RU2840258C2 - Types of immuno-oncological combination therapy using il-2 conjugates - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention relates to biotechnology and can be used in medicine. Presented is a method for immunotherapy of various proliferative diseases involving administering to a subject a combination of an IL-2 conjugate with DBCO-PEG and an anti-PD-1 antibody. Materials of the present application demonstrate the advantage of using the IL-2 conjugate in combination with a PD-1 inhibitor for treating cancer as compared to using said therapeutic agents separately.

EFFECT: disclosed are types of immuno-oncological combination therapy using il-2 conjugates.

18 cl, 27 dwg, 35 tbl, 17 ex

Description

CROSS-REFERENCES 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 hereby incorporated by reference in their entireties.

SEQUENCE LIST

This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 12, 2020, is named 2020-08-12_01183-0073-00PCT_seq_listing.txt and is 128,000 bytes in size.

BACKGROUND OF THE INVENTION

Distinct T cell populations modulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T cells (Tregs) prevent inappropriate immune responses by preventing pathological autoreactivity, while cytotoxic T cells target and destroy infected cells and/or cancer cells. In some cases, modulation of different T cell populations represents a treatment option for a disease or indication. In some cases, this is facilitated by the availability of additional agents or modalities in combination therapy.

Accordingly, in one aspect, provided herein are methods of treating cancer in a subject comprising administering to the subject an IL-2 conjugate in combination with one or more immune checkpoint inhibitors.

BRIEF DESCRIPTION OF THE INVENTION

Described herein are methods for treating cancer in certain embodiments. The following embodiments are encompassed.

Embodiment A1 is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount 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, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (I):

Formula (I);

Where:

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa;

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

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.

Embodiment A2 is the method of embodiment A1, wherein in the IL-2 conjugate, Z is CH ₂ and Y is .

Embodiment A3 is the method of embodiment A1, wherein in the IL-2 conjugate Y is CH ₂ and Z is .

Embodiment A4 is the method of embodiment A1, wherein in the IL-2 conjugate, Z is CH ₂ and Y is .

Embodiment A5 is the method of embodiment A1, wherein in the IL-2 conjugate Y is CH ₂ and Z is .

Embodiment A6 is the method of any one of embodiments A1-A5, wherein in the IL-2 conjugate the PEG group has an average molecular weight of 25 kDa, 30 kDa, or 35 kDa.

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.

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

Embodiment A9 is a method according to embodiment A1, wherein the structure of formula (I) has a structure of formula (X) or formula (XI) or is a mixture of formula (X) and formula (XI):

Formula (X);

(XI);

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Embodiment A10 is the method according to 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.

Embodiment A11 is the method of embodiments A9 or A10, wherein in the IL-2 conjugate n is an integer such that -( _OCH2CH2 ) _{n -OCH3 has} a molecular weight of about 25 kDa, 30 kDa, or 35 kDa.

Embodiment A12 is the method of embodiment A11, wherein in the IL-2 conjugate n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 30 kDa.

Embodiment A13 is a method according to embodiment A1, wherein the structure of formula (I) has a structure of formula (XII) or formula (XIII) or is a mixture of formula (XII) and formula (XIII):

Formula (XII);

(XIII);

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Embodiment A14 is the method according to 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.

Embodiment A15 is the method of embodiments A13 or A14, wherein in the IL-2 conjugate n is an integer such that - _{( OCH2CH2} ) _n - _OCH3 has a molecular weight of about 25 kDa, 30 kDa, or 35 kDa.

Embodiment A16 is the method of embodiment A15, wherein in the IL-2 conjugate n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 30 kDa.

Embodiment A17 is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount 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, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV) or formula (V) or is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa;

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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.

Embodiment A19 is a method according to embodiment A18, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment A20 is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount 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, wherein [AzK_L1_PEG30kDa] has a structure of formula (XII) or formula (XIII), or is a mixture of structures of formula (XII) and formula (XIII):

Formula (XII);

(XIII);

Where:

n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular mass of approximately 30 kDa; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 50 that are not replaced.

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

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

Embodiment A23 is the method of embodiment A22 wherein the one or more PD-1 inhibitors are pembrolizumab.

Embodiment A24 is the method of embodiment A22 wherein the one or more PD-1 inhibitors are nivolumab.

Embodiment A25 is the method of any one of Embodiments A1-A24, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer (CRC), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, esophageal squamous cell carcinoma (ESCC), glioblastoma, mesothelioma, breast cancer, triple-negative breast cancer, prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or metastatic castration-resistant prostate cancer with defects in DNA damage response (DDR), bladder cancer, ovarian cancer, tumors with intermediate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low to absent PD-L1 expression, tumors that have spread systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma.

Embodiment A26 is a method according to any one of embodiments A1-A25, wherein the IL-2 conjugate is administered to the subject once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks.

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

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

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

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

Figure 1 shows a graph of the antitumor activity of compound A, dosed IV using the QWx3 schedule, from Study 1 in Example 11. Black arrows indicate the days of compound A dosing.

Fig. 2 shows a plot of tumor volumes for Compound A, dosed IV using the QWx3 schedule, from Study 1 in Example 11.

Figure 3 shows tumor volumes at day 15 post-treatment for each animal treated with Compound A QWx3 dosing from Study 1 in Example 11. Black arrows indicate days of Compound A dosing.

Fig. 4 shows tumor volumes at day 15 post-treatment for each animal administered the Compound A Q2Wx2 doses from Study 1 in Example 11.

Fig. 5 shows the mean tumor growth curves for mice treated 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 of Compound A dosing.

Fig. 6 shows a graph of % TGI data at day 15 post-treatment in the group treated with the combination of Compound A and anti-PD-1 antibody compared to the 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; compared to vehicle control. ^┴ p < 0.05 compared to anti-PD-1 antibody. #p < 0.05 compared to Compound A. Data represent mean tumor volume ± SEM (14 mice/group).

Fig. 7 shows a graph of the Kaplan-Meier survival curves for the treatment groups from Study 2 in Example 11. *p < 0.05 versus vehicle control. ^┴ p < 0.05 versus anti-PD-1 antibody. #p < 0.05 versus Compound A.

Figure 8 shows the mean tumor growth curves for doses of Compound A as monotherapy 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 12 mice/group for 9 mg/kg Compound A). Black arrows indicate days of Compound A dosing.

Figure 9 shows individual tumor volumes at day 15 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 compared to vehicle control.

Fig. 10 shows a graph of the Kaplan-Meier survival curves for the treatment groups receiving treatment with vehicle (control), anti-PD-1 antibody alone, Compound A alone, and a combination of Compound A and anti-PD-1 antibody. *p < 0.05 versus vehicle control from Study 3 in Example 11. ^┴ p < 0.05 versus anti-PD-1 antibody. #p < 0.05 versus Compound A.

Fig. 11A and Fig. 11B show graphs of exemplary cytokine levels for IL-2 and IL-2_P65[AzK_L1_PEG30kDa]-1 alone and in combination with nivolumab (Nivo) or pembrolizumab (Pem) for one donor from Example 12. Fig. 11A shows a graph of IFN-gamma, IL-8, IL-6, TNF-alpha, IL-4, and IL-5 levels. Fig. 11B shows a graph of IL-6, TNF-alpha, and IL-5 levels.

Fig. 12 shows the release of interferon gamma in a mixed lymphocyte reaction (MLR) assay for the combination of Compound B (IL-2_P65[AzK_L1_PEG30kDa]-1) and pembrolizumab according to Example 13.

Fig. 13 and Fig. 14 show the release of interferon gamma in a mixed lymphocyte reaction (MLR) assay for the combination of Compound B (IL-2_P65[AzK_L1_PEG30kDa]-1) and nivolumab according to Example 13.

Fig. 15 shows the pharmacokinetic properties of compound B from Example 14.

Fig. 16A-16D show the number of pSTAT5+ cells among peripheral blood CD8+ T cells, memory CD8+ T cells, NK cells and Treg cells, respectively, after administration of compound B according to Example 14.

Fig. 17A-17G show the activation of Ki67 in CD8+ T, NK and Treg cell populations by Compound B according to Example 14.

Fig. 18A-18D show the analyses of tumor samples (levels of CD8+ T cells, NK cells and Treg cells and CD8+/Treg ratio) after treatment with Compound B according to Example 14.

Fig. 19 shows TCR diversity following treatment with compound B and mouse anti-PD-1 antibody according to Example 15.

Fig. 20 shows the clonality of TILs as a function of the proportion of T cells following the indicated treatments (e.g., with Compound B and/or anti-mouse PD-1 antibody) according to Example 15.

Fig. 21 shows the clonality of T cells after treatment with Compound B compared to the vehicle control according to Example 15.

Fig. 22 shows the expression heat map in CT26 tumor samples at day 8 after treatment with control (vehicle), compound B (6 mg/kg), anti-mouse PD-1 antibody (10 mg/kg), or a combination of compound B and anti-mouse PD-1 antibody (N=10 mice per group) from Example 16.

Fig. 23A-23C show key expression reporters of the tumor microenvironment status after treatment with Compound B according to Example 16: analysis of infiltration of activated CD8+ effector and memory T cells and cytolytic NK cells. CTL=control (vehicle); Compound B=compound B; aPD1=anti-mouse PD-1; Compound B aPD1=combination of Compound B and anti-mouse PD-1.

Fig. 24A-24B show profiling analysis of interferon γ signature gene expression levels in response to therapy according to Example 16. CTL=control (vehicle); Compound B=compound B; aPD1=anti-mouse PD-1 antibody; Compound B aPD1=combination of Compound B and anti-mouse PD-1 antibody.

Fig. 25 and Fig. 26 show the evaluation of tumor survival and growth in re-stimulated tumor-free animals according to Example 17.

Fig. 27A and Fig. 27B show that Compound B promotes an overall increase in the number of memory T cells (CD3+), including memory CD8+ T cells, in the peripheral blood of restimulated mice according to Example 17.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the foregoing general description and the following detailed description are illustrative and explanatory only and are not limiting of any claimed subject matter. To the extent that any material incorporated herein by reference is inconsistent with the express teachings of the present disclosure, the express teachings shall control.

Definitions

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 singular includes the plural unless specifically stated otherwise. It should be noted that the singular forms used in the description and appended claims include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless otherwise stated. In addition, the use of the term "including", as well as other forms such as "include", "includes" and "included" is not limiting.

Reference in this specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present invention.

Ranges and amounts as used herein may be expressed as "about" a particular value or range. "About" also includes an exact amount. Thus, "about 5 µL" means "about 5 µL" as well as "5 µL." Generally, the term "about" includes an amount that is expected to be within the experimental error, such as within 15%, 10%, or 5%.

The section headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter of the invention described.

The term "or" is used in an inclusive sense equivalent to "and/or" unless the context clearly requires otherwise.

As used herein, the terms "individual(s)", "subject(s)", and "patient(s)" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of the terms require or are limited to situations that are characterized by the supervision (e.g., ongoing or intermittent) of a healthcare professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, orderly, or hospice worker).

As used herein, the term "significant" or "to a significant extent" with respect to binding affinity means a change in the binding affinity of a cytokine (e.g., an IL-2 polypeptide) sufficient to affect the binding of the cytokine (e.g., an IL-2 polypeptide) to a target receptor. In some cases, the term refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some cases, the term 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.

In some cases, the term "significant" or "to a significant extent" with respect to activation of one or more cell populations by a cytokine signaling complex means a change sufficient to activate the cell population. In some cases, the change to activate the cell population is measured as receptor signaling activity. In such cases, the EC50 value may be reported. In other cases, the ED50 value may be reported. In additional cases, the concentration or dose of the cytokine may be reported.

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

As used herein, the term "unnatural amino acid" refers to an amino acid other than one of the 20 naturally occurring amino acids. Illustrative 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.

The term "antibody" is used in the broadest sense herein to encompass a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies) and antibody fragments, so long as they exhibit the desired antigen-binding activity. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains the portion of an intact antibody that binds 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 (eg, scFv) and polyspecific antibodies formed from antibody fragments. In some embodiments, the antigen is EGFR.

As used herein, the term "monoclonal antibody(ies)" 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 the same epitope, excluding possible antibody variants, for example, those containing mutations that occur naturally or that arise during the production of the monoclonal antibody preparation, such variants typically being present in minor amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or a portion of the human immunoglobulin loci, such methods and other illustrative methods for producing monoclonal antibodies being described herein.

As used herein, "nucleotide" refers to a compound containing a nucleoside moiety and a phosphate moiety. Illustrative 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 (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). Illustrative naturally occurring deoxyribonucleotides that contain deoxyribose as a sugar moiety include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Illustrative naturally occurring ribonucleotides that contain ribose as a sugar moiety include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.

As used herein, a "base" or "nucleobase" refers to at least a portion of a nucleoside or nucleotide (nucleoside and nucleotide include ribo- or deoxyribonucleotide variants) that is a nucleobase, which in some cases may contain additional modifications to the sugar portion of the nucleoside or nucleotide. In some cases, the term "base" is also used to refer to the entire nucleoside or nucleotide (e.g., a "base" may be incorporated by DNA polymerase into DNA or by RNA polymerase into RNA). However, the term "base" should not be interpreted as necessarily representing the entire nucleoside or nucleotide unless the context so requires. In the chemical structures of a base or nucleobase provided herein, only the base of the nucleoside or nucleotide is shown, and the sugar moiety and optionally any phosphate residues are omitted for clarity. As used in the chemical structures of a base or nucleobase provided herein, a wavy line represents a connection to a nucleoside or nucleotide, wherein the sugar portion of the nucleoside or nucleotide may be further modified. In some embodiments, a wavy line represents the attachment of a base or nucleobase to a sugar portion of a nucleoside or nucleotide, such as a pentose. In some embodiments, the pentose is ribose or deoxyribose.

In some embodiments, the nucleobase is typically a heterocyclic base moiety of a nucleoside. Nucleobases may be naturally occurring, may be modified, may not resemble natural bases, and/or may be synthesized, such as by organic synthesis. In certain embodiments, the nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide wherein the atom or group of atoms is capable of interacting with a base of another nucleic acid, with or without hydrogen bonding. In certain embodiments, the non-natural nucleobase is not a derivative of a natural nucleobase. It should be noted that non-natural nucleobases do not necessarily have basic properties, but for simplicity they are referred to as nucleobases. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase may be attached to deoxyribose or ribose, whereas "d" without parentheses indicates that the nucleobase is attached to deoxyribose.

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 (found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar moieties. Nucleosides include nucleosides containing any variety of substituents. A nucleoside may be a glycosidic compound formed through a glycosidic linkage between a nucleic acid base and a reducing group on a sugar.

As used herein, the term "analog" of a chemical structure refers to a chemical structure that retains substantial similarity to the parent structure, although it cannot be readily prepared synthetically from 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. A related chemical structure that is readily prepared synthetically from the parent chemical structure is called a "derivative."

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

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 cellular homeostasis of the innate and adaptive immune systems. Cytokines are produced by immune cells such as macrophages, B cells, T cells, and mast cells, endothelial cells, fibroblasts, and various stromal cells. In some cases, cytokines modulate the balance between humoral and cellular immune responses.

Interleukins are signaling proteins that modulate the development and differentiation of T and B lymphocytes, cells of the monocytic lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by CD4 helper T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue-resident cells.

Interleukin 2 (IL-2) is a type 1 pleiotropic cytokine with a 15.5 kDa, 4-helix bundle structure. The precursor form of IL-2 is 153 amino acid residues long, with the first 20 amino acids forming the signal peptide and residues 21–153 forming the mature form. IL-2 is produced primarily by CD4+ T cells following antigen stimulation and to a lesser extent by CD8+ cells, natural killer (NK) cells and natural killer T (NKT) cells, activated dendritic cells (DC), and mast cells. IL-2-mediated signaling occurs through interaction with specific combinations 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). When IL-2 interacts with IL-2Rα, a "low affinity" IL-2 receptor complex is formed with a K _d of approximately 10 ^-8 M. When IL-2 interacts with IL-2Rβ and IL-2Rγ, an "intermediate affinity" IL-2 receptor complex is formed with a K _d of approximately 10 ^-9 M. When IL-2 interacts with all three subunits of IL-2Rα, IL-2Rβ, and IL-2Rγ, a "high affinity" IL-2 receptor complex is formed with a K _d of approximately > 10 ^-11 M.

In some cases, IL-2-mediated signaling via the IL-2Rαβγ "high affinity" complex modulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells (Treg) mediate the maintenance of immune homeostasis by suppressing effector cells such as CD4 ⁺ T cells, CD8 ⁺ T cells, B cells, NK cells, and NKT cells. In some cases, Treg cells are generated in the thymus (tTreg cells) or are induced from naive T cells in the peripheral blood (pTreg cells). In some cases, Treg cells are considered mediators of peripheral tolerance. Indeed, in one study, transfer of CD25-depleted CD4 ⁺ T cells from peripheral blood induced a variety of autoimmune diseases in nude mice, whereas co-transfer of CD4 ⁺ CD25 ⁺ T cells suppressed the development of autoimmunity (Sakaguchi, et al ., "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)", J. Immunol . 155(3): 1151-1164 (1995), the disclosure of which is incorporated herein by reference). By expanding the Treg cell population, effector T cell proliferation is down-regulated and autoimmunity and T cell antitumor responses are suppressed.

IL-2-mediated signaling through the intermediate-affinity IL-2Rβγ complex modulates the activation and proliferation of CD8 ⁺ effector T cells (Teff), NK cells, and NKT cells. CD8 ⁺ Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, killer T cells, cytolytic T cells, Tcon, or killer T cells) are T lymphocytes that recognize and destroy damaged cells, cancer cells, and pathogen-infected cells. NK and NKT cells are types of lymphocytes that, like CD8 ⁺ Teff cells, target cancer cells and pathogen-infected cells.

In some cases, IL-2-mediated signaling is used to modulate T cell responses and thus to treat cancer. For example, IL-2 is administered in high doses to induce the expansion of Teff cell populations for cancer treatment. However, high-dose IL-2 also results in concomitant stimulation of Treg cells, which attenuate antitumor immune responses. High-dose IL-2 also induces toxic adverse events mediated by the recruitment of IL-2R alpha chain-expressing cells into the vasculature, including innate immune cells type 2 (ILC-2), eosinophils, and endothelial cells. This results in eosinophilia, capillary leak syndrome, and vascular leak syndrome (VLS).

Adoptive cell therapy allows doctors to effectively use a patient's own immune cells to fight diseases such as proliferative diseases (eg, cancer), as well as an infectious disease. 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 may include one or more immune checkpoint inhibitors.

