daltons, or about 15,000 daltons to about 30,000 daltons, or about 20,20,000 daltons to about 50,000 daltons, or about 20,000 daltons to about 45,000 daltons, or about 20,000 daltons to about 40,000 daltons, or about 20,000 daltons to about 35,000 daltons, or about 20,000 daltons to about 30,000 daltons.

[0145] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), and n is a number ranging from about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, or about 30,000 daltons. , about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons or about 200,000 daltons.

[0146] In some embodiments, the methods use an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), and n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, or about 50,000 daltons.

[0147] 1. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein at least one amino acid residue in the IL-2 conjugate has the structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX): [ka] (In the formula: n is an integer ranging from about 2 to about 5000; X is of the structure: [ka] and X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0148] Herein and throughout, the structure of formula (VIII) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (IX) includes pharmaceutically acceptable salts, solvates, or hydrates thereof.

[0149] In some embodiments, n in compounds of Formula (VIII) and Formula (IX) is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 2 75, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.In some embodiments, n in the compounds of Formula (VIII) and Formula (IX) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1479, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1 is an integer selected from 478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0150] In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate is relative to the position of SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is K34. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is F41. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is F43. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is K42. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37.In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is T40. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is E67. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is Y44. In some embodiments, the position of the structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is V68. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71.

[0151] In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX), including the total amount of the IL-2 conjugate, is about 1:1. In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX), including the total amount of the IL-2 conjugate, is greater than 1:1. In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX), including the total amount of the IL-2 conjugate, is less than 1:1.

[0152] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), and the replaced amino acid residues in SEQ ID NO: 3 are K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L37. 71, where n is selected from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 12 50, or an integer from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575.In some embodiments, n in the compounds of Formula (VIII) and Formula (IX) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1479, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1 is an integer selected from 478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0153] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from F41, F43, K42, E61, and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (VIII) and Formula (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0154] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from E61 and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (VIII) and Formula (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0155] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), where the replaced amino acid residue in SEQ ID NO: 3 is E61, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (VIII) and Formula (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0156] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), wherein the replaced amino acid residue in SEQ ID NO: 3 is P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (VIII) and Formula (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0157] A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formulas (VIII) and (IX), and n is a number ranging from about 1,000 daltons to about 200,000 daltons, and or about 2,000 daltons to about 150,000 daltons, or about 3,000 daltons to about 125,000 daltons, or about 4,000 daltons to about 100,000 daltons, or about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or about 5,000 daltons to about 65,000 daltons 0 daltons to about 60,000 daltons, or about 5,000 daltons to about 50,000 daltons, or about 6,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons daltons, or about 9,000 daltons to about 45,000 daltons, or about 9,000 daltons to about 40,000 daltons, or about 9,000 daltons to about 35,000 daltons, or about 9,000 daltons to about 30,000 daltons, or about 9,500 daltons to about 35,000 daltons, or about 9,500 daltons to about 30,000 daltons, or about 10,000 daltons to about 50,000 daltons, or about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 Daltons to about 35,000 Daltons, or about 10,000 Daltons to about 30,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 15,000 Daltons to about 45,000 Daltons, or about 15,000 Daltons to about 40,000 Daltons, or about 15,000 Daltons to about 35,000 Daltons, or about 15,000 Daltons to about 30 The method described herein is characterized in that the nucleotide sequence is an integer ranging from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons.

[0158] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), and n is a number representing a molecular weight of the PEG moiety of about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, or about 30,000 daltons. about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons or about 200,000 daltons. In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), and n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, or about 50,000 daltons.

[0159] 1. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein at least one amino acid residue in the IL-2 conjugate has the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI): [ka] (In the formula: n is an integer ranging from about 2 to about 5000; The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 3 that is not replaced. The method is described herein, where

[0160] Herein and throughout, the structure of formula (X) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (XI) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0161] In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is racemic, (R)-rich, (S)-rich, substantially (R), substantially (S), (R), or (S). In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is racemic. In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is (R)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is (S)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is substantially (R). In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is substantially (S). In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is (R). In some embodiments, the stereochemistry of the chiral centers in Formula (X) and Formula (XI) is (S).

[0162] In some embodiments, n in compounds of Formula (X) and Formula (XI) is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275 , or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1 710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.In some embodiments, n in the compounds of Formula (X) and Formula (XI) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 15 78, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0163] In some embodiments, the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and the position of the structure of Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI) in the amino acid sequence of the IL-2 conjugate is relative to the position of SEQ ID NO: 3. In some embodiments, the method uses an IL-2 conjugate in which the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the location of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, the location of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, the method utilizes an IL-2 conjugate in which the location of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, the method utilizes an IL-2 conjugate in which the location of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, the method uses an IL-2 conjugate in which the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is E61. In some embodiments, the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is P64.In some embodiments, the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is R37. In some embodiments, the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is T40. In some embodiments, the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is E67. In some embodiments, the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is Y44. In some embodiments, the position of the structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is V68. In some embodiments, the position of the structure of formula (X) or formula (XI), or a mixture of formula (X) and formula (XI), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71.

[0164] In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (X) to the amount of the structure of Formula (XI), including the total amount of the IL-2 conjugate, is about 1:1. In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (X) to the amount of the structure of Formula (XI), including the total amount of the IL-2 conjugate, is greater than 1:1. In some embodiments, the method uses an IL-2 conjugate, wherein the ratio of the amount of the structure of Formula (X) to the amount of the structure of Formula (XI), including the total amount of the IL-2 conjugate, is less than 1:1.

[0165] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), and the replaced amino acid residues in SEQ ID NO: 3 are selected from the group consisting of K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. and n is selected from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or about 15 0 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 125 It is an integer of 0, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.In some embodiments, n in the compounds of Formula (VI) and Formula (VII) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1479, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 1 is an integer selected from 478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0166] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from F41, F43, K42, E61, and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (X) and Formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0167] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from E61 and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (X) and Formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0168] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), where the replaced amino acid residue in SEQ ID NO: 3 is E61, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, the method uses an IL-2 conjugate in which n in the compounds of Formula (X) and Formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0169] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the replaced amino acid residue in SEQ ID NO: 3 is P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (X) and Formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0170] In some embodiments, n in the structures of Formula (X) and Formula (XI) indicates that the molecular weight of the PEG moiety is from about 1,000 daltons to about 200,000 daltons, or from about 2,000 daltons to about 150,000 daltons, or from about 3,000 daltons to about 125,000 daltons, or from about 4,000 daltons to about 100,000 daltons, or from about 5,000 daltons to about 100,000 daltons, or from about 6,000 daltons to about 90,000 daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons. daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or about 5,000 daltons to about 60,000 daltons, or about 5,000 daltons to about 50,000 daltons, or about 6,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 daltons to about 45,000 daltons, or about 9,000 daltons to about 40,000 daltons, or about 9,000 daltons to about 35,000 daltons, or about 9,000 daltons to about 30,000 daltons, or about 9,500 daltons to about 35,000 daltons, or about 9,500 daltons to about 30,000 daltons, or about 10,000 daltons to about 50,000 daltons, or about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,000 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 40,000 daltons, or about 15,000 daltons to about 35,000 daltons, or about 15,000 daltons to about 30,000 daltons, or about 20,20,000 daltons to about 50,000 daltons, or about 20,000 daltons to about 45,000 daltons, or about 20,000 daltons to about 40,000 daltons, or about 20,000 daltons to about 35,000 daltons, or about 20,000 daltons to about 30,000 daltons.

[0171] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), and n is a number ranging from about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25 The amino acid sequence is an integer such that the amino acid sequence is about 1,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons or about 200,000 daltons.

[0172] In some embodiments, the methods use an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), and n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, or about 50,000 daltons.

[0173] 1. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein at least one amino acid residue in the IL-2 conjugate has the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII): [ka] (In the formula: n is an integer ranging from about 2 to about 5000; The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 3 that is not replaced. The method is described herein, wherein the IL-2 conjugate is replaced by

[0174] Herein and throughout, the structure of formula (XII) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (XIII) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0175] In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is racemic, (R)-rich, (S)-rich, substantially (R), substantially (S), (R), or (S). In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is racemic. In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is (R)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is (S)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is substantially (R). In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is substantially (S). In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is (R). In some embodiments, the stereochemistry of the chiral centers in Formula (XII) and Formula (XIII) is (S).

[0176] In some embodiments, n in compounds of Formula (XII) and Formula (XIII) is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 2 75, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.In some embodiments, n in compounds of Formula (XII) and (XIII) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1479, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543, 1544, 1545, 15 is an integer selected from 478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0177] In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and the position of the structure of Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula (XIII) in the amino acid sequence of the IL-2 conjugate is relative to the position of SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64.In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is R37. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is T40. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is E67. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is Y44. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments, the position of the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71.

[0178] In some embodiments, the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII), including the total amount of IL-2 conjugate, is about 1:1. In some embodiments, the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII), including the total amount of IL-2 conjugate, is greater than 1:1. In some embodiments, the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII), including the total amount of IL-2 conjugate, is less than 1:1.

[0179] In some embodiments, described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and the replaced amino acid residues in SEQ ID NO: 3 are K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, , V68, and L71, and n is selected from 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 4 75, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about It is an integer of about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.In some embodiments, n in the compounds of Formula (XII) and Formula (XIII) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, is an integer selected from 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0180] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from F41, F43, K42, E61, and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (XII) and Formula (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0181] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from E61 and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (XII) and Formula (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0182] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), where the replaced amino acid residue in SEQ ID NO: 3 is E61, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (XII) and Formula (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0183] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), wherein the replaced amino acid residue in SEQ ID NO: 3 is P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, n in the compounds of Formula (XII) and Formula (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0184] In some embodiments, n in the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), is a number that indicates that the molecular weight of the PEG moiety is from about 1,000 daltons to about 200,000 daltons, or from about 2,000 daltons to about 150,000 daltons, or from about 3,000 daltons to about 125,000 daltons, or from about 4,000 daltons to about 100,000 daltons, or from about 5,000 daltons to about 100,000 daltons, or from about 6,000 daltons to about 90,000 daltons, or from about 7,000 daltons to about 80,000 daltons. 0 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or about 5,000 daltons to about 60,000 daltons, or about 5,000 daltons to about 50,000 daltons, or about 6,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons 1000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 daltons to about 45,000 daltons, or about 9,000 daltons to about 40,000 daltons, or about 9,000 daltons to about 35,000 daltons, or about 9,000 daltons to about 30,000 daltons, or about 9,500 daltons to about 35,000 daltons, or about 9,500 daltons to about 30,000 daltons, or about 10,000 daltons to about 50,000 daltons, or about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,000 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 40,000 daltons, or about 15,000 daltons to about 35,000 daltons,20,000 daltons to about 30,000 daltons, or about 20,000 daltons to about 50,000 daltons, or about 20,000 daltons to about 45,000 daltons, or about 20,000 daltons to about 40,000 daltons, or about 20,000 daltons to about 35,000 daltons, or about 20,000 daltons to about 30,000 daltons.

[0185] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and n is a number ranging from about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, or about 30,000 daltons. daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons or about 200,000 daltons. Described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, or about 50,000 daltons.

[0186] 1. A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein the amino acid residue at E61 or P64 in the IL-2 conjugate has the structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX): [ka] (In the formula: n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) The method is described herein, where

[0187] In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0188] In some embodiments, the amino acid residue E61 in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), where n is an integer such that the molecular weight of the PEG group is about 20,000 daltons to about 40,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab. In some embodiments, the one or more PD-1 inhibitors are nivolumab. In some embodiments, the one or more PD-1 inhibitors are cemiplimab.

[0189] In some embodiments, the amino acid residue of P64 in the IL-2 conjugate is replaced with a structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula (IX), where n is an integer such that the molecular weight of the PEG group is about 20,000 daltons to about 40,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab. In some embodiments, the one or more PD-1 inhibitors are nivolumab. In some embodiments, the one or more PD-1 inhibitors are cemiplimab.

[0190] A method of treating cancer in a subject, comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein an amino acid residue of E61 or P64 in the IL-2 conjugate has the structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII): [ka] (In the formula: n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; X is of the structure: [ka] having; X-1 indicates the point of attachment to the preceding amino acid residue; X+1 indicates the point of attachment to the subsequent amino acid residue) The method is described herein, where In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0191] In some embodiments, the amino acid residue E61 in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), where n is an integer such that the molecular weight of the PEG group is about 20,000 daltons to about 40,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab. In some embodiments, the one or more PD-1 inhibitors are nivolumab. In some embodiments, the one or more PD-1 inhibitors are cemiplimab.

[0192] In some embodiments, the amino acid residue of P64 in the IL-2 conjugate is replaced with a structure of Formula (VI) or Formula (VII), or a mixture of Formula (VI) and Formula (VII), where n is an integer such that the molecular weight of the PEG group is about 20,000 daltons to about 40,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the one or more PD-1 inhibitors are pembrolizumab. In some embodiments, the one or more PD-1 inhibitors are nivolumab. In some embodiments, the one or more PD-1 inhibitors are cemiplimab.

[0193]

[0010] Described herein are methods for treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4, and wherein at least one amino acid residue in the IL-2 conjugate is replaced with a cysteine covalently linked to a PEG group. In some embodiments, the PEG group has a molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa. In some embodiments, the PEG group has a molecular weight of 5 kDa. In some embodiments, the PEG group has a molecular weight of 10 kDa. In some embodiments, the PEG group has a molecular weight of 15 kDa. In some embodiments, the PEG group has a molecular weight of 20 kDa. In some embodiments, the PEG group has a molecular weight of 25 kDa. In some embodiments, the PEG group has a molecular weight of 30 kDa. In some embodiments, the PEG group has a molecular weight of 35 kDa. In some embodiments, the PEG group has a molecular weight of 40 kDa. In some embodiments, the PEG group has a molecular weight of 45 kDa. In some embodiments, the PEG group has a molecular weight of 50 kDa. In some embodiments, the PEG group has a molecular weight of 60 kDa. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate that is replaced with a cysteine is selected from K34, T36, R37, T40, F41, K42, F43, Y44, E60, E61, E67, K63, P64, V68, L71, and Y106. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate that is replaced with a cysteine is selected from K34, T40, F41, K42, Y44, E60, E61, E67, K63, P64, V68, and L71.In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 4, and at least one amino acid residue in the IL-2 conjugate replaced with a cysteine is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.

[0194] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, and wherein at least one non-lysine residue is replaced with a lysine, comprising a linker and a water-soluble polymer. In some embodiments, the water-soluble polymer is a PEG group.

[0195] In some embodiments, the IL-2 conjugate comprises a PEG group covalently attached via a non-releasable bond. In some embodiments, the IL-2 conjugate comprises a non-releasable, covalently attached PEG group.

[0196]

[0010] Described herein are methods for treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate has SEQ ID NO: 3, wherein a non-lysine amino acid in the IL-2 conjugate is replaced with a lysine residue, and the lysine residue comprises one or more water-soluble polymers and a covalent linker. In some embodiments, the lysine residue is located in the region K34-Y106 of SEQ ID NO: 3. In some embodiments, the lysine residue is located at K34. In some embodiments, the lysine residue is located at F41. In some embodiments, the lysine residue is located at F43. In some embodiments, the lysine residue is located at K42. In some embodiments, the lysine residue is located at E61. In some embodiments, the lysine residue is located at P64. In some embodiments, the lysine residue is located at R37. In some embodiments, the lysine residue is located at T40. In some embodiments, the lysine residue is located at E67. In some embodiments, the lysine residue is located at Y44. In some embodiments, the lysine residue is located at V68. In some embodiments, the lysine residue is located at L71.