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

Table 1 Name Subsequence SEQ ID NO: IL-2
(Homo sapiens)
(mature form) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 1 IL-2
(Homo sapiens)
(predecessor)
NCBI Accession No: AAB46883.1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 2 Aldesleukin PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 3 IL-2_C125S APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 4 IL-2_P65X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK X LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 5 IL-2_E62X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE X LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 6 IL-2_F42X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT X KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 7 IL-2_K43X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF X FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 8 IL-2_K35X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP X LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 9 IL-2_P65[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 10 IL-2_E62[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE[ AzK] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 11 IL-2_F42[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT[ AzK] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 12 IL-2_K43[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF[ AzK] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 13 IL-2_K35[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP[ AzK] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 14 IL-2_P65[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 15 IL-2_E62[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 16 IL-2_F42[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 17 IL-2_K43[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 18 IL-2_K35[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 19 IL-2_P65[AzK_PEG5kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG5kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 20 IL-2_E62[AzK_PEG5kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG5kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 21 IL-2_F42[AzK_PEG5kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG5kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 22 IL-2_K43[AzK_PEG5kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG5kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 23 IL-2_K35[AzK_PEG5kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG5kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 24 IL-2_P65[AzK_PEG30kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG30kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 25 IL-2_E62[AzK_PEG30kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG30kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 26 IL-2_F42[AzK_PEG30kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG30kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 27 IL-2_K43[AzK_PEG30kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG30kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 28 IL-2_K35[AzK_PEG30kDa] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG30kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 29 IL-2_P65X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK X LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 30 IL-2_E62X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE X LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 31 IL-2_F42X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT X KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 32 IL-2_K43X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF X FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 33 IL-2_K35X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP X LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 34 IL-2_P65[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 35 IL-2_E62[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE[ AzK] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 36 IL-2_F42[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT[ AzK] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 37 IL-2_K43[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF[ AzK] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 38 IL-2_K35[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP[ AzK] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 39 IL-2_P65[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 40 IL-2_E62[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 41 IL-2_F42[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 42 IL-2_K43[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 43 IL-2_K35[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 44 IL-2_P65[AzK_L1_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG5kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 45 IL-2_E62[AzK_L1_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG5kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 46 IL-2_F42[AzK_L1_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG5kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 47 IL-2_K43[AzK_L1_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG5kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 48 IL-2_K35[AzK_L1_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG5kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 49 IL-2_P65[AzK_L1_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG30kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 50 IL-2_E62[AzK_L1_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG30kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 51 IL-2_F42[AzK_L1_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG30kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 52 IL-2_K43[AzK_L1_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG30kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 53 IL-2_K35[AzK_L1_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG30kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 54 IL-2_P65[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 55 IL-2_E62[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 56 IL-2_F42[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 57 IL-2_K43[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 58 IL-2_K35[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 59 IL-2_P65[AzK_L1_PEG5kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG5kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 60 IL-2_E62[AzK_L1_PEG5kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG5kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 61 IL-2_F42[AzK_L1_PEG5kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG5kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 62 IL-2_K43[AzK_L1_PEG5kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG5kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 63 IL-2_K35[AzK_L1_PEG5kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG5kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 64 IL-2_P65[AzK_L1_PEG30kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG30kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 65 IL-2_E62[AzK_L1_PEG30kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG30kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 66 IL-2_F42[AzK_L1_PEG30kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG30kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 67 IL-2_K43[AzK_L1_PEG30kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG30kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 68 IL-2_K35[AzK_L1_PEG30kDa]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG30kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 69 IL-2_P65[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 70 IL-2_E62[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 71 IL-2_F42[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 72 IL-2_K43[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 73 IL-2_K35[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 74 IL-2_P65[AzK_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG5kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 75 IL-2_E62[AzK_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG5kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 76 IL-2_F42[AzK_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG5kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 77 IL-2_K43[AzK_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG5kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 78 IL-2_K35[AzK_PEG5kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG5kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 79 IL-2_P65[AzK_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG30kDa] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 80 IL-2_E62[AzK_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG30kDa] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 81 IL-2_F42[AzK_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG30kDa] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 82 IL-2_K43[AzK_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG30kDa] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 83 IL-2_K35[AzK_PEG30kDa]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG30kDa] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 84 IL-2_F44X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK X YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 85 IL-2_F44X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK X YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 86 IL-2_R38X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT X MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 87 IL-2_R38X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT X MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 88 IL-2_T41X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML X FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 89 IL-2_T41X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML X FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 90 IL-2_E68X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE X VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 91 IL-2_E68X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE X VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 92 IL-2_Y45X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF X MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 93 IL-2_Y45X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF X MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 94 IL-2_V69X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE X LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 95 IL-2_V69X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE X LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 96 IL-2_L72X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN X AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 97 IL-2_L72X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN X AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 98

X= site containing unnatural amino acid.

[AzK]= N6-((2-azidoethoxy)-carbonyl)-L-lysine with Chemical Abstracts Service registry number 1167421-25-1.

[AzK_PEG]=N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG by DBCO-mediated click chemistry to form a compound comprising a structure of formula (II) or formula (III). For example, when indicated, PEG5kDa refers to a linear polyethyleneglycol chain with an average molecular weight of 5 kilodaltons, capped with a methoxy group. The ratio of regioisomers formed as a result of the click reaction is approximately 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety containing a dibenzocyclooctyne group, such as that comprising the mPEG-DBCO compound illustrated in Scheme 1 in Example 2. An exemplary structure of a methoxy-PEG group is illustrated in the mPEG-DBCO structure in Scheme 1 in Example 2.

[AzK_L1_PEG]=N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG by DBCO-mediated click chemistry to form a compound comprising a structure of formula (IV) or formula (V). For example, when indicated, PEG5kDa refers to a linear polyethyleneglycol chain with an average molecular weight of 5 kilodaltons, capped with a methoxy group. The ratio of regioisomers formed as a result of the click reaction is approximately 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety containing a dibenzocyclooctyne group, such as that comprising the compound mPEG-DBCO illustrated in Scheme 1 in Example 2.

Described herein 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 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 by a structure of formula (I):

Formula (I);

Where:

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

Here and throughout this document, the term "IL-2 conjugate" includes pharmaceutically acceptable salts, solvates and hydrates of the structure indicated.

Here and throughout this document, the structure of formula (I) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a pharmaceutically acceptable salt thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a solvate thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a hydrate thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a free base.

In some embodiments of the method described herein, in the IL-2 conjugate, Z is CH ₂ and Y is In some embodiments of the method described herein, in the IL-2 conjugate, Y is CH ₂ and Z is . In some embodiments of the method described herein, Z is CH ₂ and Y is In some embodiments of the method described herein, in the IL-2 conjugate, Z is CH ₂ and Y is In some embodiments of the method described herein, in the IL-2 conjugate, Y is CH ₂ and Z is Here and throughout this document, embodiments Z and Y also encompass a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments of the method described herein, in the IL-2 conjugate, the PEG moiety has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa, and 30 kDa. In some embodiments of the method described herein, in the IL-2 conjugate, the PEG moiety has an average molecular weight of 5 kDa. In some embodiments of the method described herein, in the IL-2 conjugate, the PEG moiety has an average molecular weight of 10 kDa. In some embodiments of the method described herein, in the IL-2 conjugate, the PEG moiety has an average molecular weight of 15 kDa. In some embodiments, the methods utilize an IL-2 conjugate, wherein the PEG moiety in the IL-2 conjugate has an average molecular weight of 20 kDa. In some embodiments of the method described herein, the PEG moiety of the IL-2 conjugate has an average molecular weight of 25 kDa. In some embodiments of the method described herein, the PEG moiety of the IL-2 conjugate has an average molecular weight of 30 kDa. In some embodiments of the method described herein, the PEG moiety of the IL-2 conjugate has an average molecular weight of 35 kDa. In some embodiments of the method described herein, the PEG moiety of the IL-2 conjugate has an average molecular weight of 40 kDa. In some embodiments of the method described herein, the PEG moiety of the IL-2 conjugate has an average molecular weight of 45 kDa. In some embodiments of the method described herein, the PEG moiety of the IL-2 conjugate has an average molecular weight of 50 kDa. In some embodiments, the methods utilize an IL-2 conjugate, wherein the IL-2 conjugate has a PEG group having an average molecular weight of 60 kDa.

In some embodiments of the method 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, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3. In some embodiments of the method 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, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] has a structure of formula (II) or formula (III) or is a mixture of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

Here and throughout this document, the structure of formula (II) includes pharmaceutically acceptable salts, solvates or hydrates thereof. Here and throughout this document, the structure of formula (III) includes pharmaceutically acceptable salts, solvates or hydrates thereof.

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

In some embodiments, [AzK_PEG] has a structure of formula (II):

Formula (II)

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 moiety 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 moiety 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 moiety having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (II) is a PEG moiety having an average molecular weight of 30 kDa.

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 moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (II) is a PEG moiety 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 moiety having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (II) is a PEG moiety having an average molecular weight of 30 kDa.

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 moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (II) is a PEG moiety 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 moiety having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (II) is a PEG moiety having an average molecular weight of 30 kDa.

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

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

In some embodiments, [AzK_PEG] has a structure of formula (III):

Formula (III)

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 moiety 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 moiety 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 moiety having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (III) is a PEG moiety having an average molecular weight of 30 kDa.

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 moiety 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 moiety 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 moiety having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (III) is a PEG moiety having an average molecular weight of 30 kDa.

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 moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (III) is a PEG moiety 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 moiety having an average molecular weight of 5 kDa. In some embodiments, W in the structure of formula (III) is a PEG moiety having an average molecular weight of 30 kDa.

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 moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (III) is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (III) is a PEG moiety having an average molecular weight of 5 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (III) is a PEG moiety having an average molecular weight of 30 kDa.

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

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

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kDa] has a structure of formula (II) or formula (III) or is a mixture of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

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.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_PEG5kDa] has a structure of formula (II):

Formula (II)

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.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_PEG5kDa] has a structure of formula (III):

Formula (III);

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.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kDa] has a structure of formula (II) or formula (III) or is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

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.

In some embodiments, the methods disclosed herein utilize an IL-2 conjugate in which [AzK_PEG30kDa] has a structure of formula (II):

Formula (II);

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.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_PEG30kDa] has a structure of formula (III):

Formula (III)

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.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG] in the IL-2 conjugate is about 1:1. In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG] in the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG] in the IL-2 conjugate is less than 1:1. In some embodiments, the methods use an IL-2 conjugate wherein W is a linear or branched PEG group. In some embodiments, the methods use an IL-2 conjugate wherein W is a linear PEG group. In some embodiments, the methods use an IL-2 conjugate wherein W is a branched PEG group. In some embodiments, the methods use an IL-2 conjugate wherein W is a methoxy-PEG group. In some embodiments, the methods use an IL-2 conjugate wherein the methoxy-PEG group is linear or branched. In some embodiments, the methods use an IL-2 conjugate wherein the methoxy-PEG group is linear. In some embodiments, the methods use an IL-2 conjugate wherein the methoxy-PEG group is branched.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kDa] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG5kDa] in the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG5kDa] in the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG5kDa] in the IL-2 conjugate is less than 1:1.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kDa] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use 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) that make up the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is less than 1:1.

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

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [AzK_L1_PEG] has a structure of formula (IV) or formula (V) or is a mixture of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Here and throughout this document, the structure of formula (IV) includes pharmaceutically acceptable salts, solvates or hydrates thereof. Here and throughout this document, the structure of formula (V) includes pharmaceutically acceptable salts, solvates or hydrates thereof.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_L1_PEG] is a mixture of formula (IV) and formula (V).

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_L1_PEG] has a structure of formula (IV):

Formula (IV)

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

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

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

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

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

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_L1_PEG] has a structure of formula (V):

Formula (V)

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

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

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

In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (V) is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (V) is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (V) is a PEG moiety having an average molecular weight of 5 kDa. In some embodiments, the methods utilize an IL-2 conjugate wherein W in the structure of formula (V) is 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] comprises a PEG moiety 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, wherein [AzK_L1_PEG] comprises a PEG moiety 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, wherein [AzK_L1_PEG] comprises a PEG moiety 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] comprises a PEG moiety 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] comprises a PEG moiety 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] comprises a PEG moiety 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] comprises a PEG moiety 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] comprises a PEG moiety 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, wherein [AzK_L1_PEG] comprises a PEG moiety 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, wherein [AzK_L1_PEG] comprises a PEG moiety 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, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 50 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 60 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety 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 wherein the PEG moiety is a methoxy-PEG moiety, a linear methoxy-PEG moiety, or a branched methoxy-PEG moiety.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kDa] has a structure of formula (IV) or formula (V) or is a mixture of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

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.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_L1_PEG5kDa] has a structure of formula (IV):

Formula (IV);

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.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_L1_PEG5kDa] has a structure of formula (V):

Formula (V)

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.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV) or formula (V) or is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

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.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_L1_PEG30kDa] has a structure of formula (IV):

Formula (IV);

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.

In some embodiments, the methods utilize an IL-2 conjugate in which [AzK_L1_PEG30kDa] has a structure of formula (V):

Formula (V)

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.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [Azk_L1_PEG] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG] in the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG] in the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG] in the IL-2 conjugate is less than 1:1.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kDa] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG5kDa] in the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG5kDa] in the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG5kDa] in the IL-2 conjugate is less than 1:1.

Described herein 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 IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kDa] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is less than 1:1.

In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (IV) or formula (V) is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (IV) or formula (V) is a PEG moiety having an average molecular weight of 5 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (IV) or formula (V) is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments, the methods use an IL-2 conjugate wherein W in the structure of formula (IV) or formula (V) is a PEG moiety having an average molecular weight of 10 kDa. In some embodiments, the methods use an IL-2 conjugate wherein 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 methods use an IL-2 conjugate wherein 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 methods use an IL-2 conjugate wherein 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 methods use an IL-2 conjugate wherein 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 methods use an IL-2 conjugate wherein 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 methods use an IL-2 conjugate wherein 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 methods use an IL-2 conjugate wherein 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 wherein 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 methods use an IL-2 conjugate wherein 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 methods use an IL-2 conjugate wherein W in the structure of formula (IV) or formula (V) is a PEG group having an average molecular weight of 60 kDa.

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

With respect to the IL-2 conjugates used in the methods described herein, an exemplary structure of a methoxy-PEG moiety is illustrated in the mPEG-DBCO structure in Scheme 1 in Example 2. Exemplary structures of a methoxy-PEG moiety are illustrated in the following mPEG-DBCO structures:

Described herein 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 IL-2 conjugate 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 by a structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII):

Formula (VI),

Formula (VII),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

X has the structure ,

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

Here and throughout this document, the structure of formula (VI) includes pharmaceutically acceptable salts, solvates or hydrates thereof. Here and throughout this document, the structure of formula (VII) includes pharmaceutically acceptable salts, solvates or hydrates thereof.

In some embodiments, n in the compounds of Formula (VI) and Formula (VII) is in the range of 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 from about 100 to about 1500, or from about 100 to about 1400, or about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of Formula (VI) and Formula (VII) is an integer selected from 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, 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, wherein 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 refers to the positions 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 position 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 position 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 position 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 position 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 position 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 position 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 position 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 position 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.

In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is less than 1:1.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of Formula (VI) and Formula (VII) is an integer selected from 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, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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, 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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 methods use an IL-2 conjugate wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

In some embodiments, n in the structures of Formula (VI) and Formula (VII) is an integer such that the molecular weight of the PEG moiety is in the range of 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, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

In some embodiments, the methods utilize 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 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, about 50,000 Daltons, about 60,000 Daltons, about 70,000 daltons, approximately 80,000 daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons.

In some embodiments, the methods utilize 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 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.

Described herein 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 IL-2 conjugate 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 by a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX):

Formula (VIII),

Formula (IX),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

X has the structure ,

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

Here and throughout this document, the structure of formula (VIII) includes pharmaceutically acceptable salts, solvates or hydrates thereof. Here and throughout this document, the structure of formula (IX) includes pharmaceutically acceptable salts, solvates or hydrates thereof.

In some embodiments, n in the compounds of Formula (VIII) and Formula (IX) is in the range of 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 from about 100 to about 1500, or from about 100 to about 1400, or about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of Formula (VIII) and Formula (IX) is an integer selected from 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, 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, wherein 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 refers to the positions 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 position 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 position 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 position 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 position 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 position 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 position 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 position 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 position 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.

In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is less than 1:1.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of Formula (VIII) and Formula (IX) is an integer selected from 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, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

Described herein 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 IL-2 conjugate 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 by a structure of formula (VIII) or formula (IX), or a mixture of formula (VIII) and formula (IX), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of 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, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9000 daltons to about 30000 daltons, or from about 9500 daltons to about 35000 daltons, or from about 9500 daltons to about 30000 daltons, or from about 10000 daltons to about 50000 daltons, or from about 10000 daltons to about 45000 daltons, or from about 10000 daltons to about 40000 daltons, or from about 10000 daltons to about 35000 daltons, or from about 10000 daltons to about 30000 daltons, or from about 15000 daltons to about 50000 daltons, or from about 15000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

In some embodiments, the methods utilize 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 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, about 50,000 Daltons, about 60,000 Daltons, about 70,000 daltons, approximately 80,000 daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. In some embodiments, the methods utilize 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 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.

Described herein 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 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 by a structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI):

Formula (X);

Formula (XI);

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Here and throughout this document, the structure of formula (X) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (XI) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

In some embodiments, the stereochemical configuration of the chiral center of formula (X) and formula (XI) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides essentially (R), provides essentially (S), provides (R), or provides (S). In some embodiments, the stereochemical configuration of the chiral center of formula (X) and formula (XI) provides a racemic mixture. In some embodiments, the stereochemical configuration of the chiral center of formula (X) and formula (XI) provides a mixture enriched in (R). In some embodiments, the stereochemical configuration of the chiral center of formula (X) and formula (XI) provides a mixture enriched in (S). In some embodiments, the stereochemical configuration of the chiral center of formula (X) and formula (XI) provides essentially (R). In some embodiments, the stereochemical configuration of the chiral center of formula (X) and formula (XI) provides essentially (S). In some embodiments, the stereochemical configuration of the chiral center in formula (X) and formula (XI) is (R). In some embodiments, the stereochemical configuration of the chiral center in formula (X) and formula (XI) is (S).

In some embodiments, n in the compounds of Formula (X) and Formula (XI) ranges 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 from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of Formula (X) and Formula (XI) is an integer selected from 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, 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, wherein 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 a position in SEQ ID NO: 3. In some embodiments, the methods use an IL-2 conjugate wherein 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, T40, E67, Y44, V68 and L71. 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 K34. 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 F41. In some embodiments, the methods use an IL-2 conjugate wherein 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 F43. In some embodiments, the methods use an IL-2 conjugate wherein 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 K42. In some embodiments, the methods use an IL-2 conjugate wherein 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 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 position 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 position 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 position 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 position 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 position 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 position 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.

In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is approximately 1:1. In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, the methods use 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) comprising the total amount of the IL-2 conjugate is less than 1:1.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of Formula (VI) and Formula (VII) is an integer selected from 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, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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, 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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 methods use an IL-2 conjugate wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

In some embodiments, n in the structures of Formula (X) and Formula (XI) is an integer such that the molecular weight of the PEG moiety is in the range of 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, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

In some embodiments, the methods utilize 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 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, about 50,000 Daltons, about 60,000 Daltons, about 70,000 daltons, approximately 80,000 daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons.

In some embodiments, the methods utilize 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 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.

Described herein 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 IL-2 conjugate 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 by a structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII):

Formula (XII);

Formula (XIII);

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Here and throughout this document, the structure of formula (XII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (XIII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

In some embodiments, the stereochemical configuration of the chiral center of formula (XII) and formula (XIII) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides essentially (R), provides essentially (S), provides (R), or provides (S). In some embodiments, the stereochemical configuration of the chiral center of formula (XII) and formula (XIII) provides a racemic mixture. In some embodiments, the stereochemical configuration of the chiral center of formula (XII) and formula (XIII) provides a mixture enriched in (R). In some embodiments, the stereochemical configuration of the chiral center of formula (XII) and formula (XIII) provides a mixture enriched in (S). In some embodiments, the stereochemical configuration of the chiral center of formula (XII) and formula (XIII) provides essentially (R). In some embodiments, the stereochemical configuration of the chiral center of formula (XII) and formula (XIII) provides essentially (S). In some embodiments, the stereochemical configuration of the chiral center in formula (XII) and formula (XIII) is (R). In some embodiments, the stereochemical configuration of the chiral center in formula (XII) and formula (XIII) is (S).

In some embodiments, n in the compounds of Formula (XII) and Formula (XIII) is in the range of 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 from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of formula (XII) and (XIII) is an integer selected from 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, 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, wherein 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 refers to the positions 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 formulas (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 position 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 position 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 position 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 position 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.

In some embodiments, the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) comprising the total amount of the 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) comprising the total amount of the 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) comprising the total amount of the IL-2 conjugate is less than 1:1.

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 is replaced with a structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, n in the compounds of Formula (XII) and Formula (XIII) is an integer selected from 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, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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.

In some embodiments, the methods utilize 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 amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

In some embodiments, n in the structures of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) is an integer such that the molecular weight of the PEG moiety is in the range of 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, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

In some embodiments, the methods utilize 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 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, about 50,000 Daltons, about 60,000 Daltons, about 70,000 daltons, approximately 80,000 daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. 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 is replaced by a structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII), wherein 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.

Described herein 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 PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX):

Formula (VIII);

(IX);

Where:

n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; and

X has the structure ;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

In some embodiments, the amino acid residue at 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), and wherein n is an integer such that the molecular weight of the PEG moiety is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG moiety 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.

In some embodiments, the amino acid residue at 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), and wherein n is an integer such that the molecular weight of the PEG moiety is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG moiety 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.

Described herein 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 PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII):

Formula (VI);

(VII);

Where:

n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; and

X has the structure ;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

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

In some embodiments, the amino acid residue at 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), and wherein n is an integer such that the molecular weight of the PEG moiety is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG moiety 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.

In some embodiments, the amino acid residue at 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), and wherein n is an integer such that the molecular weight of the PEG moiety is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG moiety 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.

Described herein 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 IL-2 conjugate is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a cysteine covalently linked to a PEG moiety. In some embodiments, the PEG moiety 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 moiety has a molecular weight of 5 kDa. In some embodiments, the PEG moiety has a molecular weight of 10 kDa. In some embodiments, the PEG moiety has a molecular weight of 15 kDa. In some embodiments, the PEG moiety has a molecular weight of 20 kDa. In some embodiments, the PEG moiety has a molecular weight of 25 kDa. In some embodiments, the PEG moiety has a molecular weight of 30 kDa. In some embodiments, the PEG moiety has a molecular weight of 35 kDa. In some embodiments, the PEG moiety has a molecular weight of 40 kDa. In some embodiments, the PEG moiety has a molecular weight of 45 kDa. In some embodiments, the PEG moiety has a molecular weight of 50 kDa. In some embodiments, the PEG 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 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 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 that is replaced with cysteine is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.