[0197]

[0010] Described herein are methods for treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate has SEQ ID NO: 3, wherein a non-lysine amino acid in the IL-2 conjugate is replaced with a lysine residue, and the lysine residue comprises one or more water-soluble polymers and a covalent linker. In some embodiments, the lysine residue is located in the region K34-Y106 of SEQ ID NO: 3. In some embodiments, the lysine residue is located at K34. In some embodiments, the lysine residue is located at F41. In some embodiments, the lysine residue is located at F43. In some embodiments, the lysine residue is located at K42. In some embodiments, the lysine residue is located at E61. In some embodiments, the lysine residue is located at P64. In some embodiments, the lysine residue is located at R37. In some embodiments, the lysine residue is located at T40. In some embodiments, the lysine residue is located at E67. In some embodiments, the lysine residue is located at Y44. In some embodiments, the lysine residue is located at V68. In some embodiments, the lysine residue is located at L71.

[0198]

[0009] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate is an interleukin-2 (IL-2) variant in which non-lysine amino acids in the amino acid sequence of the IL-2 variant have been replaced with amino acids comprising (a) lysine; (b) a covalent linker; and (3) and one or more water-soluble polymers. In some embodiments, the one or more water-soluble polymers comprise a PEG group.

[0199] A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, and wherein at least one amino acid residue in the IL-2 conjugate has the structure of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV): [ka] (In the formula: m is an integer from 0 to 20; p is an integer from 0 to 20; n is an integer ranging from about 2 to about 5000; The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 3 that is not replaced), the methods described herein.

[0200] Herein and throughout, the structure of formula (XIV) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (XV) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0201] In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is racemic, (R)-rich, (S)-rich, substantially (R), substantially (S), (R), or (S). In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is racemic. In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is (R)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is (S)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is substantially (R). In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is substantially (S). In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is (R). In some embodiments, the stereochemistry of the chiral centers in Formula (XIV) and Formula (XV) is (S).

[0202] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein m in the compounds of Formula (XIV) and Formula (XV) is 0 to 20, or 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 1. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 2. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 3. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 4. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 5. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 6. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 7. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 8. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 9. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 10. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 11. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 12. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 13.In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 14. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 15. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 16. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 17. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 18. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 19. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 20.

[0203] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein p in the compounds of Formula (XIV) and Formula (XV) is 1 to 20, or 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 1. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 2. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 3. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 4. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 5. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 6. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 7. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 8. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 9. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 10. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 11. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 12. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 13.In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 14. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 15. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XIV) and Formula (XV) is 16. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 17. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 18. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 19. In some embodiments of the IL-2 conjugates described herein, p in the compounds of Formula (XIV) and Formula (XV) is 20.

[0204] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein n in the compounds of Formula (XIV) and Formula (XV) is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or about 100 to about 1750, or about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 10 0 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.

[0205] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein in the compounds of Formula (XIV) and Formula (XV), m is an integer from 1 to 6, p is an integer from 1 to 6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is an integer from 2 to 6, p is an integer from 2 to 6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is an integer from 2 to 4, p is an integer from 2 to 4, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 1, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 2, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 3, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 4, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 5, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 6, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 7, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 8, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 9, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 10, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 11, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 11, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is an integer selected from 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.

[0206] In some embodiments, the methods use an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein n in the compounds of Formula (XIV) and Formula (XV) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363 , 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV), in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate is relative to the position in SEQ ID NO: 3. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34.In some embodiments of the IL-2 conjugates described herein, the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is located at position F41. In some embodiments of the IL-2 conjugates described herein, the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is located at position F43. In some embodiments of the IL-2 conjugates described herein, the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is located at position K42. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44.In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71.

[0207] In some embodiments of the IL-2 conjugates described herein, the ratio of the amount of the structure of formula (XIV) to the amount of the structure of formula (XV), including the total amount of the IL-2 conjugate, is about 1:1. In some embodiments of the IL-2 conjugates described herein, the ratio of the amount of the structure of formula (XIV) to the amount of the structure of formula (XV), including the total amount of the IL-2 conjugate, is greater than 1:1. In some embodiments of the IL-2 conjugates described herein, the ratio of the amount of the structure of formula (XIV) to the amount of the structure of formula (XV), including the total amount of the IL-2 conjugate, is less than 1:1.

[0208] A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formulas (XIV) and (XV), and the replaced SEQ ID NO: 3 are selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and n is 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 575. about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or is an integer from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575.In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XIV) and Formula (XV) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, is an integer selected from 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0209]

[0013] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from F41, F43, K42, E61, and P64, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XIV) and Formula (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0210]

[0010] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formulas (XIV) and (XV), wherein the replaced amino acid residue in SEQ ID NO: 3 is selected from E61 and P64, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XIV) and Formula (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0211]

[0013] Described herein is a method for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formulas (XIV) and (XV), the replaced amino acid residue in SEQ ID NO: 3 is E61, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XIV) and Formula (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0212]

[0013] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formulas (XIV) and (XV), the replaced amino acid residue in SEQ ID NO: 3 is P64, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XIV) and (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0213] A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV), and n is a number ranging from about 1,000 daltons to about 200,000 daltons, or about 2,000 daltons, and to about 150,000 daltons, or about 3,000 daltons to about 125,000 daltons, or about 4,000 daltons to about 100,000 daltons, or about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or about 5,000 daltons to about 60,000 daltons daltons, or about 5,000 daltons to about 50,000 daltons, or about 6,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 Daltons to about 45,000 Daltons, or about 9,000 Daltons to about 40,000 Daltons, or about 9,000 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 30,000 Daltons, or about 9,500 Daltons to about 35,000 Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons to about 40,000 Daltons, or about 10,000 Daltons to about 35,000 Daltons.000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,000 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 40,000 daltons, or about 15,000 daltons to about 35,000 daltons, or about 15,000 daltons to about 30,000 daltons The method described herein is characterized in that the nucleotide sequence is an integer ranging from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons.

[0214] An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), and n is a number ranging from about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, or about 40,000 daltons. daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons or about 200,000 daltons.

[0215] Described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV), and n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, or about 50,000 daltons.

[0216]

[0013] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), and the replaced amino acid residue in SEQ ID NO: 3 is selected from F41, F43, K42, E61, and P64, m is an integer of 1 to 6, p is an integer of 1 to 6, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of formula (XIV) and (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0217]

[0013] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), and the replaced amino acid residue in SEQ ID NO: 3 is selected from E61 and P64, m is an integer of 1 to 6, p is an integer of 1 to 6, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0218]

[0013] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the replaced amino acid residue in SEQ ID NO: 3 is E61, m is an integer of 1 to 6, p is an integer of 1 to 6, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0219]

[0013] Described herein are methods for treating cancer in a subject, the method comprising administering to a subject in need thereof therapeutically effective amounts of (a) an IL-2 conjugate and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the replaced amino acid residue in SEQ ID NO: 3 is P64, m is an integer of 1 to 6, p is an integer of 1 to 6, and n is an integer of about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XIV) and Formula (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0220] 1. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) an IL-2 conjugate, and (b) one or more additional agents, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and wherein at least one amino acid residue in the IL-2 conjugate has the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII): [ka] (In the formula: m is an integer from 0 to 20; n is an integer ranging from about 2 to about 5000; The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 3 that is not replaced. The method is described herein, where

[0221] Herein and throughout, the structure of formula (XVI) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout, the structure of formula (XVII) includes pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0222] In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is racemic, (R)-rich, (S)-rich, substantially (R), substantially (S), (R), or (S). In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is racemic. In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is (R)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is (S)-rich. In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is substantially (R). In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is substantially (S). In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is (R). In some embodiments, the stereochemistry of the chiral centers in Formula (XVI) and Formula (XVII) is (S).

[0223] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein m in the compounds of Formula (XVI) and Formula (XVII) is 1 to 20, or 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 1. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 2. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 3. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 4. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 5. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 6. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 7. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 8. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 9. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 10. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 11. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 12.In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 13. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 14. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 15. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 16. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 17. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 18. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 19. In some embodiments of the IL-2 conjugates described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 20.

[0224] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein n in the compounds of Formula (XVI) and Formula (XVII) is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or or about 100 to about 1750, or about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 1 00 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.

[0225] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein in the compounds of Formula (XVI) and Formula (XVII), m is an integer from 1 to 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is an integer from 2 to 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is an integer from 2 to 4 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 1 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 2 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 3 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 4 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 5 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 7 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 8 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 9 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 10 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 11 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 12 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 2 and n is an integer selected from 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.