Described herein 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 IL-2 conjugate is an IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one non-lysine residue is replaced with lysine, comprising a linker and a water-soluble polymer. In some embodiments, the water-soluble polymer is a PEG moiety.

In some embodiments, the IL-2 conjugate comprises a PEG moiety covalently linked via a non-releasable linkage. In some embodiments, the IL-2 conjugate comprises a non-releasable covalently linked PEG moiety.

Described herein are methods of treating a 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 an amino acid other than lysine in the IL-2 conjugate is replaced with a lysine residue, and wherein 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.

Described herein are methods of treating a 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 an amino acid other than lysine in the IL-2 conjugate is replaced with a lysine residue, and wherein 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.

Described herein 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 IL-2 conjugate is an interleukin-2 (IL-2) variant wherein an amino acid other than lysine in the amino acid sequence of the IL-2 variant is replaced with an amino acid comprising (a) lysine; (b) a covalent linker, and (3) one or more water-soluble polymers. In some embodiments, the one or more water-soluble polymers comprise a PEG moiety.

Described herein 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 IL-2 conjugate 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 by a structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV):

Formula (XIV);

Formula (XV);

Where:

m is an integer between 0 and 20;

p is an integer between 0 and 20;

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Here and throughout this document, the structure of formula (XIV) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (XV) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

In some embodiments, the stereochemical configuration of the chiral center of formula (XIV) and formula (XV) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides essentially (R), provides essentially (S), provides (R), or provides (S). In some embodiments, the stereochemical configuration of the chiral center of formula (XIV) and formula (XV) provides a racemic mixture. In some embodiments, the stereochemical configuration of the chiral center of formula (XIV) and formula (XV) provides a mixture enriched in (R). In some embodiments, the stereochemical configuration of the chiral center of formula (XIV) and formula (XV) provides a mixture enriched in (S). In some embodiments, the stereochemical configuration of the chiral center of formula (XIV) and formula (XV) provides essentially (R). In some embodiments, the stereochemical configuration of the chiral center of formula (XIV) and formula (XV) provides essentially (S). In some embodiments, the stereochemical configuration of the chiral center in formula (XIV) and formula (XV) is (R). In some embodiments, the stereochemical configuration of the chiral center in formula (XIV) and formula (XV) is (S).

In some embodiments, the methods utilize 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 from 0 to 20, or from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XIV) and formula (XV) is 1. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XIV) and formula (XV) is 2. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 3. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 4. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 5. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 6. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 7. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and formula (XV) is 8. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XIV) and formula (XV) is 9. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XIV) and formula (XV) is 10. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XIV) and formula (XV) is 11. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XIV) and formula (XV) is 12. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XIV) and formula (XV) is 13. In some embodiments of the IL-2 conjugate described herein, document, m in the compounds of Formula (XIV) and Formula (XV) is 14. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 15. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 16. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 17. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 18. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 19. In some embodiments of the IL-2 conjugate described in this document, m in the compounds of formula (XIV) and formula (XV) is equal to 20.

In some embodiments, the methods utilize 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 from 1 to 20, or from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 1. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 2. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 3. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 4. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 5. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 6. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 7. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and formula (XV) is 8. In some embodiments of the IL-2 conjugate described herein, p in the compounds of formula (XIV) and formula (XV) is 9. In some embodiments of the IL-2 conjugate described herein, p in the compounds of formula (XIV) and formula (XV) is 10. In some embodiments of the IL-2 conjugate described herein, p in the compounds of formula (XIV) and formula (XV) is 11. In some embodiments of the IL-2 conjugate described herein, p in the compounds of formula (XIV) and formula (XV) is 12. In some embodiments of the IL-2 conjugate described herein, p in the compounds of formula (XIV) and formula (XV) is 13. In some embodiments of the IL-2 conjugate described herein, document, p in the compounds of Formula (XIV) and Formula (XV) is 14. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 15. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XIV) and Formula (XV) is 16. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 17. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 18. In some embodiments of the IL-2 conjugate described herein, p in the compounds of Formula (XIV) and Formula (XV) is 19. In some embodiments of the IL-2 conjugate described herein, p in the compounds of formula (XIV) and formula (XV) is 20.

In some embodiments, the methods utilize 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 in the range of 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 from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, the methods use 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 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate IL-2 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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.

In some embodiments, the methods utilize 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 an integer selected from 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 conjugate 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, wherein 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 refers to the positions of SEQ ID NO: 3. In some embodiments of the IL-2 conjugate 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 conjugate 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 conjugate 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 F41. In some embodiments of the IL-2 conjugate 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 F43. In some embodiments of the IL-2 conjugate 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 K42. In some embodiments of the IL-2 conjugate 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 conjugate 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 conjugate 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 conjugate 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 T40. In some embodiments of the IL-2 conjugate 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 E67. In some embodiments of the IL-2 conjugate 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 Y44. In some embodiments of the IL-2 conjugate 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 conjugate 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.

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

Described herein 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 IL-2 conjugate 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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 conjugate described herein, n in the compounds of Formula (XIV) and Formula (XV) is an integer selected from 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.

Described herein are methods of treating a 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, 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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 conjugate, 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.

Described herein are methods of treating a 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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 conjugate described herein, n in the compounds of formula (XIV) and formula (XV) is an integer chosen from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Described herein are methods of treating a 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, 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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 conjugate 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 from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

Described herein 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 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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 conjugate 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 from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

Described herein 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 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), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of 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, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

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 is replaced by a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein 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, about 50,000 Daltons, about 60,000 Daltons, about 70,000 daltons, approximately 80,000 daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons.

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 is replaced by a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein 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.

Described herein 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 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 (XV) or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, m is an integer from 1 to 6, p 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 IL-2 conjugate described herein, in compounds of formulas (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.

Described herein 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 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein m is an integer from 1 to 6, p 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 conjugate IL-2 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.

Described herein 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 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein m is an integer from 1 to 6, p 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 conjugate described herein, in 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 from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

Described herein 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 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), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein m is an integer from 1 to 6, p 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, the IL-2 conjugate described herein, in 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 from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

Described herein 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 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 by a structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII):

Formula (XVI);

Formula (XVII);

Where:

m is an integer between 0 and 20;

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Here and throughout this document, the structure of formula (XVI) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (XVII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

In some embodiments, the stereochemical configuration of the chiral center of formula (XVI) and formula (XVII) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides essentially (R), provides essentially (S), provides (R), or provides (S). In some embodiments, the stereochemical configuration of the chiral center of formula (XVI) and formula (XVII) provides a racemic mixture. In some embodiments, the stereochemical configuration of the chiral center of formula (XVI) and formula (XVII) provides a mixture enriched in (R). In some embodiments, the stereochemical configuration of the chiral center of formula (XVI) and formula (XVII) provides a mixture enriched in (S). In some embodiments, the stereochemical configuration of the chiral center of formula (XVI) and formula (XVII) provides essentially (R). In some embodiments, the stereochemical configuration of the chiral center of formula (XVI) and formula (XVII) provides essentially (S). In some embodiments, the stereochemical configuration of the chiral center in formula (XVI) and formula (XVII) is (R). In some embodiments, the stereochemical configuration of the chiral center in formula (XVI) and formula (XVII) is (S).

In some embodiments, the methods utilize 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 from 1 to 20, or from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XVI) and formula (XVII) is 1. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XVI) and formula (XVII) is 2. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 3. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 4. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 5. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 6. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 7. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 8. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 9. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 10. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 11. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 12. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and Formula (XVII) is 13. In some embodiments of the IL-2 conjugate described herein, m in the compounds of Formula (XVI) and formula (XVII) is 14. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XVI) and formula (XVII) is 15. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XVI) and formula (XVII) is 16. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XVI) and formula (XVII) is 17. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XVI) and formula (XVII) is 18. In some embodiments of the IL-2 conjugate described herein, m in the compounds of formula (XVI) and formula (XVII) is 19. In some embodiments of the IL-2 conjugate described herein, m in the compounds formula (XVI) and formula (XVII) is equal to 20.

In some embodiments, the methods utilize 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 in the range of 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 from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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, the methods use 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 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate 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 conjugate described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 7 and n is an integer a number 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 conjugate 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 conjugate 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 conjugate 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 conjugate described herein, in the compounds of Formula (XVI) and Formula (XVII), m is 11 and n is an integer selected from 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 conjugate 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 conjugate 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.

In some embodiments, the methods utilize 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 an integer selected from 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 conjugate 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, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions of SEQ ID NO: 3. In some embodiments of the IL-2 conjugate 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 conjugate 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 conjugate 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 F41. In some embodiments of the IL-2 conjugate 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 F43. In some embodiments of the IL-2 conjugate 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 K42. In some embodiments of the IL-2 conjugate 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 E61. In some embodiments of the IL-2 conjugate 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 P64. In some embodiments of the IL-2 conjugate 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 R37. In some embodiments of the IL-2 conjugate 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 T40. In some embodiments of the IL-2 conjugate 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 E67. In some embodiments of the IL-2 conjugate 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 Y44. In some embodiments of the IL-2 conjugate 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 V68. In some embodiments of the IL-2 conjugate 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 L71.

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

In some embodiments, the methods utilize 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 replaced with a structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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 conjugate described herein, n in the compounds of Formula (XVI) and Formula (XVII) is an integer selected from 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, 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 replaced with a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), is selected from F41, F43, K42, E61, and P64, and wherein 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 conjugate 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.

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 replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), is selected from E61 and P64, and wherein 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 conjugate 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, 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 replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII) that is replaced, is E61, and wherein 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 conjugate 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 from about 550 to about 800. In some embodiments, n is about 681.

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 replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII) that is replaced, is P64, and wherein 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 conjugate 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 from about 550 to about 800. In some embodiments, n is about 681.

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 is replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of 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, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

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 is replaced by a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein 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, about 50,000 Daltons, about 60,000 Daltons, about 70,000 daltons, approximately 80,000 daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. 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 is replaced by a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein 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.

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 replaced with a structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) 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 conjugate 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.

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 replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), is selected from E61 and P64, and wherein 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 conjugate 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, 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 replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII) that is replaced, is E61, and wherein 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 conjugate 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 from about 550 to about 800. In some embodiments, n is about 681.

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 replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), is P64, and wherein 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 conjugate 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 from about 550 to about 800. In some embodiments, n is about 681.

In some embodiments, described herein is a method of treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) a cytokine conjugate (e.g., an IL-2 conjugate) described in Table 1 , and (b) one or more additional agents. 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 NO: 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 conjugate IL-2 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: 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: 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 conjugate IL-2 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.

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

In some embodiments, the IL-2 conjugate comprises an 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 formula (II) to formula (XVII) or any variation thereof, is inserted into a site containing a non-natural amino acid.

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

In some cases, 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 the additional conjugation moiety to increase serum half-life, stability, or a combination thereof. Alternatively, prior to linkage to the additional conjugation moiety, the amino acid is first mutated to a naturally occurring amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine or tyrosine; or to a non-naturally occurring amino acid.

In some cases, the PEG group is not limited to a specific structure. In some cases, the PEG is linear (e.g., end-capped, such as alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., fork-shaped PEG or PEG attached to a polyol core), has a dendritic (or star-shaped) architecture, each of which has or does not have one or more degradable bonds. Moreover, the internal structure of the water-soluble polymer can be arranged in any number of different repeating patterns and can be selected from the group consisting of a homopolymer, an alternating copolymer, a random copolymer, a block copolymer, an alternating tripolymer, a random tripolymer, and a block tripolymer.

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

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

In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear or branched PEG moiety. In some embodiments, the PEG moiety is a linear PEG moiety. In some embodiments, the PEG moiety is a branched PEG moiety. In some embodiments, the PEG moiety is a methoxy-PEG moiety. In some embodiments, the PEG moiety is a linear or branched methoxy-PEG moiety. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety. In some embodiments, the PEG moiety is a linear or branched PEG moiety having an average molecular weight of from about 100 Daltons to about 150,000 Daltons. Illustrative ranges include, for example, weight average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of about 6,000 Daltons to about 90,000 Daltons, in the range of about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of about 20,000 Daltons to about 85,000 Daltons, in the range of about 53,000 Daltons to about 85,000 Daltons, in the range of about 25,000 Daltons to about 120,000 Daltons, in the range of about 29,000 Daltons to about 120,000 Daltons, in the range of about 35,000 Daltons to approximately 120,000 Daltons and in the range from approximately 40,000 Daltons to approximately 120,000 Daltons. Illustrative weight average molecular weights of the PEG group include values of 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 1000 Daltons, about 1500 Daltons, about 2000 Daltons, about 2200 Daltons, about 2500 Daltons, about 3000 Daltons, about 4000 Daltons, about 4400 Daltons, about 4500 Daltons, about 5000 Daltons, about 5500 daltons, approximately 6,000 daltons, approximately 7,000 daltons, approximately 7,500 daltons, approximately 8,000 daltons, approximately 9,000 daltons, approximately 10,000 daltons, approximately 11,000 daltons, approximately 12,000 daltons, approximately 13,000 daltons, approximately 14,000 daltons, approximately 15,000 daltons, approximately 20,000 daltons, approximately 22,500 daltons, approximately 25,000 daltons, approximately 30,000 daltons, approximately 35,000 daltons, approximately 40,000 daltons, approximately 45,000 daltons, approximately 50,000 daltons, approximately 55,000 daltons, approximately 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 moiety is a linear PEG moiety having an average molecular weight as disclosed above. In some embodiments, the PEG moiety is a branched PEG moiety having an average molecular weight as disclosed above. In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear or branched PEG moiety having a specified molecular weight of ±10%, or 15%, or 20%, or 25%. For example, included within the scope of the present invention are 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.

In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear or branched PEG moiety having an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG moiety is a linear or branched PEG moiety having an average molecular weight of 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, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 daltons, approximately 45,000 daltons, approximately 50,000 daltons, approximately 55,000 daltons, approximately 60,000 daltons, approximately 65,000 daltons, approximately 70,000 daltons, approximately 75,000 daltons, approximately 80,000 daltons, approximately 90,000 daltons, approximately 95,000 daltons and approximately 100,000 daltons. In some embodiments, the PEG moiety is a linear or branched PEG moiety 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 moiety is a linear or branched PEG moiety 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 moiety is a linear PEG moiety 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 moiety is a branched PEG moiety 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 moiety contained in the IL-2 conjugates disclosed herein is a linear methoxy-PEG moiety having an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety having an average molecular weight of 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, 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 moiety is a linear methoxy-PEG moiety 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 moiety is a linear methoxy-PEG moiety 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 moiety is a linear methoxy-PEG moiety 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 moiety is a linear methoxy-PEG moiety having an average molecular weight of about 5,000 Daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety having an average molecular weight of about 10,000 daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety having an average molecular weight of about 20,000 daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety having an average molecular weight of about 30,000 daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety having an average molecular weight of about 50,000 daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety having an average molecular weight of about 60,000 daltons. In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear methoxy-PEG moiety having a specified molecular weight of ±10%, or 15%, or 20%, or 25%. For example, included within the scope of the present invention are IL-2 conjugates comprising a linear methoxy-PEG moiety 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.

In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a branched methoxy-PEG moiety having an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety having an average molecular weight of 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, 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 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 moiety is a branched methoxy-PEG moiety 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 moiety is a branched methoxy-PEG moiety having an average molecular weight of about 5,000 Daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety having an average molecular weight of about 10,000 Daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety having an average molecular weight of about 20,000 Daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety having an average molecular weight of about 30,000 daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety having an average molecular weight of about 50,000 daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety having an average molecular weight of about 60,000 daltons. In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a branched methoxy-PEG moiety having a specified molecular weight of ±10%, or 15%, or 20%, or 25%. For example, included within the scope of the present invention are IL-2 conjugates comprising a branched 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.

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 PEGs from Nektar Therapeutics; and Y-derivatized PEGs from JenKem Technology.

Conjugation chemistry

Conjugation chemistry

Various conjugation reactions are used to conjugate 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 "biorthogonal" reactions. In some embodiments, the conjugation reactions are mediated by chemical reagents, such as catalysts, light, or reactive chemical groups found in linkers, conjugation moieties, or unnatural amino acids. In some embodiments, the conjugation reactions are mediated by enzymes. In some embodiments, a conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, a conjugation reaction used herein is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417. The disclosure of each of these literature sources is incorporated herein by reference.

In some embodiments described herein, a conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction. In some embodiments, a 1,3-dipolar cycloaddition reaction comprises a click reaction of an azide and a phosphine. In some embodiments, the conjugation reaction is copper catalyzed. In some embodiments, a conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached via a triazole. In some embodiments, a conjugation reaction described herein comprises a reaction of an azide with a strained olefin. In some embodiments, a conjugation reaction described herein comprises a reaction of an azide with a strained alkyne. In some embodiments, a conjugation reaction described herein comprises a reaction of an azide with a cycloalkyne, such as DBCO.

In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme S1, wherein X is a position in the IL-2 conjugate that contains a non-natural amino acid, such as in any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

Scheme S1

In some embodiments, the conjugating moiety comprises a water-soluble polymer. In some embodiments, the reactive group comprises an alkyne or azide.

In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme S2, wherein X is a position in the IL-2 conjugate that contains a non-natural amino acid, such as in any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

Scheme S2

In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme S3, wherein X is a position in the IL-2 conjugate that contains a non-natural amino acid, such as in any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

Scheme S3

,

In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme S4, wherein X is a position in the IL-2 conjugate that contains a non-natural amino acid, such as in any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34.

Scheme S4

In some embodiments described herein, the conjugation reaction described herein involves 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, which is a chemical moiety containing a dibenzocyclooctyne group. PEG moieties containing the DBCO moiety are commercially available or can be prepared by methods known to those of ordinary skill in the art. An exemplary reaction is shown in Schemes S5 and S6.

Scheme S5

Scheme S6

Conjugation reactions, such as the click reaction described herein, may produce a single regioisomer or a mixture of regioisomers. In some cases, the ratio of regioisomers is approximately 1:1. In some cases, the ratio of regioisomers is approximately 2:1. In some cases, the ratio of regioisomers is approximately 1.5:1. In some cases, the ratio of regioisomers is approximately 1.2:1. In some cases, the ratio of regioisomers is approximately 1.1:1. In some cases, the ratio of regioisomers is greater than 1:1.

Obtaining cytokine polypeptides

In some cases, the IL-2 conjugates described herein comprising a naturally occurring amino acid substitution mutation or a non-naturally occurring amino acid substitution mutation are recombinantly produced or chemically synthesized. In some cases, the IL-2 conjugates described herein are recombinantly produced, such as in a host cell system or in a cell-free system. In any of the embodiments or variations described herein, the amino acid can be an L-amino acid or a D-amino acid. In some embodiments, the amino acid is an L-amino acid. In other embodiments, the amino acid is a D-amino acid.

In some cases, the IL-2 conjugates are produced recombinantly using a host cell system. In some cases, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (e.g., a gram-positive bacterium or a gram-negative bacterium). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is an established cell line or cell line that contains the genetic material of interest integrated into its own genome and has the ability to express the product of the genetic material after many generations of cell division. In other cases, the host cell, which is a mammalian cell, is a transient cell line or a cell line that does not contain the genetic material of interest integrated into its own genome and does not have the ability to express the product of the genetic material after many generations of cell division.

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

In some embodiments, the eukaryotic host cell is a host cell that is an insect cell. An exemplary host cell that is an insect cell includes Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

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 a Saccharomyces cerevisiae yeast strain such as INVSc1.

In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, the plant cell comprises an algal cell. Exemplary plant cell lines include Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

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™.

In some cases, suitable polynucleic acid molecules or vectors for producing the IL-2 polypeptide described herein include any suitable vectors derived from a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors derived from a bacterial (e.g., E. coli ), insect, yeast (e.g., Pichia pastoris , K. phaffii ), algae, or mammalian source. Bacterial vectors include, for example, pACYC177, pASK75, pBAD series vectors, pBADM series vectors, pET series vectors, pETM series vectors, pGEX series vectors, pHAT, pHAT2, pMal-c2, pMal-p2, pQE series vectors, 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.

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

Yeast vectors include, for example, ^Gateway® pDEST™ 14 vector, Gateway® ^pDEST ™ 15 vector, Gateway® ^pDEST ™ 17 vector, ^Gateway® pDEST™ 24 vector, ^Gateway® pYES-DEST52 vector, ^Gateway® pBAD-DEST49 delivery vector, pAO815 Pichia vector, Pichia pastoris ( K. phaffii ) pFLD1 vector, Pichia pastoris ( K. phaffii ) pGAPZ A, B, and C vector, pPIC3.5K Pichia vector, pPIC6 A, B, and C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, and C yeast vector or yeast vector pYES3/CT.

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

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

In some cases, a cell-free system is used to produce a cytokine polypeptide (e.g., IL-2) described herein. In some cases, the cell-free system contains a mixture of cytoplasmic and/or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some cases, the cell-free system uses prokaryotic cell components. In other cases, the cell-free system uses eukaryotic cell components. The nucleic acid synthesis is achieved in a cell-free system, such as a Drosophila cell, Xenopus egg, archaea, or HeLa cell based system. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or the PURExpress®, XpressCF, and XpressCF+ system.

Cell-free translation systems comprise various components, such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and/or unnatural amino acids, and/or other components used to express the protein. Such components are optionally modified to improve yield, increase the rate of synthesis, increase the quality of the protein product, or incorporate unnatural amino acids. In some embodiments, the cytokines described herein are synthesized using the cell-free translation systems described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245, or US 8,778,631. In some embodiments, the cell-free translation systems involve modifying the release factors or even removing one or more release factors from the system. In some embodiments, the cell-free translation systems contain a reduced concentration of protease. In some embodiments, the cell-free translation systems contain modified tRNAs with reassigned codons used to encode unnatural amino acids. In some embodiments, the synthetases described herein for incorporating unnatural amino acids are used in the cell-free translation systems. In some embodiments, the tRNAs are preloaded with unnatural amino acids using enzymatic or chemical methods before being added to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or another organism.

In some embodiments, the cytokine polypeptide (e.g., IL-2) is produced in a ring-rearranged form using a host expression system or a cell-free system.

Obtaining a cytokine polypeptide containing an unnatural amino acid

The present invention may employ an orthogonal or expanded genetic code in which one or more specific codons present in the nucleic acid sequence of a cytokine polypeptide (e.g., IL-2) are designed to encode an unnatural amino acid so that it can be genetically inserted into the cytokine (e.g., IL-2) using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA synthetase/tRNA pair is capable of loading the tRNA with an unnatural amino acid and is capable of inserting this unnatural amino acid into the polypeptide chain in accordance with the codon.

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

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

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

In some cases, unnatural nucleic acids are used, resulting in the incorporation of one or more unnatural amino acids into the cytokine (eg, IL-2). Illustrative non-naturally occurring nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl- and other alkyl derivatives of adenine and guanine, 2-propyl- and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 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-, in particular 5-bromo-, 5-trifluoromethyl- and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids are provided 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 enhance the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, mixed nucleic acids, enlarged 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-, 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-, in particular 5-bromo-, 5-trifluoromethyl-, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-iodouracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine and those described in U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5093232; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121; 5596091; 5614617; 5645985; 5681941; 5750692; 5763588; 5830653 and 6005096; WO 99/62923; Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Further modifications of the bases can be found, for example, in U.S. Patent No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993. The disclosures of each of these literature sources are incorporated herein by reference.

Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acids in some cases contain one or more heterocyclic bases other than the basic five base components of naturally occurring nucleic acids. For example, a heterocyclic base includes in some cases 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, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-3-yl groups, wherein purines are attached to the sugar moiety of the nucleic acid at position 9, pyrimidines at position 1, pyrrolopyrimidines at position 7, and pyrazolopyrimidines at position 1.

In some embodiments, the nucleotide analogs are also modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, moieties with a modification at the linkage between two nucleotides and include, for example, phosphorothioate, chiral phosphorothioate, phosphodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3'-alkylenephosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidate and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It should be understood that these phosphate or modified phosphate linkages between two nucleotides are formed by a 3'-5' linkage or a 2'-5' linkage and the linkage is characterized by inverted polarity, such as from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Numerous U.S. patents teach the concept of making and using nucleotides containing modified phosphates and include, but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5453496; 5455233; 5466677; 5476925; 5519126; 5536821; 5541306; 5550111; 5563253; 5571799; 5587361 and 5625050, the disclosures of each of which are incorporated herein by reference.

In some embodiments, non-naturally occurring nucleic acids include 2′,3′-dideoxy-2′,3′-didehydronucleosides (PCT/US2002/006460), 5′-substituted derivatives of DNA and RNA (PCT/US2011/033961; Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020; PCT/JP2000/004720; PCT/JP2003/002342; PCT/JP2004/013216; PCT/JP2005/020435; PCT/JP2006/315479; PCT/JP2006/324484; PCT/JP2009/056718; PCT/JP2010/067560) or 5'-substituted monomers prepared as monophosphate with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337). The disclosure of each of these literature sources is incorporated herein by reference.

In some embodiments, the non-natural nucleic acids contain modifications at the 5'-position and 2'-position of the sugar ring (PCT/US94/02993), such as 5'-CH2 _- substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some cases, unnatural nucleic acids contain amide-linked nucleoside dimers that have been engineered for incorporation into oligonucleotides, where the 3'-linked nucleoside in the dimer (5'-3') contains 2'-OCH ₃ and 5'-(S)-CH ₃ (Mesmaeker et al., Synlett, 1997, 1287-1290). Unnatural nucleic acids may contain 2'-substituted 5'-CH ₂ - (or O-) modified nucleosides (PCT/US92/01020). Unnatural nucleic acids may comprise 5'-methylenephosphonate monomers and dimers of DNA and RNA (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Unnatural nucleic acids may comprise 5'-phosphonate monomers having a 2'-substitution (US2006/0074035) and other modified 5'-phosphonate monomers (WO1997/35869). Unnatural nucleic acids may contain 5'-modified methylenephosphonate monomers (EP614907 and EP629633). Unnatural nucleic acids may contain analogs of 5'- or 6'-phosphonate ribonucleosides containing a hydroxyl group at the 5'- and/or 6'-position (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033). Unnatural nucleic acids may contain 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Unnatural nucleic acids may contain nucleosides having a 6'-phosphonate group in which the 5'- and/or 6'-position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH ₃ ) ₃ ) (and its analogues); methyleneamino group (CH ₂ NH ₂ ) (and its analogues) or cyano group (CN) (and its analogues) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640; and Hampton et al., J. Am. Chem. Soc, 1973, 95(13), 4404-4414). The disclosure of each of these literature sources is incorporated herein by reference.

In some embodiments, the non-natural nucleic acids also comprise modifications of the sugar moiety. In some cases, the nucleic acids comprise one or more nucleosides wherein the sugar group has been modified. Such sugar-modified nucleosides are capable of imparting increased stability to nucleases, increased binding affinity, or some other useful biological properties. In certain embodiments, the nucleic acids comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituent groups (including 5' and/or 2' substituent groups); bridging two ring atoms to form bicyclic nucleic acids (BNAs), replacing the oxygen atom of the ribosyl ring with S, N(R) or C(R ₁ )(R ₂ ) (R=H, C ₁ -C ₁₂ alkyl or a protecting group), and combinations thereof. Examples of chemically modified sugars can be found in WO2008/101157, US2005/0130923 and WO2007/134181, the disclosures of each of which are incorporated herein by reference.

In some cases, the modified nucleic acid contains modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety may be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a cyclopentyl group representing a sugar "analog." The sugar may be in the pyranosyl or furanosyl form. The sugar moiety may be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar may be attached to the corresponding heterocyclic bases in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy RNA analogs, 2'-amino RNA analogs, 2'-fluoro DNA, and 2'-alkoxy or amino RNA/DNA chimeric forms. For example, a sugar modification may include 2'-O-methyluridine or 2'-O-methylcytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. It is known to prepare these sugars or sugar analogs and the corresponding "nucleosides" wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base). Sugar modifications may also be prepared and combined with other modifications.

Modifications of the sugar moiety include naturally occurring modifications of ribose and deoxyribose, as well as non-naturally occurring modifications. Sugar modifications include, but are not limited to, the following modifications at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl may be substituted or unsubstituted _C1 - _C10 alkyl or _C2 - _C10 alkenyl and -alkynyl. 2'-Sugar modifications also include, but are not limited to, -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n ONH ₂ , and -O(CH ₂ ) _n ON[(CH ₂ )nCH ₃ )] ₂ , where n and m are from 1 to about 10.

Other modifications at the 2'-position include, but are not limited to, lower _C1 - _C10 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 group, reporter group, intercalator, group for improving the pharmacokinetic properties _of the oligonucleotide or group for improving the pharmacodynamic properties of the oligonucleotide, and other substituents with similar properties. Similar modifications can also be made at other positions in the sugar, particularly at the 3' position of the sugar in the 3' terminal nucleotide or in oligonucleotides with 2'-5' linkages and at the 5' position of the 5' terminal nucleotide. Modified sugars also include sugars that contain modifications at the bridging ring oxygen such as _CH2 and S. Sugar analog nucleotides can also contain sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous U.S. patents that teach the concept of making such modified sugar structures and that detail and describe a variety of base modifications, such as U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; 4845205; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121, 5596091; 5614617; 5681941 and 5700920, each of which is incorporated herein by reference in its entirety.

Examples of nucleic acids having 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 substituent groups} . The substituent at the 2'-position can also be selected from allyl, amino, azido, thio, O-allyl, O-( _C1 - _C10 alkyl), _OCF3 , O( _CH2 ) _2SCH3 , O( _CH2 ) ₂ - _ON ( _Rm )( _Rn ), and O- _CH2 -C(=O)-N( _Rm )( _Rn ), wherein each _Rm and _Rn is independently H or substituted or _{unsubstituted C1-C10 alkyl} .

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' atoms of the ribosyl ring. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge forms a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, but are not limited to, one of the formulas: 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4'-(CH ₂ ) ₂ -O-2'(ENA);4'-CH(CH ₃ )-O-2' and 4'-CH(CH ₂ OCH ₃ )-O-2' and their analogs (see U.S. Patent No. 7,399,845); 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see WO2009/006478, WO2008/150729, US2004/0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134 and WO2008/154401). See also, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patents 4,849,513, 5,015,733, 5,118,800, 5,118,802, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; International Publication Nos. WO2004/106356, WO1994/14226, WO2005/021570, WO2007/090071, and WO2007/134181; U.S. Patent Application Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. Provisional Patent Application Nos. 60/989574, 61/026995, 61/026998, 61/056564, 61/086231, 61/097787, and 61/099844; and International Application Nos. PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922 and PCT/DK98/00393, the disclosures of each of which are incorporated herein by reference.

In certain embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids may be linked to one another by any internucleic acid linkage. Two general classes of nucleic acid interlinkage groups are defined by the presence or absence of a phosphorus atom. Exemplary phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Exemplary non-phosphorus-containing internucleic acid interlinkage groups include, but are not limited to, methylenemethylimino (-CH₂-N(CH₃)-O-CH₂-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)₂-O-) and N, N*-dimethylhydrazine (-CH₂-N(CH₃)-N(CH₃)). In certain embodiments, bonds between nucleic acids having a chiral atom can be obtained as a racemic mixture, as individual enantiomers, for example, alkyl phosphonates and phosphorothioates. Unnatural nucleic acids may contain one modification. Unnatural nucleic acids may contain several modifications in one of the fragments or between different fragments.

Modifications of the phosphate backbone in a nucleic acid include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridged or unbridged), phosphotriester, phosphodithioate, dithiophosphate, and boranophosphate, and may be used in any combination. Other non-phosphate linkages may also be used.

In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphodithioate internucleotide linkages) may impart immunomodulatory activity to the modified nucleic acid and/or enhance its stability in vivo .

In some cases, a phosphorus-containing derivative (or modified phosphate group) is attached to the sugar moiety or sugar analog moiety and may be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphodithioate, phosphoramidate, or the like. Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found in Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; and Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview” in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.), John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; US Patent No. 5453496 and Micklefield, 2001, Curr. Med. Chem. 8: 1157-1179, the disclosures of each of which are incorporated herein by reference.

In some cases, the backbone modification involves replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral, or cationic group. Examples of such modifications include an anionic internucleoside linkage; a phosphoramidate N3'-P5' modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide-linked DNA; methylene(methylimine) linkages; formacole and thioformacetal linkages; sulfonyl group-containing backbones; morpholine oligonucleotides; peptide nucleic acids (PNA), and positively charged deoxyribonucleic guanidine (DNG) oligonucleotides (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179, the disclosure of which is incorporated herein by reference). The modified nucleic acid may comprise a chimeric or mixed backbone containing one or more modifications, such as, a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

Substituents for the phosphate include, for example, short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. They include those having morpholine 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; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones and others having mixed constituents of N, O, S, and CH₂. Numerous U.S. patents disclose the preparation and use of these types of phosphate replacements and include, without limitation, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the disclosures of each of which are incorporated herein by reference. In the case of a substituent for a nucleotide, it is also to be understood that both the sugar and phosphate moieties of the nucleotide can be replaced, for example, by an amide-type (aminoethylglycine) linkage (PNA). U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach the preparation and use of PNA molecules, and each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates may be chemically linked to a nucleotide or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioether, for example, 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), an aliphatic chain, for example, 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), phospholipid, for example, di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237) or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous U.S. patents have disclosed the concept of preparing such conjugates and include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5414077; 5486603; 5512439; 5578718; 5608046; 4587044; 4605735; 4667025; 4762779; 4789737; 4824941; 4835263; 4876335; 4904582; 4958013; 5082830; 5112963; 5214136; 5082830; 5112963; 5214136; 5245022; 5254469; 5258506; 5262536; 5272250; 5292873; 5317098; 5371241; 5391723; 5416203; 5451463; 5510475; 5512667; 5514785; 5565552; 5567810; 5574142; 5585481; 5587371; 5595726; 5597696; 5599923; 5599928 and 5688941. The disclosures of each of these literature sources are incorporated herein by reference.

In some cases, the non-natural nucleic acids further form non-natural base pairs. Exemplary non-natural nucleotides capable of forming a non-natural DNA or RNA base pair (UBP) in vivo 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 non-natural nucleotides include:

Illustrative 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.

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, the non-naturally occurring nucleotides include:

In some embodiments, non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be prepared from a compound of the formula

where R ₂ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano and azido; and

the wavy line indicates a bond with ribosyl or 2'-deoxyribosyl, where the 5'-hydroxy group of the ribosyl or 2'-deoxyribosyl moiety is in free form, optionally linked to a monophosphate, diphosphate or triphosphate group, or incorporated into RNA or DNA or into an RNA analogue or DNA analogue.

In some embodiments, non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be prepared from a compound of the formula

Where:

each X independently represents carbon or nitrogen;

R ₂ is absent if X is nitrogen and is present if X is carbon and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano or azide;

Y represents sulfur, oxygen, selenium, or a secondary amine;

E represents oxygen, sulfur, or selenium; and

the wavy line indicates the point of attachment to a ribosyl, deoxyribosyl or dideoxyribosyl moiety or an analogue thereof, where the ribosyl, deoxyribosyl or dideoxyribosyl moiety or an analogue thereof is in free form, linked to a monophosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate or γ-thiotriphosphate group, or incorporated into RNA or DNA or into an RNA analogue or a DNA analogue.

In some embodiments, each X is carbon. In some embodiments, at least one X is carbon. In some embodiments, one X is carbon. In some embodiments, at least two X are carbon. In some embodiments, two X are carbon. In some embodiments, at least one X is nitrogen. In some embodiments, one X is nitrogen. In some embodiments, at least two X are nitrogen. In some embodiments, two X are nitrogen.

In some embodiments, Y is sulfur. In some embodiments, Y is oxygen. In some embodiments, Y is selenium. In some embodiments, Y is a secondary amine.

In some embodiments, E is sulfur. In some embodiments, E is oxygen. In some embodiments, E is selenium.

In some embodiments, R ₂ is present when X is carbon. In some embodiments, R ² is absent when X is nitrogen. In some embodiments, each R ₂ , when present, is hydrogen. In some embodiments, R ₂ is alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ₂ is alkenyl, such as -CH ₂ =CH ₂ . In some embodiments, R ₂ is alkynyl, such as ethynyl. In some embodiments, R ₂ is methoxy. In some embodiments, R ₂ is methanethiol. In some embodiments, R ₂ is methaneseleno. In some embodiments, R ₂ is a halogen, such as chloro, bromo, or fluoro. In some embodiments, R ₂ is cyano. In some embodiments, R ₂ is azide.

In some embodiments, E is sulfur, Y is sulfur, and each X is independently carbon or nitrogen. In some embodiments, E is sulfur, Y is sulfur, and each X is carbon.

In some embodiments, non-natural nucleotides that can be used to produce the IL-2 conjugates disclosed herein can be obtained from

In some embodiments, non-natural nucleotides that can be used to produce the IL-2 conjugates disclosed herein include

In some embodiments, the unnatural base pair forms a unnatural amino acid as described in Dumas et al., "Designing logical codon reassignment - Expanding the chemistry in biology," Chemical Science , 6 : 50-69 (2015), the disclosure of which is incorporated herein by reference.

In some embodiments, a non-natural amino acid is incorporated into a cytokine (e.g., an IL polypeptide) using a synthetic codon containing a non-natural nucleic acid. In some cases, a non-natural amino acid is incorporated into a cytokine using a modified synthetase/tRNA orthogonal pair. Such orthogonal pairs comprise a non-natural synthetase that is capable of loading a non-natural tRNA with the non-natural amino acid while minimizing the loading of a) other endogenous amino acids onto the non-natural tRNA and b) non-natural amino acids onto other endogenous tRNAs. Such orthogonal pairs comprise tRNAs that are capable of being loaded by the non-natural synthetase while avoiding the loading of a) other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacterial, yeast, archaeal, or human sources. In some embodiments, the orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, the orthogonal synthetase/tRNA pair comprises components from two different organisms. In some embodiments, the orthogonal synthetase/tRNA pair comprises components that, prior to modification, facilitate the translation of two different amino acids. In some embodiments, the orthogonal synthetase is a modified alanine synthetase. In some embodiments, the orthogonal synthetase is a modified arginine synthetase. In some embodiments, the orthogonal synthetase is a modified asparagine synthetase. In some embodiments, the orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, the orthogonal synthetase is a modified cysteine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamic acid synthetase. In some embodiments, the orthogonal synthetase is a modified alanine/glycine synthetase. In some embodiments, the orthogonal synthetase is a modified histidine synthetase. In some embodiments, the orthogonal synthetase is a modified leucine synthetase. In some embodiments, the orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, the orthogonal synthetase is a modified lysine synthetase. In some embodiments, the orthogonal synthetase is a modified methionine synthetase. In some embodiments, the orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, the orthogonal synthetase is a modified proline synthetase. In some embodiments, the orthogonal synthetase is a modified serine synthetase. In some embodiments, the orthogonal synthetase is a modified threonine synthetase. In some embodiments, the orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, the orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, the orthogonal synthetase is a modified valine synthetase. In some embodiments, the orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, the orthogonal tRNA is a modified tRNA for alanine. In some embodiments, the orthogonal tRNA is a modified tRNA for arginine. In some embodiments, the orthogonal tRNA is a modified tRNA for asparagine. In some embodiments, the orthogonal tRNA is a modified tRNA for aspartic acid. In some embodiments, the orthogonal tRNA is a modified tRNA for cysteine. In some embodiments, the orthogonal tRNA is a modified tRNA for glutamine. In some embodiments, the orthogonal tRNA is a modified tRNA for glutamic acid. In some embodiments, the orthogonal tRNA is a modified tRNA for alanine/glycine. In some embodiments, the orthogonal tRNA is a modified tRNA for histidine. In some embodiments, the orthogonal tRNA is a modified tRNA for leucine. In some embodiments, the orthogonal tRNA is a modified tRNA for isoleucine. In some embodiments, the orthogonal tRNA is a modified tRNA for lysine. In some embodiments, the orthogonal tRNA is a modified tRNA for methionine. In some embodiments, the orthogonal tRNA is a modified tRNA for phenylalanine. In some embodiments, the orthogonal tRNA is a modified tRNA for proline. In some embodiments, the orthogonal tRNA is a modified tRNA for serine. In some embodiments, the orthogonal tRNA is a modified tRNA for threonine. In some embodiments, the orthogonal tRNA is a modified tRNA for tryptophan. In some embodiments, the orthogonal tRNA is a modified tRNA for tyrosine. In some embodiments, the orthogonal tRNA is a modified tRNA for valine. In some embodiments, the orthogonal tRNA is a modified tRNA for phosphoserine.