[0226] In some embodiments, the methods use an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein n in the compounds of Formula (XVI) and Formula (XVII) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 136 is an integer selected from 3, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is relative to the position of SEQ ID NO: 3. In some embodiments of the IL-2 conjugates described herein, the position of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments of the IL-2 conjugates described herein, the position of the structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34.In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67.In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments of the IL-2 conjugates described herein, the location of the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71.

[0227] In some embodiments of the IL-2 conjugates described herein, the ratio of the amount of the structure of formula (XVI) to the amount of the structure of formula (XVII), including the total amount of the IL-2 conjugate, is about 1:1. In some embodiments of the IL-2 conjugates described herein, the ratio of the amount of the structure of formula (XVI) to the amount of the structure of formula (XVII), including the total amount of the IL-2 conjugate, is greater than 1:1. In some embodiments of the IL-2 conjugates described herein, the ratio of the amount of the structure of formula (XVI) to the amount of the structure of formula (XVII), including the total amount of the IL-2 conjugate, is less than 1:1.

[0228] In some embodiments, the method uses an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), and is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and n is 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 34 0, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or an integer from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575.In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XVI) and Formula (XVII) is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364 , 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0229] In some embodiments, described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), and wherein n is selected from F41, F43, K42, E61, and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XVI) and Formula (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0230]

[0013] In some embodiments, described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), and wherein n is selected from E61 and P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XVI) and (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0231] Described herein in some embodiments is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), where the replacement is E61, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XVI) and Formula (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0232] Described herein in some embodiments is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), where the replacement is P64, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, n in the compounds of Formula (XVI) and Formula (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0233] In some embodiments, an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), and n is an integer from 1 to 2, ... 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or about 5,000 daltons to about 60,000 daltons, or about 5,000 daltons to about 50,000 daltons, or about 6,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 50,000 daltons 00 daltons, or about 7,000 daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 daltons to about 45,000 daltons, or about 9,000 daltons to about 40,000 daltons, or about 9,000 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons Daltons to about 30,000 Daltons, or about 9,500 Daltons to about 35,000 Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons to about 40,000 Daltons, or about 10,000 Daltons to about 35,000 Daltons, or about 10,000 Daltons to about 30,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 15,000 Daltons to about 45,000 DaltonsThe IL-2 conjugates described herein are those in which the IL-2 conjugate has a molecular weight of about 1,0 ...

[0234] Described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), and n is a number representing a molecular weight of the PEG moiety of about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, or about 30,000 daltons. , about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons or about 200,000 daltons. Described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), and n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, or about 50,000 daltons.

[0235] In some embodiments, described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), and is selected from F41, F43, K42, E61, and P64; m is an integer from 1 to 6; and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0236] In some embodiments, described herein is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), wherein the at least one amino acid residue in the IL-2 conjugate is selected from E61 and P64, m is an integer from 1 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0237] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, where at least one amino acid residue in the replaced IL-2 conjugate is E61, m is an integer from 1 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0238] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate, which is replaced by a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), is P64, m is an integer from 1 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-2 conjugates described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is about 550 to about 800. In some embodiments, n is about 681.

[0239] Described herein, in some embodiments, are methods of treating a proliferative disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) a cytokine conjugate (e.g., an IL-2 conjugate) and (b) one or more additional agents described in Table 1. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 1-98. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 1-84. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 15-29. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 40-54. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 55-69. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 70-84. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 85-98. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 1. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 2. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 3. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 4. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 5. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 6. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 7. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 8. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 9. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 10. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 11. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 12. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 13. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 14. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 15. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 16. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 17. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 18.In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 19. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 20. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 21. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 22. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 23. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 24. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 25. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 28 ... In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 29. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 30. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 31. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 32. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 33. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 34. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 35. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 36. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 37. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 38. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 39. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 40. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 41. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 42. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 43.In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 44. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 45. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 46. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 47. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 48. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 49. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 50. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 51. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 52. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 53. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 54. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 55. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 56. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 57. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 58. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 59. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 60. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 61. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 62. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 63. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 64. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 65. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 66. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 67. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 68. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 69. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 70. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 71. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 72. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 73.In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 74. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 75. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 76. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 77. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 78. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 79. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 80. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 81. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 82. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 83. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 84. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 85. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 86. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 87. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 88. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 89. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 90. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 91. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 92. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 93. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 94. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 95. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 96. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 97. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 98.

[0240] In some embodiments, the IL-2 conjugate comprises the structure of Formula (I). In some embodiments, the IL-2 conjugate comprises the structure of Formula (II). In some embodiments, the IL-2 conjugate comprises the structure of Formula (III). In some embodiments, the IL-2 conjugate comprises the structure of Formula (IV). In some embodiments, the IL-2 conjugate comprises the structure of Formula (V). In some embodiments, the IL-2 conjugate comprises the structure of Formula (VI). In some embodiments, the IL-2 conjugate comprises the structure of Formula (VII). In some embodiments, the IL-2 conjugate comprises the structure of Formula (VIII). In some embodiments, the IL-2 conjugate comprises the structure of Formula (IX). In some embodiments, the IL-2 conjugate comprises the structure of Formula (X). In some embodiments, the IL-2 conjugate comprises the structure of Formula (XI). In some embodiments, the IL-2 conjugate comprises the structure of Formula (XII). In some embodiments, the IL-2 conjugate comprises the structure of Formula (XIII). In some embodiments, the IL-2 conjugate comprises the structure of formula (XIV). In some embodiments, the IL-2 conjugate comprises the structure of formula (XV). In some embodiments, the IL-2 conjugate comprises the structure of formula (XVI). In some embodiments, the IL-2 conjugate comprises the structure of formula (XV). In some embodiments, the IL-2 conjugate comprises the structure of formula (XVI). In some embodiments, the IL-2 conjugate comprises the structure of formula (XVII).

[0241] In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 86, 88, 90, 92, 94, 96, and 98. In any of these embodiments, the structure of Formula (I), or any variation thereof, such as Formulas (II) through (XVII), or any variation thereof, is incorporated at a site that includes an unnatural amino acid.

[0242] In some embodiments, described herein are IL-2 conjugates modified at an amino acid position. In some examples, the modification is to a natural amino acid. In some examples, the modification is to a non-natural amino acid. In some examples, described herein are isolated and modified IL-2 polypeptides comprising at least one non-natural amino acid. In some cases, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-84. In some cases, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3-98.

[0243] In some examples, the IL-2 conjugate further comprises an additional mutation. In some cases, the additional mutation is at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In such cases, the amino acid is conjugated to an additional conjugation moiety for increased serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a natural amino acid, such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine; or to a non-natural amino acid before being attached to the additional conjugation moiety.

[0244] In some cases, the PEG group is not limited to a particular structure. In some cases, the PEG is linear (e.g., end-capped, e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG, or PEG attached to a polyol core), dendritic (or star) structure, each of which may or may not have one or more degradable linkages. 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 tripolymers, random tripolymers, and block tripolymers.

[0245] PEG typically comprises multiple (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how PEG is defined]. As used herein, the number of repeating units is referred to as "(OCH2CH2) n " is identified by the subscript "n." Thus, the value of (n) typically falls within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, about 225 to about 1930, about 450 to about 1930, about 1200 to about 1930, about 568 to about 2727, about 660 to about 2730, about 795 to about 2730, about 795 to about 2730, about 909 to about 2730, and about 1,200 to about 1,900. For any given polymer whose molecular weight is known, the number of repeat units (i.e., "n") can be determined by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0246] In some instances, PEG is an end-capped polymer, i.e., a polymer end-capped with a relatively inert group, e.g., a lower C 1-6It is a polymer having at least one end capped with an alkoxy group or a hydroxyl group. When the polymer is PEG, for example, methoxy-PEG (commonly referred to as mPEG) can be used, which is a linear form of PEG in which one end of the polymer is a methoxy (-OCH3) group, and the other end is a hydroxyl or other functional group that can be optionally chemically modified.