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

In some cases, an unnatural amino acid is incorporated into a cytokine (e.g., an IL polypeptide) via an Ec-Tyr /tRNA _CUA or an Ec-Leu /tRNA _CUA pair. Exemplary UAAs that can be incorporated via an Ec-Tyr /tRNA _CUA or an Ec-Leu /tRNA _CUA pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O -propargyl tyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine, and 3-(naphthalen-2-ylamino)-2-aminopropanoic acid.

In some cases, the unnatural amino acid is incorporated into a cytokine (e.g., an IL polypeptide) via a pyrrolyl-tRNA pair. In some cases, PylRS is derived from an archaebacterium, such as a methanogenic archaebacterium. In some cases, PylRS is derived from Methanosarcina barkeri , Methanosarcina mazei , or Methanosarcina acetivorans . Illustrative UAAs that can be incorporated via a pyrrolyl-tRNA pair include, but are not limited to, amide- and carbamate-substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N -ε- _D -prolyl- _L- lysine, and N -ε-cyclopentyloxycarbonyl- _L -lysine; N -ε-acryloyl- _L -lysine; N -ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl] _-L- lysine and N -ε-(1-methylcycloprop-2-ene-carboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein can be prepared by using M. mazei tRNA that is selectively loaded with a non-natural amino acid, such as N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), using M. barkeri pyrrolizyl-tRNA synthetase ( Mb PylRS). Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647, the disclosure of which is incorporated herein by reference.

In some cases, the unnatural amino acid is incorporated into a cytokine described herein (e.g., an IL polypeptide) using the synthetase disclosed in US 9988619 and US 9938516, the disclosures of each of which are incorporated herein by reference.

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

An orthogonal tRNA synthetase/tRNA pair loads a tRNA with an unnatural amino acid and inserts the unnatural amino acid into a polypeptide chain according to a codon. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii aaRS ( Mj-Tyr )/tRNA pairs, E. coli TyrRS ( Ec-Tyr )/ _B . stearothermophilus tRNACUA pairs, E. coli LeuRS ₍ Ec-Leu )/ B. stearothermophilus tRNACUA pairs, and pyrrolizyl-tRNA pairs. Other aaRS-tRNA pairs that can be used in accordance with the present invention include those derived from M. mazei , the pairs described in Feldman et al., J Am Chem Soc., 2018 140:1447-1454; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322, the disclosures of each of which are incorporated herein by reference.

In some embodiments, methods for producing IL-2 conjugates disclosed herein are provided in a cellular system that expresses NTT and a tRNA synthetase. In some embodiments described herein, NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , and M. mazei . In some embodiments, NTT is PtNTT1 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT2 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT4 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT5 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei .

In some embodiments, the IL-2 conjugates disclosed herein can be produced in a cell, such asE. colicontaining (a) a nucleotide triphosphate transporter PtNTT2 (including a truncated variant in which the first 65 amino acid residues of the full-length protein are deleted), (b) a plasmid containing a double-stranded oligonucleotide that encodes an IL-2 variant having the desired amino acid sequence and that contains a non-natural base pair comprising a first non-natural nucleotide and a second non-natural nucleotide to provide a codon at the desired position into which the non-natural amino acid will be inserted, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), (c) plasmid encoding tRNA, obtained fromM. mazei, which contains an unnatural nucleotide to provide a recognizable anticodon (for the IL-2 variant codon) in place of its native sequence, and (d) a plasmid encoding pyrrolizyl-tRNA synthetase derived fromM. barkeri(MbPylRS), which may be the same plasmid that encodes the tRNA or a different plasmid. Furthermore, in some embodiments, the cell is supplemented with deoxyribotriphosphates containing one or more unnatural bases. Furthermore, in some embodiments, the cell is supplemented with ribotriphosphates containing one or more unnatural bases. Furthermore, in some embodiments, the cells are supplemented with one or more unnatural amino acids, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, a double-stranded oligonucleotide that encodes the amino acid sequence of a desired IL-2 variant comprises an AXC codon, such as at position 34, 37, 40, 41, 42, 43, 44, 61, 64, 68, or 71 of the sequence that encodes the protein of SEQ ID NO: 3, or at position 35, 38, 41, 42, 43, 45, 62, 65, 69, or 72 of the sequence that encodes the protein of SEQ ID NO: 4, wherein X is a non-natural nucleotide. In some embodiments, the cell further comprises a plasmid, which can be a protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene fromM. mazei, which comprises a GYT anticodon corresponding to AXC instead of its native sequence, wherein Y is a non-natural nucleotide that is complementary to the non-natural nucleotide in the codon and may be the same as or different from it. In some embodiments, the non-natural nucleotide in the codon is different from and complementary to the non-natural nucleotide in the anticodon. In some embodiments, the non-natural nucleotide in the codon is the same as the non-natural nucleotide in the anticodon. In some embodiments, the first and second non-natural nucleotides that comprise the non-natural base pair in the double-stranded oligonucleotide may be derived from . In some embodiments, the first and second non-natural nucleotides comprising the non-natural base pair in the double-stranded oligonucleotide may be derived from And . In some embodiments, the first and second non-natural nucleotides comprising the non-natural base pair in the double-stranded oligonucleotide may be derived from And In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise, And or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise And or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise And or salts thereof. In some embodiments, mRNA derived from a double-stranded oligonucleotide comprising a first non-natural nucleotide and a second non-natural nucleotide may comprise a codon comprising a non-natural nucleotide derived from,,,, And . In some embodiments, the tRNA M. mazeimay contain an anticodon containing an unnatural nucleotide that recognizes a codon containing an unnatural nucleotide in mRNA. Anticodon in tRNAM. mazeimay contain an unnatural nucleotide derived from,,,, And . In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases that are necessary for the replication of the plasmid(s) encoding the cytokine gene and carrying the codon, (b) ribonucleoside triphosphates containing one or more unnatural bases necessary for the transcription of (i) mRNA corresponding to the cytokine encoding sequence and containing a codon containing one or more unnatural bases, and (ii) tRNA containing an anticodon containing one or more unnatural bases, and (c) unnatural amino acid(s) to be inserted into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that allow expression of the protein of interest.

The resulting expressed protein comprising AzK can be purified by methods known to those of ordinary skill in the art and then reacted with an alkyne, such as DBCO, containing a PEG chain having a desired average molecular weight as disclosed herein under conditions known to those of ordinary skill in the art to produce the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO 2019014262; WO2019014267; WO2019028419 and WO2019/028425, the disclosures of each of which are incorporated herein by reference.

The resulting expressed protein comprising one or more unnatural amino acids, such as AzK, can be purified by methods known to those of ordinary skill in the art and then reacted with an alkyne, such as DBCO, comprising a PEG chain having a desired average molecular weight as disclosed herein, under conditions known to those of ordinary skill in the art to produce the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO2019014262; WO2019014267; WO2019028419 and WO2019/028425, the disclosures of each of which are incorporated herein by reference.

Alternatively, a cytokine polypeptide (e.g., IL-2) comprising a non-natural amino acid(s) is produced by introducing into a host cell the nucleic acid constructs described herein comprising a tRNA and aminoacyl-tRNA synthetase and comprising a nucleic acid sequence of interest with one or more orthogonal (stop) codons in frame. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene and carrying the novel codon and anticodon, (b) ribonucleoside triphosphates required for transcription of mRNA corresponding to (i) the cytokine sequence containing the codon and (ii) the orthogonal tRNA containing the anticodon, and (c) unnatural amino acid(s). The host cells are then maintained under conditions that allow expression of the protein of interest. The unnatural amino acid(s) is(are) incorporated into the polypeptide chain corresponding to the unnatural codon. For example, one or more unnatural amino acids are incorporated into a cytokine polypeptide (e.g., IL-2). Alternatively, two or more unnatural amino acids may be incorporated into a cytokine polypeptide (e.g., IL-2) at two or more sites in the protein.

Once produced in a host cell, the cytokine polypeptide (e.g., IL-2) into which the unnatural amino acid(s) is(are) incorporated can be extracted therefrom by a number of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. The cytokine polypeptide (e.g., IL-2) can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or any other suitable technique known to those of ordinary skill in the art.

Suitable host cells may include bacterial cells (e.g., E. coli, BL21(DE3)), but most suitably the host cells are eukaryotic cells, such as insect cells (e.g., a member of the genus Drosophila such as Drosophila melanogaster ), yeast cells, nematode cells (e.g., C. elegans ), mouse cells (e.g., Mus musculus ) or mammalian cells (such as Chinese hamster ovary (CHO) cells or COS cells, human 293T cells, HeLa cells, NIH/3T3 cells, and mouse erythroleukemia (MEL) cells) or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Suitably, the host cell is a mammalian cell, such as a human cell, or an insect cell. In some embodiments, suitable host cells include E. coli .

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

In the generation of cell lines, it is generally preferred to obtain stable cell lines. For example, it is known that in the case of stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of the cells can integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., for antibiotic resistance) is usually introduced into the host cells along with the gene of interest. Preferred selectable markers include markers that confer resistance to drugs such as G418, hygromycin, or methotrexate. Nucleic acid molecules encoding a selectable marker can be introduced into the host cell in the same vector or can be introduced in a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by selection for drug sensitivity (e.g., cells that contain the inserted selectable marker gene survive while other cells die).

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

Additional funds

In some embodiments, described herein is a method of treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein. In some embodiments, described herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein in combination with one or more additional agents. In some embodiments, described herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein in combination with one or more immune checkpoint inhibitors.

In some embodiment, the one or more additional agents include one or more immune checkpoint inhibitors selected from PD-1 inhibitors. In some embodiment, the one or more additional agents include one or more PD-1 inhibitors. In some embodiments, the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (MacroGenics/Incyte), GLS-010 (Arcus/WuXi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer), and genolimzumab. (Apollomics/Genor BioPharma). 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. In some embodiments, the one or more PD-1 inhibitors are lambrolizumab. In some embodiments, the one or more PD-1 inhibitors are AMP-224. In some embodiments, the one or more PD-1 inhibitors are sintilimab. In some embodiments, the one or more PD-1 inhibitors are toripalimab. In some embodiments, the one or more PD-1 inhibitors are camrelizumab. In some embodiments, the one or more PD-1 inhibitors are tislelizumab.

In some embodiments, the one or more additional agents comprise immune checkpoint inhibitors selected from PD-L1 inhibitors. In some embodiments, the one or more PD-L1 inhibitors are selected from atezolizumab, avelumab, and durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (CytomX), CS1001 (CStone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics). In some embodiments, the one or more PD-L1 inhibitors are atezolizumab. In some embodiments, the one or more PD-L1 inhibitors are avelumab. In some embodiments, the one or more PD-L1 inhibitors are durvalumab. In some embodiments, the one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors. In some embodiments, the one or more CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus). In some embodiments, the one or more CTLA-4 inhibitors are tremelimumab. In some embodiments, the one or more CTLA-4 inhibitors are ipilimumab.

In some embodiments, the one or more additional agents include immune checkpoint inhibitors selected from CTLA-4 inhibitors. In some embodiments, the CTLA-4 inhibitor is selected from tremelimumab and ipilimumab. In some embodiments, the CTLA-4 inhibitor is tremelimumab. In some embodiments, the CTLA-4 inhibitor is ipilimumab.

Treatment methods

Described herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of (a) an IL-2 conjugate described herein and (b) one or more additional agents. In some embodiments, described herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of (a) an IL-2 conjugate described herein and (b) one or more immune checkpoint inhibitors.

Types of cancer

Described herein are methods for treating cancer in a subject comprising administering to the subject in need thereof an effective amount of an IL-2 conjugate described herein. In some embodiments of the method of treating cancer described herein, the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stability cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, glioblastoma, mesothelioma, breast cancer, triple-negative breast cancer, prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or metastatic castration-resistant prostate cancer with defects in DNA damage response (DDR), bladder cancer, ovarian cancer, tumors with intermediate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low to absent PD-L1 expression, tumors that have spread systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma.

In some embodiments of the method of treating cancer described herein, the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), urothelial carcinoma, melanoma, Merkel cell carcinoma (MCC), and head and neck squamous cell carcinoma (HNSCC). In one embodiment, the cancer is renal cell carcinoma (RCC). In one embodiment, the cancer is non-small cell lung cancer (NSCLC). In one embodiment, the cancer is urothelial carcinoma. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is Merkel cell carcinoma (MCC). In one embodiment, the cancer is head and neck squamous cell carcinoma (HNSCC).

In some embodiments, the methods described herein are provided, wherein the one or more additional agents comprise one or more immune checkpoint inhibitors.

In some embodiments, one or more immune checkpoint inhibitors are selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, OX40 agonists, and 4-1BB agonists.

In some embodiments, one or more immune checkpoint inhibitors are selected from PD-1 inhibitors. In some embodiments, the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (Macrogenics/Incyte), GLS-010 (Arcus/WuXi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer), and genolimzumab. (Apollomics/Genor BioPharma). 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. In some embodiments, the one or more PD-1 inhibitors are lambrolizumab. In some embodiments, the one or more PD-1 inhibitors are AMP-224. In some embodiments, the one or more PD-1 inhibitors are sintilimab. In some embodiments, the one or more PD-1 inhibitors are toripalimab. In some embodiments, the one or more PD-1 inhibitors are camrelizumab. In some embodiments, the one or more PD-1 inhibitors are tislelizumab.

In some embodiments, the one or more PD-L1 inhibitors are selected from atezolizumab, avelumab, and durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (CytomX), CS1001 (CStone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics). In some embodiments, the one or more PD-L1 inhibitors are atezolizumab. In some embodiments, the one or more PD-L1 inhibitors are avelumab. In some embodiments, the one or more PD-L1 inhibitors are durvalumab. In some embodiments, the one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors. In some embodiments, the one or more CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus). In some embodiments, the one or more CTLA-4 inhibitors are tremelimumab. In some embodiments, the one or more CTLA-4 inhibitors are ipilimumab.

In some embodiments, the one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors. In some embodiments, the CTLA-4 inhibitor is selected from tremelimumab and ipilimumab. In some embodiments, the CTLA-4 inhibitor is tremelimumab. In some embodiments, the CTLA-4 inhibitor is ipilimumab.

In some embodiments, the cancer is in the form of a solid tumor. In some embodiments, the cancer is in the form of a liquid tissue tumor.

In some embodiments, the IL-2 conjugate is administered to the subject prior to administration of the one or more additional agents to the subject. In some embodiments, the one or more additional agents are administered to the subject prior to administration of the IL-2 conjugate to the subject. In some embodiments, the IL-2 conjugate and the one or more additional agents are administered to the subject concurrently.

In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of one or more vascular endothelial cell growth factor (VEGF) or mammalian target of rapamycin (mTOR) signaling pathway inhibitors in addition to one or more checkpoint inhibitors. In some embodiments, the subject is administered one or more VEGF signaling pathway inhibitors. In some embodiments, the one or more VEGF signaling pathway inhibitors are selected from the group consisting of vascular endothelial cell growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) and monoclonal antibodies to VEGF. In some embodiments, the one or more VEGF signaling pathway inhibitors are selected from one or more VEGFR TKIs. In some embodiments, the one or more VEGFR TKIs are selected from the group consisting of cabozantinib, axitinib, pazopanib, sunitinib, or sorafenib. In some embodiments, the one or more VEGFR TKIs are cabozantinib. In some embodiments, the one or more VEGFR TKIs are axitinib. In some embodiments, the one or more VEGFR TKIs are pazopanib. In some embodiments, the one or more VEGFR TKIs are sunitinib. In some embodiments, wherein the one or more VEGFR TKIs are sorafenib. In some embodiments, the one or more VEGF signaling pathway inhibitors are selected from one or more anti-VEGF monoclonal antibodies. In some embodiments, the one or more anti-VEGF monoclonal antibodies are bevacizumab.

In some embodiments, the one or more mTOR inhibitors are selected from the group consisting of rapamycin, everolimus, temsirolimus, ridaforolimus, and deforolimus. In some embodiments, the one or more mTOR inhibitors are rapamycin. In some embodiments, the one or more mTOR inhibitors are everolimus. In some embodiments, the one or more mTOR inhibitors are temsirolimus. In some embodiments, the one or more mTOR inhibitors are ridaforolimus. In some embodiments, the one or more mTOR inhibitors are deforolimus. In some embodiments, the cancer in the subject is renal cell carcinoma (RCC).

In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of one or more poly ADP ribose polymerase (PARP) inhibitors in addition to the one or more checkpoint inhibitors. In some embodiments, the PARP inhibitors are selected from the group consisting of olaparib, niraparib, rucaparib, talazoparib, veliparib, CEP-9722, and E7016. In some embodiments, the PARP inhibitor is olaparib. In some embodiments, the PARP inhibitor is niraparib. In some embodiments, the PARP inhibitor is rucaparib. In some embodiments, the PARP inhibitor is talazoparib. In some embodiments, the PARP inhibitor is veliparib. In some embodiments, the PARP inhibitor is CEP-9722. In some embodiments, the PARP inhibitor is E7016.

In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of a nonsteroidal antiandrogen compound (NSAA) in addition to one or more checkpoint inhibitors. In some embodiments, the NSAA is flutamide, nilutamide, bicalutamide, topilutamide, apalutamide, or enzalutamide. In some embodiments, the NSAA is flutamide. In some embodiments, the NSAA is nilutamide. In some embodiments, the NSAA is bicalutamide. In some embodiments, the NSAA is topilutamide. In some embodiments, the NSAA is apalutamide. In some embodiments, the NSAA is enzalutamide.

In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of one or more poly-ADP-ribose polymerase (PARP) inhibitors and a non-steroidal antiandrogen compound (NSAA) in addition to the one or more checkpoint inhibitors, wherein the PARP and NSAA inhibitors can be independently selected from those set forth above.

In some embodiments, the one or more additional agents further comprise one or more chemotherapeutic agents in addition to the one or more checkpoint inhibitors. In some embodiments, the one or more chemotherapeutic agents comprise one or more platinum-based chemotherapeutic agents. In some embodiments, the one or more chemotherapeutic agents comprise carboplatin and pemetrexed. In some embodiments, the one or more chemotherapeutic agents comprise carboplatin and nab-paclitaxel. In some embodiments, the one or more chemotherapeutic agents comprise carboplatin and docetaxel. In some embodiments, the cancer in the subject is non-small cell lung cancer (NSCLC).

In some embodiments, the one or more additional agents are one or more chemotherapeutic agents. In some embodiments, the one or more chemotherapeutic agents include one or more platinum-based chemotherapeutic agents. In some embodiments, the subject tests positive for human papillomavirus (HPV) prior to administration of the IL-2 conjugate and the one or more additional agents. In some embodiments, the cancer in the subject is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the method further comprises the subject testing positive for human papillomavirus (HPV+) followed by administration of the IL-2 conjugate and the one or more additional agents.

Introduction

In some embodiments, following administration of the IL-2 conjugate and one or more additional agents, the subject experiences a response as measured by the immune-mediated Response Evaluation Criteria in Solid Tumors (iRECIST).

In some embodiments, the response is a complete response, a partial response, or stabilization of the disease. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, or intramuscular administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous administration. In some embodiments, the IL-2 conjugate is administered to the subject by subcutaneous administration. In some embodiments, the IL-2 conjugate is administered to the subject by intramuscular administration.

In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, once every 16 weeks, once every 17 weeks, once every 18 weeks, once every 19 weeks, once every 20 weeks, once every 21 weeks, once every 22 weeks, once every 23 weeks, once every 24 weeks, once every 25 weeks, once every 26 weeks, once every 27 weeks, or once every 28 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once a week. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every two weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every three weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 4 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 5 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 6 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 7 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 8 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 9 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 10 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 11 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 12 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 13 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 14 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 15 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 16 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 17 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 18 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 19 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 20 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 21 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 22 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 23 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 24 weeks.

In some embodiments, the amount of said agent that corresponds to such an amount varies depending on factors such as the particular compound, the severity of the disease, the individual characteristics (e.g., weight) of the subject or host in need of treatment, but is nevertheless usually determined in a manner known in the art, in accordance with the specific circumstances relating to a particular case, including, for example, the particular agent administered, the route of administration, and the subject or host receiving treatment. In some cases, the desired dose is conveniently presented as a single dose or divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example, as two, three, four or more portions of a dose per day.