[0247] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 100 daltons to about 150,000 daltons. Exemplary ranges include, for example, weight-average molecular weights in the range of greater than 5,000 daltons to about 100,000 daltons, the range of about 6,000 daltons to about 90,000 daltons, the range of about 10,000 daltons to about 85,000 daltons, the range of greater than 10,000 daltons to about 85,000 daltons, the range of about 20,000 daltons to about 85,000 daltons, the range of about 53,000 daltons to about 85,000 daltons, the range of about 25,000 daltons to about 120,000 daltons, the range of about 29,000 daltons to about 120,000 daltons, the range of about 35,000 daltons to about 120,000 daltons, and the range of about 40,000 daltons to about 120,000 daltons.Exemplary weight-average molecular weights for PEG groups include about 100 daltons, about 200 daltons, about 300 daltons, about 400 daltons, about 500 daltons, about 600 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 900 daltons, about 1,000 daltons, about 1,500 daltons, about 2,000 daltons, about 2,200 daltons, about 2,500 daltons, about 3,000 daltons, about 4,000 daltons, about 4,400 daltons, about 4,500 daltons, about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, and about 9,000 daltons. about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG group is a linear PEG group having an average molecular weight as disclosed above. In some embodiments, the PEG group is a branched PEG group having an average molecular weight as disclosed above. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group having a defined molecular weight ± 10%, or 15%, or 20%, or 25%. For example, IL-2 conjugates comprising a 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 are within the scope of the present disclosure.

[0248] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear or branched PEG group having an average molecular weight of about 5,000 daltons to about 60,000 daltons. In some embodiments, the PEG group is about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, or about 25,000 daltons. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight of about 5,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons.

[0249] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear methoxy PEG group having an average molecular weight of about 5,000 daltons to about 60,000 daltons. In some embodiments, the PEG group is about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, or about 25,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 5,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 10,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 20,000 daltons.In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 30,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 50,000 daltons. In some embodiments, the PEG group is a linear methoxy PEG group having an average molecular weight of about 60,000 daltons. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a linear methoxy PEG group having a defined molecular weight ± 10%, or 15%, or 20%, or 25%. For example, IL-2 conjugates comprising a linear 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 are within the scope of the present disclosure.

[0250] In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons to about 60,000 daltons. In some embodiments, the PEG group is about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, or about 25,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons.In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 10,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 20,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 30,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 50,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 60,000 daltons. In some embodiments, the PEG group comprising the IL-2 conjugates disclosed herein is a branched methoxy PEG group having a defined molecular weight ± 10%, or 15%, or 20%, or 25%. For example, IL-2 conjugates containing branched methoxy PEG groups having molecular weights of 30,000 Da±3000 Da, or 30,000 Da±4,500 Da, or 30,000 Da±6,000 Da are included within the scope of this disclosure.

[0251] In some embodiments, exemplary PEG groups 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.

[0252] Conjugation Chemistry Conjugation Chemistry Various conjugation reactions are used to conjugate the linkers, conjugation moieties, and unnatural amino acids incorporated into the cytokine peptides described herein. Such conjugation reactions are often compatible with aqueous conditions, such as "bioorthogonal" reactions. In some embodiments, the conjugation reaction is mediated by a chemical reagent, such as a catalyst, light, or a reactive chemical group found on the linker, conjugation moiety, or unnatural amino acid. In some embodiments, the conjugation reaction is mediated by an enzyme. In some embodiments, the conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, the conjugation reaction used herein is described in Chen, X.; Wu, Y. W. Org. Biomol. Chem. 2016, 14, 5417. The disclosures of each of these references are incorporated herein by reference.

[0253] In some embodiments described herein, the conjugation reactions described herein comprise a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises the reaction of an azide with a phosphine (a "click" reaction). In some embodiments, the conjugation reaction is catalyzed by copper. In some embodiments, the conjugation reactions described herein result in cytokine peptides comprising a linker or conjugation moiety attached via a triazole. In some embodiments, the conjugation reactions described herein comprise the reaction of an azide with a strained olefin. In some embodiments, the conjugation reactions described herein comprise the reaction of an azide with a strained alkyne. In some embodiments, the conjugation reactions described herein comprise the reaction of an azide with a cycloalkyne, e.g., DBCO.

[0254] In some embodiments described herein, the conjugation reactions described herein include those outlined in Scheme S1, where X is a position in an IL-2 conjugate that includes an unnatural amino acid, e.g., in any one of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

[0255] [ka]

[0256] In some embodiments, the conjugating moiety comprises a water soluble polymer, hi some embodiments, the reactive group comprises an alkyne or an azide.

[0257] In some embodiments described herein, the conjugation reactions described herein include those outlined in Scheme S2, where X is a position in an IL-2 conjugate that includes an unnatural amino acid, e.g., in any one of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

[0258] [ka]

[0259] In some embodiments described herein, the conjugation reactions described herein include those outlined in Scheme S3, where X is a position in an IL-2 conjugate that includes an unnatural amino acid, e.g., in any one of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

[0260] [ka]

[0261] In some embodiments described herein, the conjugation reactions described herein include those outlined in Scheme S4, where X is a position in an IL-2 conjugate comprising an unnatural amino acid, e.g., in any one of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

[0262] [ka]

[0263] In some embodiments described herein, the conjugation reactions described herein involve a cycloaddition reaction between an azide moiety, such as one contained in a protein containing an amino acid residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as one derived from DBCO, a chemical moiety containing a dibenzocyclooctyne group. PEG groups containing DBCO moieties are commercially available or can be prepared by methods known to those skilled in the art. Exemplary reactions are shown in Schemes S5 and S6.

[0264] [ka] [ka]

[0265] Conjugation reactions, such as the click reactions described herein, can produce a single positional isomer or a mixture of positional isomers. In some examples, the ratio of positional isomers is about 1:1. In some examples, the ratio of positional isomers is about 2:1. In some examples, the ratio of positional isomers is about 1.5:1. In some examples, the ratio of positional isomers is about 1.2:1. In some examples, the ratio of positional isomers is about 1.1:1. In some examples, the ratio of positional isomers is greater than 1:1.

[0266] Cytokine Polypeptide Production In some examples, the IL-2 conjugates described herein contain natural or non-natural amino acid mutations and are recombinantly produced or chemically synthesized. In some examples, the IL-2 conjugates described herein are recombinantly produced, for example, by a host cell system or in a cell-free system. In any of the embodiments or variations described herein, the amino acids can be L-amino acids or D-amino acids. In some embodiments, the amino acids are L-amino acids. In other embodiments, the amino acids are D-amino acids.

[0267] In some instances, the IL-2 conjugate is recombinantly produced via a host cell system. In some instances, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (e.g., a gram-positive or gram-negative bacterium). In some instances, the eukaryotic host cell is a mammalian host cell. In some instances, the mammalian host cell is a stable cell line, or a cell line that has incorporated the genetic material of interest into its genome and is capable of expressing the product of the genetic material after many generations of cell division. In other instances, the mammalian host cell is a transient cell line, or a cell line that has not incorporated the genetic material of interest into its genome and is not capable of expressing the product of the genetic material after many generations of cell division.

[0268] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F(TM) cells, Flp-In(TM) T-REx(TM) 293 cell line, Flp-In(TM)-293 cell line, Flp-I n(TM)-3T3 cell line, Flp-In(TM)-BHK cell line, Flp-In(TM)-CHO cell line, Flp-In(TM)-CV-1 cell line, Flp-In(TM)-Jurkat cell line , FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[0269] 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.

[0270] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris (K. phaffii) yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0271] In some embodiments, the eukaryotic host cell is a plant host cell. In some examples, the plant cell comprises a cell derived from algae. Exemplary plant cell lines include Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942 strains.

[0272] 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™.

[0273] In some examples, polynucleic acid molecules or vectors suitable for producing the IL-2 polypeptides described herein include any suitable vector derived from a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include those derived from bacterial (e.g., E. coli), insect, yeast (e.g., Pichia pastoris, K. phaffii), algae, or mammalian sources. Examples of bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0274] Examples of insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2 or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0275] Examples of yeast vectors include the Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris (K. phaffii) vector, pGAPZA,B,&C Pichia pastoris (K. phaffii) vector, pPIC3.5K Pichia vector, pPIC6 Examples include the A, B, & C Pichia vector, the pPIC9K Pichia vector, pTEF1 / Zeo, the pYES2 yeast vector, the pYES2 / CT yeast vector, the pYES2 / NT A, B, & C yeast vector, or the pYES3 / CT yeast vector.

[0276] Examples of algal vectors include the pChlamy-4 vector or the MCS vector.

[0277] Examples of mammalian vectors include transient expression vectors or stable expression vectors.Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, b, c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3 or pBICEP-CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1 or pBICEP-CMV 2.

[0278] In some examples, cell-free systems are used to produce the cytokine (e.g., IL-2) polypeptides described herein. In some cases, the cell-free system comprises a mixture of cytoplasmic and / or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some examples, the cell-free system utilizes prokaryotic components. In other examples, the cell-free system utilizes eukaryotic 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+.

[0279] Cell-free translation systems include a variety of components, such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and / or unnatural amino acids, and / or other components used in protein expression. Such components are optionally modified to improve yield, increase synthesis rate, increase protein product fidelity, or incorporate unnatural amino acids. In some embodiments, the cytokines described herein are synthesized using a cell-free translation system described in U.S. Patent No. 8,778,631; U.S. Patent Application Publication No. 2017 / 0283469; U.S. Patent Application Publication No. 2018 / 0051065; U.S. Patent Application Publication No. 2014 / 0315245; or U.S. Patent No. 8,778,631. In some embodiments, the cell-free translation system includes modified release factors or even the removal of one or more release factors from the system. In some embodiments, the cell-free translation system comprises a reduced concentration of proteases. In some embodiments, the cell-free translation system comprises a modified tRNA in which the codon used to encode the unnatural amino acid has been reassigned. In some embodiments, a synthetase described herein for incorporating an unnatural amino acid is used in the cell-free translation system. In some embodiments, the unnatural amino acid is preloaded onto the tRNA using enzymatic or chemical methods before the tRNA is added to the cell-free translation system. In some embodiments, components for the cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organisms.

[0280] In some embodiments, the cytokine (eg, IL-2) polypeptide is produced in a circularly permuted form via an expression host system or via a cell-free system.

[0281] Production of cytokine polypeptides containing unnatural amino acids Orthogonal or extended genetic codes can be used in the present disclosure, in which one or more specific codons present within the nucleic acid sequence of a cytokine (e.g., IL-2) polypeptide are assigned to encode an unnatural amino acid, which can be genetically incorporated into the cytokine (e.g., IL-2) by using an orthogonal tRNA synthetase / tRNA pair that can add a tRNA with the unnatural amino acid, which can incorporate the unnatural amino acid into the polypeptide chain depending on the codon.

[0282] In some instances, the codon is an amber codon, an ochre codon, an opal codon, or a quadruplet codon. In some instances, the codon corresponds to an orthogonal tRNA that will be used to carry the unnatural amino acid. In some instances, the codon is an amber codon. In other instances, the codon is an orthogonal codon.

[0283] In some instances, the codon is a quadruplet codon, which is decoded by the orthogonal ribosome ribo-Q1. In some instances, the quadruplet codon is as shown in Neumann et al., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature, 464(7287):441-444 (2010), the disclosure of which is incorporated herein by reference.

[0284] In some examples, the codons used in the present disclosure are recoded codons, e.g., synonymous codons, or rare codons replaced with alternative codons. In some cases, the recoded codons are as described in Napolitano et al., "Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli," PNAS, 113(38):E5588-5597 (2016). In some cases, the recoded codons are as described in Ostrov et al., "Design, synthesis, and testing toward a 57-codon genome," Science, 353(6301):819-822 (2016). The disclosures of each of these references are incorporated herein by reference.

[0285] In some examples, unnatural nucleic acids are utilized to provide for the incorporation of one or more unnatural amino acids into a cytokine (e.g., IL-2). Exemplary unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine. uracil, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 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-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-aza-guanine 7-deazaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), pyrido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil , 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine,and U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5,093,232; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711. ; U.S. Patent No. 5,552,540; U.S. Patent No. 5,587,469; U.S. Patent No. 5,594,121; U.S. Patent No. 5,596,091; U.S. Patent No. 5,614,617; U.S. Patent No. 5,645,985; U.S. Patent No. 5,681,941; U.S. Patent No. 5,750,692; U.S. Patent No. 5,763,588; U.S. Patent No. 5,830,653 and U.S. Patent No. 6,005,096; WO 99 / 62923; Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, ed., John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, 1993, pp. 273-288. Additional base modifications can be found, for example, in U.S. Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, 1993, pp. 289-302. The disclosures of each of these references are incorporated herein by reference.

[0286] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and nucleic acids sometimes contain one or several heterocyclic bases other than the five main base components of naturally occurring nucleic acids. For example, heterocyclic bases sometimes include uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, and 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-3-yl groups, where purines are linked to the sugar moiety of nucleic acids via the 9-position, pyrimidines via the 1-position, pyrrolopyrimidines via the 7-position, and pyrazolopyrimidines via the 1-position.

[0287] In some embodiments, the nucleotide analogue is modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those with modifications at the bond between two nucleotides, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate bonds or modified phosphate bonds between two nucleotides are via a 3'-5' or 2'-5' bond, and the bond contains an inverted polarity, for example, 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Numerous U.S. patents teach methods for making and using nucleotides containing modified phosphates, including, but not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; No. 5,405,939; U.S. Pat. No. 5,453,496; U.S. Pat. No. 5,455,233; U.S. Pat. No. 5,466,677; U.S. Pat. No. 5,476,925; U.S. Pat. No. 5,519,126; U.S. Pat. No. 5,536,821; U.S. Pat. No. 5,541,306; U.S. Pat. No. 5,550,111; U.S. Pat. No. 5,563,253; U.S. Pat. No. 5,571,799; U.S. Pat. No. 5,587,361; and U.S. Pat. No. 5,625,050, the disclosures of each of which are incorporated herein by reference.

[0288] In some embodiments, the non-naturally occurring nucleic acids include 2',3'-dideoxy-2',3'-didehydronucleosides (International Application No. PCT / US2002 / 006460), 5'-substituted DNA and RNA derivatives (International Application No. PCT / US2011 / 033961; Saha et al., J. Org Chem., 1995, Vol. 60, pp. 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, Vol. 9, pp. 885-890; Mikhailov et al., Nucleosides & Nucleotides, 1991, Vol. 10(Is. 1-3), pp. 339-343; Leonid et al., 1995, Vol. 14(Is. 3-5), pp. 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, Vol. 87, pp. 3004-3020; International Application No. PCT / JP2000 / 004720; International Application No. PCT / JP2003 / 002342; International Application No. PCT / JP2004 / 013216; International Application No. PCT / JP2005 / 020435; International Application No. PCT / JP2006 / 315479; International Application No. PCT / JP2006 / 324484; International Application No. PCT / JP2009 / 056718; International Application No. PCT / JP2010 / 067560), or 5'-substituted monomers prepared as monophosphates with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, Vol. 23(1 & 2), pp. 317-337). The disclosure of each of these references is incorporated herein by reference.