In some embodiments, the methods comprise administering a dose of an IL-2 conjugate to a subject in need thereof in a dose range of from 1 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 2 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 4 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 6 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 8 μg of IL-2 conjugate per kg of subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 10 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 12 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 14 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 16 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 18 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 20 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 22 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 24 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 26 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 28 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 32 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 34 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 36 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 40 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 45 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 50 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 55 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 60 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 65 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 70 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 75 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 80 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 85 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 90 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 95 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 100 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 110 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 120 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 130 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 140 μg IL-2 conjugate per kg body weight subject to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 150 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 160 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 170 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 180 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 190 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight. The above ranges are only suggestive since the number of variables related to an individual treatment regimen is large and significant deviations from these recommended values are not uncommon. Such dosages vary depending on a number of variables not limited to the activity of the compound being used, the disease or condition being treated, the route of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner. In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or in experimental animals, including but not limited to determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and is expressed as the ratio between the LD50 and the ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within the range of circulating concentrations that includes the ED50 with minimal toxicity. The dosage varies within this range depending on the dosage form used and the route of administration used.

In some embodiments, the methods comprise administering a dose of an IL-2 conjugate to a subject in need thereof at a dose of about 1 μg of IL-2 conjugate per kg of subject body weight, or about 2 μg of IL-2 conjugate per kg of subject body weight, about 4 μg of IL-2 conjugate per kg of subject body weight, about 6 μg of IL-2 conjugate per kg of subject body weight, about 8 μg of IL-2 conjugate per kg of subject body weight, about 10 μg of IL-2 conjugate per kg of subject body weight, about 12 μg of IL-2 conjugate per kg of subject body weight, about 14 μg of IL-2 conjugate per kg of subject body weight, about 16 μg of IL-2 conjugate per kg of subject body weight, about 18 μg IL-2 conjugate per kg subject body weight, approximately 20 μg IL-2 conjugate per kg subject body weight, approximately 22 μg IL-2 conjugate per kg subject body weight, approximately 24 μg IL-2 conjugate per kg subject body weight, approximately 26 μg IL-2 conjugate per kg subject body weight, approximately 28 μg IL-2 conjugate per kg subject body weight, approximately 30 μg IL-2 conjugate per kg subject body weight, approximately 32 μg IL-2 conjugate per kg subject body weight, approximately 34 μg IL-2 conjugate per kg subject body weight, approximately 36 μg IL-2 conjugate per kg subject body weight, approximately 38 μg IL-2 conjugate per kg subject body weight, approximately 40 μg IL-2 conjugate per kg subject body weight, approximately 42 μg IL-2 conjugate per kg subject body weight, approximately 44 μg IL-2 conjugate per kg subject body weight, approximately 46 μg IL-2 conjugate per kg subject body weight, approximately 48 μg IL-2 conjugate per kg subject body weight, approximately 50 μg IL-2 conjugate per kg subject body weight, approximately 55 μg IL-2 conjugate per kg subject body weight, approximately 60 μg IL-2 conjugate per kg subject body weight, approximately 65 μg IL-2 conjugate per kg subject body weight, approximately 70 μg IL-2 conjugate per kg subject body weight, approximately 75 μg IL-2 conjugate per kg subject body weight, approximately 80 μg IL-2 conjugate per kg subject body weight, approximately 85 μg IL-2 conjugate per kg subject body weight, approximately 90 μg IL-2 conjugate per kg subject body weight, approximately 95 μg IL-2 conjugate per kg subject body weight, approximately 100 μg IL-2 conjugate per kg subject body weight, approximately 110 μg IL-2 conjugate per kg subject body weight, approximately 120 μg IL-2 conjugate per kg subject body weight, approximately 130 μg IL-2 conjugate per kg subject body weight, approximately 140 μg IL-2 conjugate per kg subject body weight, approximately 150 μg IL-2 conjugate per kg subject body weight, approximately 160 mcg IL-2 conjugate per kg subject body weight, approximately 170 mcg IL-2 conjugate per kg subject body weight, approximately 180 mcg IL-2 conjugate per kg subject body weight, approximately 190 mcg IL-2 conjugate per kg subject body weight, or approximately 200 mcg IL-2 conjugate per kg subject body weight. The above ranges are only suggestive since the number of variables related to an individual treatment regimen is large and significant deviations from these recommended values are not uncommon. Such dosages will vary depending on a number of variables not limited to the activity of the compound being administered, the disease or condition being treated, the route of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner. In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens are determined using standard pharmaceutical procedures in cell cultures or in experimental animals, including but not limited to determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population). The dose ratio for toxic and therapeutic effects is the therapeutic index and is expressed as the ratio between the LD50 and the ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a dose range for use in humans. The dose of such compounds is preferably within a range of circulating concentrations that includes the ED50 with minimal toxicity. The dose varies within this range depending on the dosage form used and the route of administration used.

In some embodiments, the additional agent can be administered at a dose and using a dosing regimen that has been determined to be safe and effective for the additional agent. For example, pembrolizumab can be administered to a subject in need thereof according to the methods described herein at a dose of about 200 mg every 3 weeks. In another example, nivolumab can be administered to a subject in need thereof according to the methods described herein at a dose of about 240 mg every 2 weeks or at a dose of about 480 mg every 4 weeks. In another example, cemiplimab can be administered to a subject in need thereof according to the methods described herein at a dose of about 350 mg as an intravenous infusion over 30 minutes every 3 weeks. In another example, atezolizumab can be administered to a subject according to the methods described herein at a dose of 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every 4 weeks. In another example, avelumab can be administered to a subject according to the methods described herein at a dose of 800 mg every 2 weeks. In another example, durvalumab can be administered to a subject according to the methods described herein at a dose of 10 mg per kg of the subject's body weight every 2 weeks. In another example, ipilimumab can be administered to a subject for the treatment of melanoma according to the methods described herein at a dose of about 3 mg per kg of the subject's body weight over 90 minutes every three weeks for a total of 4 doses, or about 10 mg per kg of the subject's body weight over 90 minutes for a total of 4 doses, and then 10 mg per kg of the subject's body weight for up to 3 years. For advanced renal cell carcinoma, ipilimumab may be administered according to the methods described herein at a dose of 1 mg per kg of subject body weight over 30 minutes.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not require an intensive care unit or skilled cardiopulmonary or critical care physicians. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not require an intensive care unit or skilled cardiopulmonary or critical care physicians. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not require an intensive care unit. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not require skilled cardiopulmonary or critical care physicians.

Effects of administration

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause vascular leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 2, grade 3, or grade 4 vascular leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 2 vascular leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 3 vascular leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 4 vascular leak syndrome in the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause a loss of vascular tone in the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause extravasation of blood plasma proteins and fluid into the extravascular space of the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause hypotension and decreased organ perfusion in the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause a disruption of neutrophil function in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause a decrease in chemotaxis in the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not associated with an increased risk of disseminated infection in the subject. In some embodiments of the method of treating cancer described herein, the disseminated infection is sepsis or bacterial endocarditis. In some embodiments of the method of treating cancer described herein, the disseminated infection is sepsis. In some embodiments of the method of treating cancer described herein, the disseminated infection is bacterial endocarditis. In some embodiments of the method of treating cancer described herein, the subject is treated for any pre-existing bacterial infections prior to administration of the IL-2 conjugate. In some embodiments of the method of treating cancer described herein, the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not worsen a pre-existing or initially manifesting autoimmune disease or inflammatory disorder in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not worsen a pre-existing or initially manifesting autoimmune disease in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not worsen a pre-existing or initially manifesting inflammatory disorder in the subject. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, oculobulbar myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is Crohn's disease. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is scleroderma. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is thyroiditis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is inflammatory arthritis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is diabetes mellitus. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is oculobulbar myasthenia gravis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is crescentic IgA glomerulonephritis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is cholecystitis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is cerebral vasculitis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is Stevens-Johnson syndrome. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is bullous pemphigoid.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause mental status changes, speech difficulties, cortical blindness, limb or gait ataxia, hallucinations, agitation, sensory dullness, or coma in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause a seizure in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not contraindicated in subjects who have a confirmed seizure disorder.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause capillary leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 2, grade 3, or grade 4 capillary leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 2 capillary leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 3 capillary leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause grade 4 capillary leak syndrome in the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause a decrease in mean arterial blood pressure in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause hypotension in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause the subject to experience a systolic blood pressure below 90 mmHg or a decrease in systolic pressure by 20 mmHg from baseline after administering the IL-2 conjugate to the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause edema in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause renal or hepatic dysfunction in the subject after administering the IL-2 conjugate to the subject.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause eosinophilia in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause the subject to have a peripheral blood eosinophil count greater than 500 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause the subject to have a peripheral blood eosinophil count greater than 500 per μL to 1500 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause the subject to have a peripheral blood eosinophil count greater than 1500 per μL to 5000 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause the subject to have a peripheral blood eosinophil count greater than 5000 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not contraindicated in subjects on a psychotropic drug regimen.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects receiving a treatment regimen of nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects receiving a treatment regimen of aminoglycosides, cytotoxic chemotherapy agents, doxorubicin, methotrexate, or asparaginase. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects receiving treatment in combination regimens that include antineoplastic agents. In some embodiments of the method of treating cancer described herein, the antineoplastic agent is selected from dacarbazine, cisplatin, tamoxifen, and interferon alpha.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not cause one or more grade 4 adverse events in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, the one or more grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; second degree atrioventricular block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function test abnormalities; gastrointestinal hemorrhage; hematemesis; hemorrhagic diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; increased alkaline phosphatase; increased blood urea nitrogen (BUN); hyperuricemia; increased non-protein nitrogen (NPN); respiratory acidosis; somnolence; agitation; neuropathy; paranoid reaction; spasm; grand mal seizure; delirium; asthma; pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; pupillary abnormalities; renal function abnormalities; renal failure; and acute tubular necrosis. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a group of subjects does not cause one or more grade 4 adverse events in more than 1% of the subjects following administration of the IL-2 conjugate to the subjects. In some embodiments of the method of treating cancer described herein, the one or more grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; second degree atrioventricular block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function test abnormalities; gastrointestinal hemorrhage; hematemesis; hemorrhagic diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; increased alkaline phosphatase; increased blood urea nitrogen (BUN) concentration; hyperuricemia; increased non-protein nitrogen (NPN) concentration; respiratory acidosis; drowsiness; agitation; neuropathy; paranoid reaction; spasm; grand mal seizure; delirium; asthma; pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; pupillary abnormalities; renal function abnormalities; renal failure and acute tubular necrosis.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a group of subjects does not cause one or more adverse events in more than 1% of the subjects after administering the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from duodenal ulcer; intestinal necrosis; myocarditis; supraventricular tachycardia; permanent or transient blindness secondary to optic neuritis; transient ischemic attack; meningitis; cerebral edema; pericarditis; allergic interstitial nephritis and tracheoesophageal fistula.

In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a group of subjects does not cause one or more adverse events in more than 1% of the subjects after administering the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary embolism; stroke; intestinal perforation; liver or kidney failure; severe depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure.

In some embodiments of the method of treating cancer described herein, administering an IL-2 conjugate to a subject results in an increase in the number of CD8+ T and NK cells in the subject's peripheral blood without increasing the number of CD4+ regulatory T cells in the subject's peripheral blood. In some embodiments of the method of treating cancer described herein, administering an IL-2 conjugate to a subject results in an increase in the number of CD8+ T and NK cells in the subject's peripheral blood without increasing the number of eosinophils in the subject's peripheral blood. In some embodiments of the method of treating cancer described herein, administering an IL-2 conjugate to a subject results in an increase in the number of CD8+ T and NK cells in the subject's peripheral blood without increasing the number of intratumoral CD8+ T and NK cells in the subject and without increasing the number of intratumoral CD4+ regulatory T cells in the subject.

Pharmaceutical compositions and formulations

Described herein are pharmaceutical compositions comprising an effective amount of an IL-2 conjugate described herein and one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical composition and formulations comprising a cytokine conjugate (e.g., an IL-2 conjugate) described herein are administered to a subject via multiple routes of administration, including, but not limited to, parenteral, oral, buccal, rectal, sublingual, or transdermal routes of administration. In some cases, parenteral administration includes intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration. In some cases, the pharmaceutical composition is formulated for topical administration. In other cases, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via intravenous, subcutaneous, and intramuscular administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via intravenous administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via intramuscular administration.

In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.

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

In some cases, the pharmaceutical composition is formulated as an immunoliposome that comprises a plurality of IL-2 conjugates directly or indirectly bound to a lipid bilayer of the liposomes. Exemplary lipids include, but are not limited to, fatty acids; phospholipids; sterols such as cholesterols; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, globosides and cerebrosides; amine surfactants such as stearyl, oleyl and linoleylamines. In some cases, the lipid includes a cationic lipid. In some cases, the lipid includes a phospholipid. Illustrative phospholipids include, but are not limited to, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phosphatidylethanolamine ("PE"), phosphatidylinositol ("PI"), and phosphatidylserine ("PS"), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-alpha-phosphatidylcholine ("DDPC"), dielaidoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleoylphosphatidylcholine ("DOPC"), dipalmitoylphosphatidylcholine ("DPPC"), Distearoylphosphatidylcholine ("DSPC"), 1-palmitoyl-2-oleoylphosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol ("DAPG"), didecanoyl-L-alpha-phosphatidylglycerol ("DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DMPG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), 1-palmitoyl-2-oleoylphosphatidylglycerol ("POPG"), diarachidoylphosphatidylethanolamine ("DAPE"), didecanoyl-L-alpha-phosphatidylethanolamine ("DDPE"), Dielaidoylphosphatidylethanolamine ("DEPE"), Dilauroylphosphatidylethanolamine ("DLPE"), Dilinoleoylphosphatidylethanolamine, Dimyristoylphosphatidylethanolamine ("DMPE"), Dioleoylphosphatidylethanolamine ("DOPE"), Dipalmitoylphosphatidylethanolamine ("DPPE"), Distearoylphosphatidylethanolamine ("DSPE"), 1-Palmitoyl-2-oleoylphosphatidylethanolamine ("POPE"), Diarachidoylphosphatidylinositol ("DAPI"), Didecanoyl-L-alpha-phosphatidylinositol ("DDPI"), Dielaidoylphosphatidylinositol ("DEPI"), Dilauroylphosphatidylinositol ("DLPI"), Dilinoleoylphosphatidylinositol, Dimyristoylphosphatidylinositol ("DMPI"), Dioleoylphosphatidylinositol ("DOPI"), Dipalmitoylphosphatidylinositol ("DPPI"), Distearoylphosphatidylinositol ("DSPI"), 1-Palmitoyl-2-oleoylphosphatidylinositol ("POPI"), Diarachidoylphosphatidylserine ("DAPS"), Didecanoyl-L-alpha-phosphatidylserine ("DDPS"), Dielaidoylphosphatidylserine ("DEPS"), Dilauroylphosphatidylserine ("DLPS"), Dilinoleoylphosphatidylserine, Dimyristoylphosphatidylserine ("DMPS"), Dioleoylphosphatidylserine ("DOPS"), Dipalmitoylphosphatidylserine ("DPPS"), Distearoylphosphatidylserine ("DSPS"), 1-Palmitoyl-2-oleoylphosphatidylserine ("POPS"), Diarachidoylsphingomyelin, Didecanoylsphingomyelin, dialidoylsphingomyelin, dilauroylsphingomyelin, dilinoleoylsphingomyelin, dimyristoylsphingomyelin, sphingomyelin, dioleoylsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin and 1-palmitoyl-2-oleoylsphingomyelin.

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

In some cases, the pharmaceutical composition contains one or more salts in an amount required to bring the osmolality of the composition into an acceptable range. Such salts include salts containing sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, with suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some embodiments, the pharmaceutical formulations contain, but are not limited to, sugars such as trehalose, sucrose, mannitol, maltose, glucose, or salts such as potassium phosphate, sodium citrate, ammonium sulfate, and/or other agents such as heparin to increase the solubility and stability of the polypeptides in vivo .

In some cases, pharmaceutical formulations additionally contain a diluent, which is used to stabilize the compounds because it is capable of providing a more stable environment. Salts dissolved in buffer solutions (which are also capable of providing pH control or maintenance) are used as diluents in the art, including but not limited to phosphate-buffered saline. In some cases, diluents increase the volume of the composition to facilitate compression or to create sufficient volume to obtain a homogeneous mixture for filling capsules. Such compounds may include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as ^Avicel® , dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar such as Di- ^Pac® (Amstar), mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like. In some embodiments, the IL-2 conjugates disclosed herein can be used in pharmaceutical formulations comprising histidine, sorbitol, and polysorbate 80, or any combination thereof that results in a stable formulation, and can be administered to subjects in need thereof. In one embodiment, the IL-2 conjugates disclosed herein can be presented as a finished dosage form in a suitable container, such as a vial, as follows: IL-2 conjugate (about 2 mg to about 10 mg); L-histidine (about 0.5 mg to about 2 mg); L-histidine hydrochloride (about 1 mg to about 2 mg); sorbitol (about 20 mg to about 80 mg), and polysorbate 80 (about 0.1 mg to about 0.2 mg); with a sufficient amount of water for injection to provide a liquid formulation suitable for use in the disclosed methods.

In some cases, pharmaceutical formulations contain disintegrating agents or disintegrants to facilitate the disintegration or disintegration of the substance. The term "disintegration" includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal juices. Examples of disintegrating agents include starch, e.g. natural starch such as corn starch or potato starch, pregelatinized starch such as National 1551 or Amijel ^® , or sodium starch glycolate such as Promogel ^® or Explotab ^® , cellulose such as a wood product, crystalline methylcellulose such as Avicel ^® , Avicel ® PH101, Avicel ^® PH102, Avicel ^® PH105, Elcema ^® P100, Emcocel ^® , Vivacel ^® , Ming Tia ^® and Solka-Floc ^® , methylcellulose, croscarmellose or cross-linked cellulose such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol ^® ), cross-linked carboxymethylcellulose or cross ^- linked croscarmellose, cross-linked starch such as sodium starch glycolate, cross-linked polymer such as crospovidone, cross-linked polyvinylpyrrolidone, alginate such as alginic acid or an alginic acid salt such as sodium alginate, clay such as ^Veegum® HV (magnesium aluminum silicate), gum such as agar, guar gum, locust bean gum, karaya gum, pectin or tragacanth gum, sodium starch glycolate, bentonite, natural sponge, surfactant, resin such as cation exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination with starch, etc.

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

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

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

Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E-TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol and dimethyl isosorbide, etc.

Stabilizers include such compounds as any antioxidant agents, buffers, acids, preservatives, etc. Illustrative stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium diphosphate dihydrate, propylene glycol, meta-cresol or m-cresol, zinc acetate, polysorbate 20 or Tween® 20, or trometamol.

Suspending agents include compounds such as polyvinylpyrrolidone, for example polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, a copolymer of vinylpyrrolidone and vinyl acetate (S630), polyethylene glycol, for example polyethylene glycol which may have a molecular weight of from about 300 to about 6,000, or from about 3,350 to about 4,000, or from about 7,000 to about 5,400, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxymethylcellulose acetate stearate, polysorbate 80, hydroxyethylcellulose, sodium alginate, gums such as, for example, gum tragacanth and gum acacia, guar gum, xanthans, in including xanthan gum, sugars, cellulose compounds such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, etc.

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

Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans, and combinations thereof.

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

In some cases, a pharmaceutical composition comprising an IL-2 conjugate (such as an IL-2 conjugate of formulas (I-XVII)) and an immune checkpoint inhibitor is administered as a formulation containing both drugs. In some cases, the weight percentage ratio of the IL-2 conjugate to the immune checkpoint inhibitor, or vice versa, is from 10:1 to 1:10. In some cases, the weight percentage ratio of the IL-2 conjugate to the immune checkpoint inhibitor, or vice versa, is from 7:1 to 1:2. In some cases, the weight percentage ratio of the IL-2 conjugate to the immune checkpoint inhibitor, or vice versa, is from 5:1 to 1:5. In some cases, the weight percentage ratio of the IL-2 conjugate to the immune checkpoint inhibitor, or vice versa, is from 3:1 to 1:3. In some cases, the weight percentage ratio of the IL-2 conjugate to the immune checkpoint inhibitor, or vice versa, is about 10:1, or about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some cases, the weight percentage ratio of the IL-2 conjugate to the immune checkpoint inhibitor, or vice versa, is from 10:1 to 1:1, from 7:1 to 2:1, from 5:1 to 1:1, or from 3:1 to 1:1.

In certain embodiments, the combination of an immune checkpoint inhibitor (such as a PD-1 inhibitor) and an IL-2 conjugate (such as the conjugates of Formulas (I-XVII)) described herein is administered as the pure chemical compound. In other embodiments, the combination of an immune checkpoint inhibitor and an IL-2 conjugate described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, a physiologically suitable (or acceptable) excipient, or a physiologically suitable (or acceptable) carrier) selected based on the selected route of administration and standard pharmaceutical practice, as described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, ^21st Ed. Mack Pub. Co., Easton, PA (2005), the disclosure of which is incorporated herein by reference). In some embodiments, the immune checkpoint inhibitor and the IL-2 conjugate described herein are each administered as separate compositions. In some embodiments, the separate immune checkpoint inhibitor and/or IL-2 conjugate compositions described herein are combined with a suitable or acceptable excipient. In some embodiments, the immune checkpoint inhibitor and the IL-2 conjugate described herein are administered as a single combined composition.

Provided herein are pharmaceutical compositions comprising an IL-2 conjugate described herein and an immune checkpoint inhibitor together with one or more pharmaceutically acceptable carriers. The carrier(s) (or excipient(s)) are acceptable or suitable if the carrier is compatible with the other ingredients of the composition and is not deleterious to the person receiving the composition (i.e. subject or patient). In certain embodiments, the immune checkpoint inhibitor and IL-2 conjugate described herein are substantially pure in that they contain less than about 5%, or less than about 1%, or less than about 0.1% of other organic small molecules, such as unreacted intermediates or synthetic byproducts that are formed, for example, in one or more steps of a synthetic process. In some cases, the pharmaceutical compositions comprise the immune checkpoint inhibitor and IL-2 conjugate described herein and one or more pharmaceutically acceptable excipients. In some cases, the pharmaceutical compositions comprising the immune checkpoint inhibitor and IL-2 conjugate described herein, or a combination thereof, comprise (by way of non-limiting example) excipients such as 0.9% sodium chloride injection USP, dehydrated alcohol,dl-alpha-tocopherol, anhydrous citric acid, polysorbate 80, polyethyleneglycol 400, propylene glycol, benzyl alcohol, sodium citrate, sodium sulfite, Cremophor EL, albumin, or any combination thereof. In some cases, the pharmaceutical compositions comprise nanoparticles. In some cases, the pharmaceutical compositions comprise other excipients conventionally used in injectable compositions. In some cases, the pharmaceutical compositions comprise a contrast agent to facilitate visualization of the delivery of the pharmaceutical composition. In some cases, the pharmaceutical compositions comprise a liquid, suspension, solution, or gel. In some cases, the pharmaceutical compositions comprising an immune checkpoint inhibitor and an IL-2 conjugate described herein, or a combination thereof, are injectable. In some cases, the pharmaceutical compositions comprise excipients that solubilize the immune checkpoint inhibitor and the IL-2 conjugate described herein, or a combination thereof. In another embodiment, pharmaceutical compositions comprising an immune checkpoint inhibitor and an IL-2 conjugate described herein are presented in a parenteral dosage form that contains one or more pharmaceutically acceptable excipients or carriers. In some cases, pharmaceutical compositions comprising an immune checkpoint inhibitor and an IL-2 conjugate described herein, or a combination thereof, are injectable. When pharmaceutical compositions are formulated for intravenous, cutaneous, or subcutaneous injection, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. Those skilled in the art are well versed in preparing suitable solutions using, for example, isotonic vehicle media such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. In some embodiments, preservatives, stabilizers, fillers, buffers, antioxidants and/or other additives are included.

Kits/finished products

In certain embodiments, kits and articles of manufacture for use with one or more of the methods and compositions described herein are disclosed herein. Such kits comprise a carrier, package, or container that is divided into compartments to accommodate one or more containers, such as vials, tubes, and the like, wherein each of the containers contains one of the individual elements to be used in the method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are made of a variety of materials, such as glass or plastic.

The kit usually contains labels with a list of contents and/or instructions for use, as well as package inserts with instructions for use. An instruction set is also usually included.

In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other symbols that form the label are attached, molded, or etched onto the container itself, and the label is associated with the container when it is located within a receptacle or holder that also holds the container, such as in a package insert. In one embodiment, the label is used to indicate that the contents are to be used for a specific therapeutic application. The label also provides directions for using the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are provided in a package or dispenser device that contains one or more unit dosage forms containing a compound as provided herein. The package, for example, comprises metal foil or plastic film, such as a blister pack. In one embodiment, the package or dispenser device is accompanied by instructions for administration. In one embodiment, the package or dispenser device is also accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice reflecting approval by the agency of the drug form for administration to humans or animals. Such a notice is, for example, a drug label approved by the U.S. Food and Drug Administration or an approved product package insert. In one embodiment, compositions containing a compound as provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of the indicated condition.

Illustrative embodiments

The present invention will now be described using the following embodiments. The features of each embodiment are combined with any of the other embodiments where appropriate and practicable.

Embodiment 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, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (I):

Formula (I);

Where:

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 1.1. 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, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (I):

Formula (I);

Where:

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 2. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate Z is CH ₂ and Y is .

Embodiment 3. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate Y is CH ₂ and Z is .

Embodiment 4. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate Z is CH ₂ and Y is .

Embodiment 5. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate Z is CH ₂ and Y is .

Embodiment 6. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate Y is CH ₂ and Z is .

Embodiment 7. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate the PEG group has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa and 30 kDa.

Embodiment 8. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate or pharmaceutically acceptable salt, solvate or hydrate thereof, the PEG group has an average molecular weight of 10 kDa, 20 kDa or 30 kDa.

Embodiment 9. The method according to embodiments 1 or 1.1, wherein in the IL-2 conjugate the PEG group has an average molecular weight of 30 kDa.

Embodiment 10. The method according to embodiments 1 or 1.1, wherein 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.

Embodiment 11. The method according to embodiments 1 or 1.1, wherein 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.

Embodiment 12. 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 any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] has a structure of formula (II) or formula (III) or is a mixture of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 12.1: 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 any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] has a structure of formula (II) or formula (III) or is a mixture of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 13. The method according to embodiments 12 or 12.1, wherein [AzK_PEG] is a mixture of formula (II) and formula (III).

Embodiment 14. The method according to embodiments 12 or 12.1, wherein [AzK_PEG] has a structure of formula (II):

Formula (II);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 15. The method according to embodiment 14, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15.

Embodiment 16. The method of embodiment 15, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 17. The method of embodiment 16, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 18. The method of embodiment 17, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 19. The method of embodiment 17, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 20. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16.

Embodiment 21. The method of embodiment 20, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 22. The method of embodiment 21, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 23. The method of embodiment 22, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 24. The method of embodiment 22, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 25. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17.

Embodiment 26. The method of embodiment 25, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 27. The method of embodiment 26, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 28. The method of embodiment 27, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 29. The method of embodiment 27, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 30. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18.

Embodiment 31. The method of embodiment 30, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 32. The method of embodiment 31, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 33. The method of embodiment 32, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 34. The method of embodiment 32, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 35. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19.

Embodiment 36. The method of embodiment 35, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 37. The method of embodiment 36, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 38. The method of embodiment 37, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 39. The method of embodiment 37, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 40. The method according to embodiments 12 or 12.1, wherein [AzK_PEG] has a structure of formula (III):

Formula (III);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 41. The method according to embodiment 40, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15.

Embodiment 42. The method of embodiment 41, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 43. The method of embodiment 42, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 44. The method of embodiment 43, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 45. The method of embodiment 43, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 46. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16.

Embodiment 47. The method of embodiment 46, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 48. The method of embodiment 47, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 49. The method of embodiment 48, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 50. The method of embodiment 48, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 51. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17.

Embodiment 52. The method of embodiment 51, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 53. The method of embodiment 52, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 54. The method of embodiment 53, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 55. The method of embodiment 53, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 56. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18.

Embodiment 57. The method of embodiment 56, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 58. The method of embodiment 57, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 59. The method of embodiment 58, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 60. The method of embodiment 58, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 61. The method according to embodiments 12 or 12.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19.

Embodiment 62. The method of embodiment 61, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 63. The method of embodiment 62, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 64. The method of embodiment 63, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 65. The method of embodiment 63, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 66. The method according to any one of embodiments 1-65, wherein W is a linear or branched PEG group.

Embodiment 67. The method according to any one of embodiments 1-65, wherein W is a linear PEG group.

Embodiment 68. The method according to any one of embodiments 1-65, wherein W is a branched PEG group.

Embodiment 69. The method according to any one of embodiments 1-65, wherein W is a methoxy-PEG group.

Embodiment 70. The method of embodiment 69, wherein the methoxy-PEG group is linear or branched.

Embodiment 71. The method of embodiment 70 wherein the methoxy-PEG group is linear.

Embodiment 72. The method of embodiment 70 wherein the methoxy-PEG group is branched.

Embodiment 73. 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 any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kDa] has a structure of formula (II) or formula (III) or is a mixture of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 73.1: 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 any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kDa] has a structure of formula (II) or formula (III) or is a mixture of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 74. The method according to embodiments 73 or 73.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20.

Embodiment 75. The method according to embodiments 73 or 73.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21.

Embodiment 76. The method according to embodiments 73 or 73.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22.

Embodiment 77. The method according to embodiments 73 or 73.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23.

Embodiment 78. The method according to embodiments 73 or 73.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24.

Embodiment 79. The method of embodiments 73 or 73.1, wherein [AzK_PEG5kDa] has a structure of formula (II):

Formula (II);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 80. The method according to embodiment 79, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20.

Embodiment 81. The method of embodiment 79, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21.

Embodiment 82. The method of embodiment 79, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22.

Embodiment 83. The method according to embodiment 79, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23.

Embodiment 84. The method according to embodiment 79, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24.

Embodiment 85. The method of embodiments 73 or 73.1, wherein [AzK_PEG5kDa] has a structure of formula (III):

Formula (III);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 86. The method according to embodiment 85, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20.

Embodiment 87. The method according to embodiment 85, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21.

Embodiment 88. The method according to embodiment 85, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22.

Embodiment 89. The method according to embodiment 85, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23.

Embodiment 90. The method according to embodiment 85, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24.

Embodiment 91. 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 any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kDa] has a structure of formula (II) or formula (III) or is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 91.1: 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 any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kDa] has a structure of formula (II) or formula (III) or is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 92. The method according to embodiments 91 or 91.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25.

Embodiment 93. The method according to embodiments 91 or 91.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26.

Embodiment 94. The method according to embodiments 91 or 91.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27.

Embodiment 95. The method according to embodiments 91 or 91.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28.

Embodiment 96. The method according to embodiments 91 or 91.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29.

Embodiment 97. The method of embodiments 91 or 91.1, wherein [AzK_PEG30kDa] has a structure of formula (II):

Formula (II);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 98. The method according to embodiment 97, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25.

Embodiment 99. The method according to embodiment 97, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26.

Embodiment 100. The method according to embodiment 97, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27.

Embodiment 101. The method of embodiment 97, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28.

Embodiment 102. The method of embodiment 97, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29.

Embodiment 103. The method of embodiments 91 or 91.1, wherein [AzK_PEG30kDa] has a structure of formula (III):

Formula (III);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 104. The method according to embodiment 103, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25.

Embodiment 105. The method according to embodiment 103, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26.

Embodiment 106. The method according to embodiment 103, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27.

Embodiment 107. The method of embodiment 103, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28.

Embodiment 108. The method of embodiment 103, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29.

Embodiment 109. 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 any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 109.1: 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 any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 110. The method of embodiments 109 or 109.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG] in the IL-2 conjugate is about 1:1.

Embodiment 111. The method of embodiments 109 or 109.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG] in the IL-2 conjugate is greater than 1:1.

Embodiment 112. The method of embodiments 109 or 109.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG] in the IL-2 conjugate is less than 1:1.

Embodiment 113. The method according to any one of embodiments 109-112, wherein W is a linear or branched PEG group.

Embodiment 114. The method according to any one of embodiments 109-112, wherein W is a linear PEG group.

Embodiment 115. The method according to any one of embodiments 109-112, wherein W is a branched PEG group.

Embodiment 116. The method according to any one of embodiments 109-112, wherein W is a methoxy-PEG group.

Embodiment 117. The method of embodiment 116 wherein the methoxy-PEG group is linear or branched.

Embodiment 118. The method of embodiment 117 wherein the methoxy-PEG group is linear.

Embodiment 119. The method of embodiment 117 wherein the methoxy-PEG group is branched.

Embodiment 120. 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 any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kDa] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 120.1: 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 any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kDa] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 121. The method of embodiments 120 or 120.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG5kDa] in the IL-2 conjugate is approximately 1:1.

Embodiment 122. The method of embodiments 120 or 120.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG5kDa] in the IL-2 conjugate is greater than 1:1.

Embodiment 123. The method of embodiments 120 or 120.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG5kDa] in the IL-2 conjugate is less than 1:1.

Embodiment 124. 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 any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kDa] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 124.1: 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 any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kDa] is a mixture of structures of formula (II) and formula (III):

Formula (II);

Formula (III);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 125. The method of embodiments 124 or 124.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is approximately 1:1.

Embodiment 126. The method of embodiments 124 or 124.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is greater than 1:1.

Embodiment 127. The method of embodiments 124 or 124.1, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG30kDa] in the IL-2 conjugate is less than 1:1.

Embodiment 128. 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 any one of SEQ ID NOs: 40-44, wherein [AzK_L1_PEG] has a structure of formula (IV) or formula (V) or is a mixture of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 128.1: 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 any one of SEQ ID NOs: 40-44, wherein [AzK_L1_PEG] has a structure of formula (IV) or formula (V) or is a mixture of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 129. The method of embodiments 128 or 128.1, wherein [AzK_L1_PEG] is a mixture of formula (IV) and formula (V).

Embodiment 130. The method of embodiments 128 or 128.1, wherein [AzK_L1_PEG] has a structure of formula (IV):

Formula (IV);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 131. The method according to embodiments 128 or 128.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40.

Embodiment 132. The method of embodiment 131, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 133. The method of embodiment 132, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 134. The method of embodiment 133, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 135. The method of embodiment 133, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 136. The method according to embodiments 128 or 128.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41.

Embodiment 137. The method of embodiment 136, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 138. The method of embodiment 137, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 139. The method of embodiment 138, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 140. The method of embodiment 138, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 141. The method of embodiments 128 or 128.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42.

Embodiment 142. The method of embodiment 141, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 143. The method of embodiment 142, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 144. The method of embodiment 143, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 145. The method of embodiment 143, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 146. The method according to embodiments 128 or 128.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43.

Embodiment 147. The method of embodiment 146, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 148. The method of embodiment 147, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 149. The method of embodiment 148, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 150. The method of embodiment 148, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 151. The method of embodiments 128 or 128.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 44.

Embodiment 152. The method of embodiment 151, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 153. The method of embodiment 152, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 154. The method of embodiment 153, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 155. The method of embodiment 153, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 156. The method of embodiments 128 or 128.1, wherein [AzK_L1_PEG] has a structure of formula (V):

Formula (V);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 157. The method of embodiment 156, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40.

Embodiment 158. The method of embodiment 156, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 159. The method of embodiment 158, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 160. The method of embodiment 159, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 161. The method of embodiment 159, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 162. The method of embodiment 156, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41.

Embodiment 163. The method of embodiment 162, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 164. The method of embodiment 163, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 165. The method of embodiment 164, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 166. The method of embodiment 164, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 167. The method of embodiment 156, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42.

Embodiment 168. The method of embodiment 167, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 169. The method of embodiment 168, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 170. The method of embodiment 169, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 171. The method of embodiment 169, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 172. The method of embodiment 156, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43.

Embodiment 173. The method of embodiment 172, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 174. The method of embodiment 173, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 175. The method of embodiment 174, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 176. The method of embodiment 174, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 177. The method of embodiment 156, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 44.

Embodiment 178. The method of embodiment 177, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa.

Embodiment 179. The method of embodiment 178, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa.

Embodiment 180. The method of embodiment 179, wherein W is a PEG group having an average molecular weight of 5 kDa.

Embodiment 181. The method of embodiment 179, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment 182. The method of any one of embodiments 128-181, wherein W is a linear or branched PEG group.

Embodiment 183. The method according to any one of embodiments 128-181, wherein W is a linear PEG group.

Embodiment 184. The method of any one of embodiments 128-181, wherein W is a branched PEG group.

Embodiment 185. The method according to any one of embodiments 128-181, wherein W is a methoxy-PEG group.

Embodiment 186. The method of embodiment 185 wherein the methoxy-PEG group is linear or branched.

Embodiment 187. The method of embodiment 186 wherein the methoxy-PEG group is linear.

Embodiment 188. The method of embodiment 186 wherein the methoxy-PEG group is branched.

Embodiment 189. 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 any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kDa] has a structure of formula (IV) or formula (V) or is a mixture of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 189.1: 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 any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kDa] has a structure of formula (IV) or formula (V) or is a mixture of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 190. The method according to embodiments 189 or 189.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45.

Embodiment 191. The method according to embodiments 189 or 189.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46.

Embodiment 192. The method according to embodiments 189 or 189.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47.

Embodiment 193. The method according to embodiments 189 or 189.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48.

Embodiment 194. The method according to embodiments 189 or 189.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49.

Embodiment 195. The method of embodiments 189 or 189.1, wherein [AzK_L1_PEG5kDa] has a structure of formula (IV):

Formula (IV);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 196. The method of embodiment 195, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45.

Embodiment 197. The method of embodiment 195, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46.

Embodiment 198. The method of embodiment 195, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47.

Embodiment 199. The method of embodiment 195, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48.

Embodiment 200. The method of embodiment 195, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49.

Embodiment 201. The method of embodiments 189 or 189.1, wherein [AzK_L1_PEG5kDa] has a structure of formula (V):

Formula (V);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 202. The method of embodiment 201, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45.

Embodiment 203. The method of embodiment 201, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46.

Embodiment 204. The method of embodiment 201, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47.

Embodiment 205. The method of embodiment 201, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48.

Embodiment 206. The method of embodiment 201, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49.

Embodiment 207. 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 any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV) or formula (V) or is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 207.1: 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 any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV) or formula (V) or is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 208. The method of embodiments 207 or 207.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50.

Embodiment 209. The method of embodiments 207 or 207.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51.

Embodiment 210. The method of embodiments 207 or 207.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52.

Embodiment 211. The method of embodiments 207 or 207.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53.

Embodiment 212. The method of embodiments 207 or 207.1, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54.

Embodiment 213. The method of embodiments 207 or 207.1, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV):

Formula (IV);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 214. The method of embodiment 213, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50.

Embodiment 215. The method of embodiment 213, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51.

Embodiment 216. The method of embodiment 213, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52.

Embodiment 217. The method of embodiment 213, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53.

Embodiment 218. The method of embodiment 213, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54.

Embodiment 219. The method of embodiments 207 or 207.1, wherein [AzK_L1_PEG30kDa] has a structure of formula (V):

Formula (V);

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 220. The method of embodiment 219, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50.

Embodiment 221. The method of embodiment 219, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51.

Embodiment 222. The method of embodiment 219, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52.

Embodiment 223. The method of embodiment 219, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53.

Embodiment 224. The method of embodiment 219, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54.

Embodiment 225. 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 any one of SEQ ID NOs: 40-44, wherein [Azk_L1_PEG] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 225.1: 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 any one of SEQ ID NOs: 40-44, wherein [Azk_L1_PEG] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 226. The method of embodiments 225 or 225.1, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) making up the total amount of [AzK_L1_PEG] in the IL-2 conjugate is approximately 1:1.

Embodiment 227. The method of embodiments 225 or 225.1, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) making up the total amount of [AzK_L1_PEG] in the IL-2 conjugate is greater than 1:1.

Embodiment 228. The method of embodiments 225 or 225.1, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) making up the total amount of [AzK_L1_PEG] in the IL-2 conjugate is less than 1:1.

Embodiment 229. The method of any one of embodiments 225-228, wherein W is a linear or branched PEG group.

Embodiment 230. The method of any one of embodiments 225-228, wherein W is a linear PEG group.

Embodiment 231. The method of any one of embodiments 225-228, wherein W is a branched PEG group.

Embodiment 232. The method of any one of embodiments 225-228, wherein W is a methoxy-PEG group.

Embodiment 233. The method of embodiment 232, wherein the methoxy-PEG group is linear or branched.

Embodiment 234. The method of embodiment 233 wherein the methoxy-PEG group is linear.

Embodiment 235. The method of embodiment 233 wherein the methoxy-PEG group is branched.

Embodiment 236. 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 any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kDa] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 236.1: 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 any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kDa] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 237. The method of embodiments 236 or 236.1, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) making up the total amount of [AzK_L1_PEG5kDa] in the IL-2 conjugate is approximately 1:1.

Embodiment 238. The method of embodiments 236 or 236.1, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) making up the total amount of [AzK_PEG5kDa] in the IL-2 conjugate is greater than 1:1.

Embodiment 239. The method of embodiments 236 or 236.1, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) making up the total amount of [AzK_L1_PEG5kDa] in the IL-2 conjugate is less than 1:1.