[0289] In some embodiments, the non-natural nucleic acid comprises modifications at the 5'- and 2'-positions of the sugar ring (International Application PCT / US94 / 02993), such as 5'-CH2-substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, Vol. 83, pp. 1127-1143 and Wu et al., Bioconjugate Chem. 1999, Vol. 10, pp. 921-924). In some cases, the non-natural nucleic acid comprises an amide-linked nucleoside dimer prepared for incorporation into an oligonucleotide, wherein the 3'-linked nucleoside (5' to 3') in the dimer comprises a 2'-OCH3 and a 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, pp. 1287-1290). Non-natural nucleic acids may include 2'-substituted 5'-CH2 (or O) modified nucleosides (International Application PCT / US92 / 01020). Non-natural nucleic acids may include 5'-methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids may include 5'-phosphonate monomers with 2'-substitutions (U.S. Patent Application Publication No. 2006 / 0074035) and other modified 5'-phosphonate monomers (WO 1997 / 35869). Non-natural nucleic acids may include 5'-modified methylene phosphonate monomers (EP 614907 and EP 629633). Non-natural nucleic acids may include 5'- or 6'-phosphonate ribonucleoside analogs containing hydroxyl groups at the 5'- and / or 6'-positions (Chen et al., Phosphorus, Sulfur and Silicon, 2002, Vol. 777, pp. 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, Vol. 8, pp. 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, pp. 925-933; and Hampton et al., J. Med. Chem., 1976, Vol. 19(8), pp. 1029-1033).Non-natural nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers with a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), pp. 68-82). Non-natural nucleic acids may include nucleosides with a 6'-phosphonate group, where the 5'- and / or 6'-positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH)) (and its analogs), a methyleneamino group (CHNH) (and its analogs), or a cyano group (CN) (and its analogs) (Fairhurst et al., Synlett, 2001, 4, pp. 467-472; Kappler et al., J. Med. Chem., 1986, 29, pp. 1030-1039). 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). The disclosures of each of these references are incorporated herein by reference.

[0290] Also, in some embodiments, non-natural nucleic acids contain modifications of the sugar moiety. In some cases, nucleic acids contain one or more nucleosides, and the sugar group is modified. Such sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, nucleic acids contain chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (5' and / or 2' substituents; bridging of two ring atoms to form bicyclic nucleic acids (BNAs); S, N(R), or C(R1)(R2) (R = H, C1-C 12and combinations thereof. Examples of chemically modified sugars can be found in WO 2008 / 101157, U.S. Patent Application Publication No. 2005 / 0130923, and WO 2007 / 134181, the disclosures of each of which are incorporated herein by reference.

[0291] In some examples, modified nucleic acids contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, with the sugar attached to each heterocyclic base in either the alpha or beta anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, the sugar modification 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. Preparation of these sugars or sugar analogs, and their respective "nucleosides" (where such sugars or analogs are attached to heterocyclic bases (nucleobases)), is known. Sugar modifications can also be combined with other modifications.

[0292] Modifications to the sugar moiety include 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6.10 , alkyl or C2-C 10 The 2' sugar modifications may be, 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 from 1 to about 10.

[0293] Other modifications at the 2' position include, but are not limited to: C1 to C 10Modified sugars include lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalating agents, groups that improve the pharmacokinetic properties of oligonucleotides, or groups that improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications are also made at other positions on the sugar on the 3'-terminal nucleotide or in 2'-5'-linked oligonucleotides, particularly the 3'-position of the sugar and the 5'-position of 5'-terminal nucleotide. Modified sugars also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar.Numerous U.S. patents exist which teach the preparation of such modified sugar structures and which detail and describe a wide range of base modifications, e.g., U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 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. ;U.S. Patent No. 5,658,873;U.S. Patent No. 5,670,633;U.S. Patent No. 4,845,205;U.S. Patent No. 5,130,302;U.S. Patent No. 5,134,066;U.S. Patent No. 5,175,273;U.S. Patent No. 5,367,066;U.S. Patent No. 5,432,272;U.S. Patent No. 5,457,187;U.S. Patent No. 5,459,255;U.S. Patent No. 5,484,908;U.S. Patent No. 5,502,177;U.S. Patent No. 5,525,711;U.S. Patent No. 5,552,540;U.S. Patent No. 5,587,469;U.S. Patent No. 5,594,121;U.S. Patent No. 5,596,091;U.S. Patent No. 5,614,617;U.S. Patent No. 5,681,941; and U.S. Patent No. 5,700,920. Each of these patents is incorporated herein by reference in its entirety.

[0294] Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Additionally, the substituent at the 2' position can be allyl, amino, azido, thio, O-allyl, O-(C1-C2). 10 alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ) and O-CH2-C(=O)-N(R m )(R n), wherein R m and R n are each independently H or a substituted or unsubstituted C1 to C 10 It is alkyl.

[0295] In certain embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge comprises a 4' to 2' bicyclic nucleic acid. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, those of the formula: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2', and analogs thereof (see U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH 3) -O-2' and analogs thereof (see WO 2009 / 006478, WO 2008 / 150729, U.S. Patent Application Publication No. 2004 / 0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., Vol. 209, No. 74, pp. 118-134, and WO 2008 / 154401). Also, the disclosures of each of these are incorporated herein by reference, e.g., Singh et al., Chem. Commun., 1998, Vol. 4, pp. 455-456; Koshkin et al., Tetrahedron, 1998, Vol. 54, pp. 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, pp. 5633-5635. 638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, Vol. 8, pp. 2219-2222; Singh et al., J. Org. Chem., 1998, Vol. 63, pp. 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, Vol. 129 (No. 26), pp. 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, Vol. 2, pp. 558-561; Braasch et al., Chem. Biol., 2001, Vol. 8, pp. 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, Vol. 3, pp. 239-243; U.S. Patent No. 4,849,513; U.S. Patent No. 5,015,733; U.S. Patent No. 5,118,800; U.S. Patent No. 5,118,802; U.S. Patent No. 7,053,207; U.S. Patent No. 6,268,490; U.S. Patent No. 6,770,748; U.S. Patent No. 6,794,499; U.S. Patent No. 7,034,133; U.S. Patent No. 6,525,191; U.S. Patent No. 6,670,461; and U.S. Patent No. 7,399,845; WO 2004 / 106356, WO 1994 / 14226, WO 2005 / 021570, WO 2007 / 090071, and WO 2007 / 134181; U.S. Patent No. Patent Application Publication No. 2004 / 0171570, U.S. Patent Application Publication No. 2007 / 0287831 and U.S. Patent Application Publication No. 2008 / 0039618; U.S. Provisional Patent Application No. 60 / 989,574, U.S. Provisional Patent Application No. 61 / 026,995, U.S. Provisional Patent Application No. 61 / 026,998, U.S. Provisional Patent Application No. 61 / 056,564, U.S. Provisional Patent See U.S. Provisional Patent Application No. 61 / 086,231, U.S. Provisional Patent Application No. 61 / 097,787, and U.S. Provisional Patent Application No. 61 / 099,844; and International Application No. PCT / US2008 / 064591, International Application No. PCT / US2008 / 066154, International Application No. PCT / US2008 / 068922, and International Application No. PCT / DK98 / 00393.

[0296] In certain embodiments, the nucleic acid comprises a linked nucleic acid. Nucleic acids can be linked together using any internucleic acid linkage. Two major classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); N,N *-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleic acid linkages with chiral atoms can be prepared as racemic mixtures or as separate enantiomers, such as alkylphosphonates and phosphorothioates. Non-natural nucleic acids may contain a single modification. Non-natural nucleic acids may contain multiple modifications within one of the moieties or between different moieties.

[0297] Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate linkages may also be used.

[0298] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity to the modified nucleic acid and / or enhance its stability in vivo.

[0299] In some examples, a phosphorus derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, and the like. Exemplary polynucleotides containing modified phosphate or non-phosphate linkages are described in Peyrottes et al., 1996, Nucleic Acids Res. 24:1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; Schultz et al. (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, "Oligonucleotide Analogs: an Overview" (Oligonucleotides as Therapeutic Agents (Chadwick and Cardew, eds.), John Wiley and Sons, New York, NY); Zon, 1993, "Oligonucleoside Phosphorothioates" (Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190); Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Pat. No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem. 8:1157-1179, the disclosures of each of which are incorporated herein by reference.

[0300] In some cases, backbone modifications include replacing phosphodiester bonds with alternative moieties, such as anionic, neutral, or cationic groups. Examples of such modifications include anionic internucleoside linkages; N3' to P5' phosphoramidate modifications; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages, such as methylphosphonate; amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8:1157-1179, the disclosure of which is incorporated herein by reference). Modified nucleic acids may contain chimeric or mixed backbones containing one or more modifications, for example, combinations of phosphate linkages, such as combinations of phosphodiester and phosphorothioate linkages.

[0301] Alternatives to phosphates include short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties. Numerous U.S. patents disclose methods of making and using these types of phosphate substitutes, including but not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; US Patent No. 5,541,307; US Patent No. 5,561,225; US Patent No. 5,596,086; US Patent No. 5,602,240; US Patent No. 5,610,289; US Patent No. 5,602,240; US Patent No. 5,608,046; US Patent No. 5,610,289; US Patent No. 5,618,704; US Patent No. 5,623,070; US Patent No. 5,663,312; US Patent No. 5,633,360; US Patent No. 5,677,437; and US Patent No. 5,677,439, the disclosures of each of which are incorporated herein by reference.It is also understood that in nucleotide substitution, both the sugar moiety and the phosphate moiety of the nucleotide can be replaced, for example, by an amide-type bond (aminoethylglycine) (PNA).U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262 teach methods for forming and using PNA molecules, each of which is incorporated herein by reference. Also see Nielsen et al., Science, 1991, vol. 254, pp. 1497-1500. Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs, for example, to enhance cellular absorption. Conjugates can be chemically linked to nucleotides or nucleotide analogs.Such conjugates may include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, vol. 18, pp. 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, vol. 14, pp. 969-973), adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, vol. 36, pp. 3651-3654), palmityl moieties (Mishra et al., Biochem. Biophys. Acta, 1995, vol. 1264, pp. 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, vol. 277, pp. 923-937).Numerous U.S. patents teach the preparation of such conjugates, including but not limited to U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; and 5,118,802. No.; U.S. Patent No. 5,138,045; U.S. Patent No. 5,414,077; U.S. Patent No. 5,486,603; U.S. Patent No. 5,512,439; U.S. Patent No. 5,578,718; U.S. Patent No. 5,608,046; U.S. Patent No. 4,587,044; U.S. Patent No. 4,605,735; U.S. Patent No. 4,667,025; U.S. Patent No. 4,762,779; U.S. Patent No. 4,789,737; U.S. Patent No. 4,824,941; U.S. Patent No. 4,835,263; U.S. Patent No. 4,876,335; U.S. Patent No. 4,904,582; U.S. Patent No. 4, No. 958,013; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,245,022; U.S. Patent No. 5,254,469; U.S. Patent No. 5,258,506; U.S. Patent No. 5,262,536; U.S. Patent No. 5,272,250; U.S. Patent No. 5,292,873; U.S. Patent No. 5,317,098; U.S. Patent No. 5,371,241; U.S. Patent No. 5,391,723 ; U.S. Patent No. 5,416,203; U.S. Patent No. 5,451,463; U.S. Patent No. 5,510,475; U.S. Patent No. 5,512,667; U.S. Patent No. 5,514,785; U.S. Patent No. 5,565,552; U.S. Patent No. 5,567,810; U.S. Patent No. 5,574,142; U.S. Patent No. 5,585,481; U.S. Patent No. 5,587,371; U.S. Patent No. 5,595,726; U.S. Patent No. 5,597,696; U.S. Patent No. 5,599,923; U.S. Patent No. 5,599,928 and U.S. Patent No. 5,688,941.The disclosure of each of these references is incorporated herein by reference.

[0302] In some cases, the unnatural nucleic acid further forms unnatural base pairs. Exemplary unnatural nucleotides that can form unnatural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. In some embodiments, the unnatural nucleotide is: [ka] Exemplary unnatural base pairs include (d)TPT3-(d)NaM; (d)5SICS-(d)NaM; (d)CNMO-(d)TAT1; (d)NaM-(d)TAT1; (d)CNMO-(d)TPT3; and (d)5FM-(d)TAT1.

[0303] Other examples of unnatural nucleotides capable of forming unnatural UBPs that can be 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. 2013, 135:5408-5419; and Malyshev et al., Proc Natl Acad Sci USA 2012, 109:12005-12010, the disclosures of each of which are incorporated herein by reference. In some embodiments, non-naturally occurring nucleotides include: [ka]

[0304] In some embodiments, the non-naturally occurring nucleotide that can be used to prepare the IL-2 conjugates disclosed herein has the formula [ka] and wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, meth...

Claims

1. 1. A pharmaceutical composition comprising a therapeutically effective amount of an IL-2 conjugate for use in a method of treating cancer in a subject in need thereof, the method further comprising administering to the subject one or more PD-1 inhibitors; The IL-2 conjugate comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:50, and [AzK_L1_PEG30kD] is located at position 64 relative to the position of SEQ ID NO:50, wherein [AzK_L1_PEG30kD] has the structure of formula (XII) or formula (XIII): 【Chemistry 1】 (In the formula: n is -(OCH 2 CH 2 ) n -OCH 3 is an integer such that the PEG group having the structure has a molecular weight of 30 kDa ± 15%; The wavy lines indicate the covalent bond to the first amino acid residue preceding and following [AzK_L1_PEG30kD] in SEQ ID NO:50, respectively. , the pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the IL-2 conjugate comprises amino acids that are at least 98% identical in sequence to the amino acid sequence of SEQ ID NO:50, and [AzK_L1_PEG30kD] is located at position 64 relative to the position of SEQ ID NO:

50.

3. 2. The pharmaceutical composition of claim 1, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO:50, in which [AzK_L1_PEG30kD] is located at position 64 relative to the position of SEQ ID NO:

50.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the IL-2 conjugate is provided as a mixture, the mixture comprising: (i) an IL-2 conjugate, wherein [AzK_L1_PEG30kD] has the structure of formula (XII); and (ii) an IL-2 conjugate, wherein [AzK_L1_PEG30kD] has the structure of formula (XIII).

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the IL-2 conjugate and the one or more PD-1 inhibitors are formulated as a single combination composition.

6. The pharmaceutical composition of any one of claims 1 to 4, wherein the IL-2 conjugate and the one or more PD-1 inhibitors are formulated as individual compositions.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the one or more PD-1 inhibitors comprise pembrolizumab, nivolumab, or cemiplimab.

8. 8. The pharmaceutical composition of claim 7, wherein the one or more PD-1 inhibitors comprise pembrolizumab.

9. 8. The pharmaceutical composition of claim 7, wherein the one or more PD-1 inhibitors comprise nivolumab.

10. 8. The pharmaceutical composition of claim 7, wherein the one or more PD-1 inhibitors comprise cemiplimab.

11. The cancers are renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable carcinoma, microsatellite stable carcinoma, gastric cancer, colon cancer, colorectal cancer (CRC), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal, esophageal squamous cell carcinoma (ESCC), glioblastoma, mesothelioma, breast cancer, triplicate, The pharmaceutical composition according to any one of claims 1 to 10, wherein the cancer is selected from: primary negative breast cancer, prostate cancer, castration resistant prostate cancer, metastatic castration resistant prostate cancer or metastatic castration resistant prostate cancer with DNA damage response (DDR) deficiency, bladder cancer, ovarian cancer, tumors with moderate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), tumors systemically disseminated beyond their primary anatomical site of origin to the liver and CNS, and diffuse large B-cell lymphoma.

12. 12. The pharmaceutical composition of claim 11, wherein the cancer is selected from non-small cell lung cancer (NSCLC), classical Hodgkin's lymphoma (cHL), head and neck squamous cell carcinoma (HNSCC), colon cancer, colorectal cancer, and melanoma.

13. The pharmaceutical composition of claim 12, wherein the cancer is colon cancer.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the IL-2 conjugate is administered to a subject once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks.

15. The pharmaceutical composition of claim 14, wherein the IL-2 conjugate is administered to the subject once weekly.

16. The pharmaceutical composition of claim 14, wherein the IL-2 conjugate is administered to the subject once every two weeks.

17. The pharmaceutical composition of claim 14, wherein the IL-2 conjugate is administered to the subject once every three weeks.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

19. The pharmaceutical composition according to any one of claims 1 to 17, wherein the IL-2 conjugate is administered to the subject by subcutaneous administration.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the IL-2 conjugate is a pharma- ceutically acceptable salt, solvate, or hydrate.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein the IL-2 conjugate is administered at a dose of 10 μg ± 15% IL-2 conjugate per kg of subject body weight to 200 μg ± 15% IL-2 conjugate per kg of subject body weight.

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