Embodiment 240. 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 any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kDa] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 240.1: 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 any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kDa] is a mixture of structures of formula (IV) and formula (V):

Formula (IV);

Formula (V);

Where:

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 241. The method of embodiments 240 or 240.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is approximately 1:1.

Embodiment 242. The method of embodiments 240 or 240.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is greater than 1:1.

Embodiment 243. The method of embodiments 240 or 240.1, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) making up the total amount of [AzK_L1_PEG30kDa] in the IL-2 conjugate is less than 1:1.

Embodiment 244. 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 by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII):

Formula (VI),

(VII),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

X has the structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 244.1: 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, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII):

Formula (VI),

(VII),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 245. The method of Embodiments 244 or 244.1 wherein n in the compounds of Formula (VI) and (VII) ranges 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 from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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.

Embodiment 246. The method of Embodiments 244 or 244.1 wherein n in the compounds of formula (VI) and (VII) is an integer selected from 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.

Embodiment 247. The method according to any one of embodiments 244-246, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (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.

Embodiment 248. The method of embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is K34.

Embodiment 249. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is F41.

Embodiment 250. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is F43.

Embodiment 251. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is K42.

Embodiment 252. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is E61.

Embodiment 253. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is P64.

Embodiment 254. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is R37.

Embodiment 255. The method of embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is T40.

Embodiment 256. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is E67.

Embodiment 257. The method of embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is Y44.

Embodiment 258. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is V68.

Embodiment 259. The method according to embodiment 247, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is L71.

Embodiment 260. The method according to any one of embodiments 244-259, wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) making up the total amount of the IL-2 conjugate is greater than 1:1.

Embodiment 261. The method according to any one of embodiments 244-259, wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) making up the total amount of the IL-2 conjugate is less than 1:1.

Embodiment 262. The method of embodiments 244 or 244.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

Embodiment 263. The method of embodiments 244 or 244.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, 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.

Embodiment 264. The method of embodiments 244 or 244.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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.

Embodiment 265. The method of embodiment 264, wherein n in the compounds of formula (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 266. The method of embodiments 244 or 244.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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.

Embodiment 267. The method of embodiment 266, wherein n in the compounds of formula (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 268. The method of embodiment 266, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 500 to about 1000.

Embodiment 269. The method of embodiment 266, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 550 to about 800.

Embodiment 270. The method of embodiment 267, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is 681.

Embodiment 271. The method of embodiments 244 or 244.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

Embodiment 272. The method of embodiment 271, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n in the compounds of formula (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 273. The method of embodiment 271, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 500 to about 1000.

Embodiment 274. The method of embodiment 273, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 550 to about 800.

Embodiment 275. The method of embodiment 271, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is 681.

Embodiment 276. The method of embodiments 244 or 244.1, wherein n is an integer such that the molecular weight of the PEG moiety is in the range of 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, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

Embodiment 277. The method of embodiments 244 or 244.1, wherein 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, 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, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons.

Embodiment 278. The method of embodiments 244 or 244.1, wherein 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.

Embodiment 279. 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 by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX):

Formula (VIII),

(IX),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

X has the structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 279.1: 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, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX):

Formula (VIII),

(IX),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 280. The method of Embodiments 279 or 279.1 wherein n in the compounds of formula (VIII) and (IX) ranges 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 from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or 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.

Embodiment 281. The method of Embodiments 279 or 279.1 wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 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.

Embodiment 282. The method according to any one of embodiments 279-281, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (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.

Embodiment 283. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is K34.

Embodiment 284. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is F41.

Embodiment 285. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is F43.

Embodiment 286. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is K42.

Embodiment 287. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is E61.

Embodiment 288. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is P64.

Embodiment 289. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is R37.

Embodiment 290. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is T40.

Embodiment 291. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is E67.

Embodiment 292. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is Y44.

Embodiment 293. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is V68.

Embodiment 294. The method according to embodiment 282, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is L71.

Embodiment 295. The method according to any one of embodiments 279-294, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) making up the total amount of the IL-2 conjugate is greater than 1:1.

Embodiment 296. The method according to any one of embodiments 279-294, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) making up the total amount of the IL-2 conjugate is less than 1:1.

Embodiment 297. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

Embodiment 298. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61 and P64, 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.

Embodiment 299. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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.

Embodiment 300. The method of embodiment 299, wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 301. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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.

Embodiment 302. The method of embodiment 300, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 303. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 500 to about 1000.

Embodiment 304. The method of embodiment 303, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 550 to about 800.

Embodiment 305. The method of embodiment 302, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is 681.

Embodiment 306. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

Embodiment 307. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 308. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 500 to about 1000.

Embodiment 309. The method of embodiments 279 or 279.1, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 550 to about 800.

Embodiment 310. The method of embodiment 307, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and where n is 681.

Embodiment 311. 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 by a structure of formula (X) or (XI) or a mixture of (X) and (XI):

Formula (X),

(XI),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Embodiment 312. The method of Embodiment 311 wherein n in the compounds of formula (X) and (XI) ranges 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 from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or 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.

Embodiment 313. The method of Embodiment 311 wherein n in the compounds of formula (X) and (XI) is an integer selected from 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.

Embodiment 314. The method according to any one of embodiments 311-313, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (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.

Embodiment 315. The method of embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is K34.

Embodiment 316. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is F41.

Embodiment 317. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is F43.

Embodiment 318. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is K42.

Embodiment 319. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is E61.

Embodiment 320. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is P64.

Embodiment 321. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is R37.

Embodiment 322. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is T40.

Embodiment 323. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is E67.

Embodiment 324. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is Y44.

Embodiment 325. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is V68.

Embodiment 326. The method according to embodiment 314, wherein the position of the structure of formula (X), formula (XI), or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is L71.

Embodiment 327. The method according to any one of embodiments 311-326, wherein the ratio of the amount of the structure of formula (X) to the amount of the structure of formula (XI) making up the total amount of the IL-2 conjugate is greater than 1:1.

Embodiment 328. The method according to any one of embodiments 311-326, wherein the ratio of the amount of the structure of formula (X) to the amount of the structure of formula (XI) making up the total amount of the IL-2 conjugate is less than 1:1.

Embodiment 329. The method of embodiment 311, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

Embodiment 330. The method of embodiment 311, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61 and P64, 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.

Embodiment 331. The method of embodiment 311, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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.

Embodiment 332. The method of embodiment 330, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61 and P64, and wherein n in the compounds of formula (X) and (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

Embodiment 333. The method of embodiment 311, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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.

Embodiment 334. The method of embodiment 311, wherein n in compounds of formula (X) and (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 335. The method of embodiment 311, wherein n is from about 500 to about 1000.

Embodiment 336. The method of embodiment 335, wherein n is from about 550 to about 800.

Embodiment 337. The method of embodiment 332, wherein n is 681.

Embodiment 338. The method of embodiment 311, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

Embodiment 339. The method of embodiment 311, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n in the compounds of formula (X) and (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 340. The method of embodiment 311, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 500 to about 1000.

Embodiment 341. The method of embodiment 340, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 550 to about 800.

Embodiment 342. The method of embodiment 339, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and where n is 681.

Embodiment 343. The method of embodiment 311, wherein n is an integer such that the molecular weight of the PEG moiety is in the range of 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, or about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

Embodiment 344. The method of Embodiment 311, wherein 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, 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, approximately 175,000 daltons, or approximately 200,000 daltons.

Embodiment 345. The method of embodiment 311, wherein 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.

Embodiment 346. 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 by a structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII):

Formula (XII),

(XIII),

Where:

n is an integer in the range from approximately 2 to approximately 5000; and

Wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not replaced.

Embodiment 347. The method of Embodiment 346 wherein n in the compounds of formula (XII) and (XIII) ranges 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 from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 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 from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or 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.

Embodiment 348. The method of Embodiment 346 wherein n in the compounds of formula (XII) and (XIII) is an integer selected from 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.

Embodiment 349. The method according to any one of embodiments 346-348, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (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.

Embodiment 350. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is K34.

Embodiment 351. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is F41.

Embodiment 352. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is F43.

Embodiment 353. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is K42.

Embodiment 354. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is E61.

Embodiment 355. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is P64.

Embodiment 356. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is R37.

Embodiment 357. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is T40.

Embodiment 358. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is E67.

Embodiment 359. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is Y44.

Embodiment 360. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is V68.

Embodiment 361. The method according to embodiment 349, wherein the position of the structure of formula (XII), formula (XIII), or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is L71.

Embodiment 362. The method according to any one of embodiments 346-361, wherein the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) making up the total amount of the IL-2 conjugate is greater than 1:1.

Embodiment 363. The method according to any one of embodiments 346-361, wherein the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) making up the total amount of the IL-2 conjugate is less than 1:1.

Embodiment 364. The method of embodiment 346, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein 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.

Embodiment 365. The method of embodiment 346, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61 and P64, 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.

Embodiment 366. The method of embodiment 346, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein 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.

Embodiment 367. The method of embodiment 365, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61 and P64, and wherein n in the compounds of formula (XII) and (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

Embodiment 368. The method of embodiment 346, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein 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.

Embodiment 369. The method of embodiment 346, wherein n in the compounds of formula (XII) and (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 370. The method of embodiment 346, wherein n is from about 500 to about 1000.

Embodiment 371. The method of embodiment 370, wherein n is from about 550 to about 800.

Embodiment 372. The method of embodiment 369, wherein n is 681.

Embodiment 373. The method of embodiment 346, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein 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.

Embodiment 374. The method of embodiment 346, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n in the compounds of formula (XII) and (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

Embodiment 375. The method of embodiment 346, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 500 to about 1000.

Embodiment 376. The method of embodiment 375, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 550 to about 800.

Embodiment 377. The method of embodiment 374, wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is 681.

Embodiment 378. The method of Embodiment 346, wherein n is an integer such that the molecular weight of the PEG moiety is in the range of 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, or about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or 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.

Embodiment 379. The method of Embodiment 346, wherein 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, 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, approximately 175,000 daltons, or approximately 200,000 daltons.

Embodiment 380. The method of embodiment 346, wherein 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.

Embodiment 381. The method of any one of embodiments 1-380, wherein the one or more additional agents is one or more immune checkpoint inhibitors selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, OX40 agonists, and 4-1BB agonists.

Embodiment 382. The method of embodiment 381, wherein one or more immune checkpoint inhibitors are selected from PD-1 inhibitors.

Embodiment 383. The method of embodiment 382, wherein the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (Macrogenics/Incyte), GLS-010 (Arcus/WuXi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer), and genolimzumab (Apollomics/Genor BioPharma).

Embodiment 384. The method of embodiment 383, wherein the one or more PD-1 inhibitors is pembrolizumab.

Embodiment 385. The method of embodiment 383, wherein the one or more PD-1 inhibitors is nivolumab.

Embodiment 386. The method of embodiment 383, wherein the one or more PD-1 inhibitors are cemiplimab.

Embodiment 387. The method of embodiment 383, wherein the one or more PD-1 inhibitors are lambrolizumab.

Embodiment 388. The method of embodiment 383, wherein the one or more PD-1 inhibitors are AMP-224.

Embodiment 389. The method of embodiment 383, wherein the one or more PD-1 inhibitors is sintilimab.

Embodiment 390. The method of embodiment 383, wherein the one or more PD-1 inhibitors is toripalimab.

Embodiment 391. The method of embodiment 383, wherein the one or more PD-1 inhibitors is camrelizumab.

Embodiment 392. The method of embodiment 383, wherein the one or more PD-1 inhibitors are tislelizumab.

Embodiment 393. The method of embodiment 381, wherein one or more immune checkpoint inhibitors are selected from PD-L1 inhibitors.

Embodiment 394. The method of embodiment 393, wherein the one or more PD-L1 inhibitors are selected from atezolizumab, avelumab, durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (CytomX), CS1001 (CStone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics).

Embodiment 395. The method of embodiment 394, wherein the one or more PD-L1 inhibitors is atezolizumab.

Embodiment 396. The method of embodiment 394, wherein the one or more PD-L1 inhibitors are avelumab.

Embodiment 397. The method of embodiment 394, wherein the one or more PD-L1 inhibitors are durvalumab.

Embodiment 398. The method of embodiment 381, wherein one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors.

Embodiment 399. The method of embodiment 398, wherein the one or more CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus).

Embodiment 400. The method of embodiment 399, wherein the one or more CTLA-4 inhibitors is tremelimumab.

Embodiment 401. The method of embodiment 399, wherein the one or more CTLA-4 inhibitors is ipilimumab.

Embodiment 402. The method of any one of embodiments 1-401, wherein the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, glioblastoma, mesothelioma, breast cancer, triple-negative breast cancer, prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or metastatic castration-resistant prostate cancer with defects in DNA damage response (DDR), bladder cancer, ovarian cancer, tumors with moderate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low to absent PD-L1 expression, tumors that have spread systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma.

Embodiment 403. The method of embodiment 402, wherein the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), urothelial carcinoma, and melanoma.

Embodiment 404. The method according to any one of embodiments 1-403, wherein the IL-2 conjugate is administered to a subject in need thereof once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks.

Embodiment 405. The method of embodiment 404, wherein the IL-2 conjugate is administered to a subject in need thereof once a week, once every two weeks, or once every three weeks.

Embodiment 406. The method of embodiment 405, wherein the IL-2 conjugate is administered to a subject in need thereof once every two weeks.

Embodiment 407. The method of embodiment 405, wherein the IL-2 conjugate is administered to a subject in need thereof once every three weeks.

Embodiment 408. The method of any one of embodiments 1-407, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause vascular leak syndrome in the subject.

Embodiment 409. The method of embodiment 408, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 2, grade 3, or grade 4 vascular leak syndrome in the subject.

Embodiment 410. The method of embodiment 409, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 2 vascular leak syndrome in the subject.

Embodiment 411. The method of embodiment 409, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 3 vascular leak syndrome in the subject.

Embodiment 412. The method of embodiment 409, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 4 vascular leak syndrome in the subject.

Embodiment 413. The method of any one of embodiments 1-412, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause a loss of vascular tone in the subject.

Embodiment 414. The method according to any one of embodiments 1-413, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause extravasation of blood plasma proteins and fluid into the extravascular space of the subject.

Embodiment 415. The method according to any one of embodiments 1-414, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause hypotension and decreased organ perfusion in the subject.

Embodiment 416. The method of any one of embodiments 1-415, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause impairment of neutrophil function in the subject.

Embodiment 417. The method of any one of embodiments 1-415, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause a decrease in chemotaxis in the subject.

Embodiment 418. The method of any one of embodiments 1-417, wherein administering an effective amount of the IL-2 conjugate to the subject is not associated with an increased risk of disseminated infection in the subject.

Embodiment 419. The method of embodiment 418, wherein the disseminated infection is sepsis or bacterial endocarditis.

Embodiment 420. The method of embodiment 419, wherein the disseminated infection is sepsis.

Embodiment 421. The method of embodiment 419, wherein the disseminated infection is bacterial endocarditis.

Embodiment 422. A method of treating cancer in a subject according to any one of embodiments 1-421, wherein the subject receives treatment for any pre-existing bacterial infections prior to administration of the IL-2 conjugate.

Embodiment 423. The method of embodiment 422, wherein the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate.

Embodiment 424. The method of any one of embodiments 1-423, wherein administering an effective amount of the IL-2 conjugate to the subject does not worsen a pre-existing or initially manifesting autoimmune disease or inflammatory disorder in the subject.

Embodiment 425. The method of embodiment 424, wherein administering an effective amount of the IL-2 conjugate to the subject does not worsen a pre-existing or initially manifesting autoimmune disease in the subject.

Embodiment 426. The method of embodiment 424, wherein administering an effective amount of the IL-2 conjugate to the subject does not worsen a pre-existing or initially manifesting inflammatory disorder in the subject.

Embodiment 427. The method of embodiment 424, wherein the autoimmune disease or inflammatory disorder in the subject is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, oculobulbar myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid.

Embodiment 428. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is Crohn's disease.

Embodiment 429. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is scleroderma.

Embodiment 430. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is thyroiditis.

Embodiment 431. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is inflammatory arthritis.

Embodiment 432. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is diabetes mellitus.

Embodiment 433. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is oculobulbar myasthenia gravis.

Embodiment 434. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is crescentic IgA glomerulonephritis.

Embodiment 435. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is cholecystitis.

Embodiment 436. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is cerebral vasculitis.

Embodiment 437. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is Stevens-Johnson syndrome.

Embodiment 438. The method of embodiment 427, wherein the autoimmune disease or inflammatory disorder in the subject is bullous pemphigoid.

Embodiment 439. The method of any one of embodiments 1-438, wherein administering an effective amount of an IL-2 conjugate to a subject does not cause mental status changes, speech difficulties, cortical blindness, limb or gait ataxia, hallucinations, agitation, sensory dullness, or coma in the subject.

Embodiment 440. The method of any one of embodiments 1-439, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause a seizure in the subject.

Embodiment 441. The method of any one of embodiments 1-440, wherein administering an effective amount of the IL-2 conjugate to the subject is not contraindicated in subjects who have a confirmed seizure disorder.

Embodiment 442. The method of any one of embodiments 1-441, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause capillary leak syndrome in the subject.

Embodiment 443. The method of embodiment 442, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 2, grade 3, or grade 4 capillary leak syndrome in the subject.

Embodiment 444. The method of embodiment 443, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 2 capillary leak syndrome in the subject.

Embodiment 445. The method of embodiment 443, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 3 capillary leak syndrome in the subject.

Embodiment 446. The method of embodiment 443, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause grade 4 capillary leak syndrome in the subject.

Embodiment 447. The method of any one of embodiments 1-446, wherein administering an effective amount of an IL-2 conjugate to a subject does not cause a decrease in mean arterial blood pressure in the subject after administering the IL-2 conjugate to the subject.

Embodiment 448. The method of any one of embodiments 1-447, wherein administering an effective amount of an IL-2 conjugate to the subject does not cause hypotension in the subject after administering the IL-2 conjugate to the subject.

Embodiment 449. The method of embodiment 448, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause the subject to experience a systolic blood pressure below 90 mmHg or a decrease in systolic pressure of 20 mmHg from baseline after administering the IL-2 conjugate to the subject.

Embodiment 450. The method according to any one of embodiments 1-449, wherein administering an effective amount of an IL-2 conjugate to the subject does not cause edema in the subject after administering the IL-2 conjugate to the subject.

Embodiment 451. The method of any one of embodiments 1-450, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause impairment of renal or hepatic function in the subject after administering the IL-2 conjugate to the subject.

Embodiment 452. The method of any one of embodiments 1-451, wherein administering an effective amount of an IL-2 conjugate to the subject does not cause eosinophilia in the subject after administering the IL-2 conjugate to the subject.

Embodiment 453. The method of embodiment 452, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause the subject to have a peripheral blood eosinophil count greater than 500 per μL after administering the IL-2 conjugate to the subject.

Embodiment 454. The method of embodiment 452, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the subject having a peripheral blood eosinophil count greater than 500 per μL to 1500 per μL after administering the IL-2 conjugate to the subject.

Embodiment 455. The method of embodiment 452, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the subject having a peripheral blood eosinophil count greater than 1500 per μL to 5000 per μL after administering the IL-2 conjugate to the subject.

Embodiment 456. The method of embodiment 452, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause the subject to have a peripheral blood eosinophil count greater than 5,000 per μL after administering the IL-2 conjugate to the subject.

Embodiment 457. The method according to any one of embodiments 1-456, wherein administering an effective amount of the IL-2 conjugate to the subject is not contraindicated in subjects on a psychotropic drug treatment regimen.

Embodiment 458. The method of any one of embodiments 1-457, wherein administering an effective amount of the IL-2 conjugate to the subject is not contraindicated in subjects on a treatment regimen with nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs.

Embodiment 459. The method of embodiment 458, wherein administering an effective amount of the IL-2 conjugate to the subject is not contraindicated in subjects on a treatment regimen of aminoglycosides, cytotoxic chemotherapy agents, doxorubicin, methotrexate, or asparaginase.

Embodiment 460. The method according to any one of embodiments 1-459, wherein administering an effective amount of the IL-2 conjugate to the subject is not contraindicated in subjects who are receiving treatment in combination regimens that include antineoplastic agents.

Embodiment 461. The method of embodiment 460, wherein the antineoplastic agent is selected from dacarbazine, cisplatin, tamoxifen, and interferon alpha.

Embodiment 462. The method of any one of embodiments 1-461, wherein administering an effective amount of the IL-2 conjugate to the subject does not cause one or more grade 4 adverse events in the subject after administering the IL-2 conjugate to the subject.

Embodiment 463. The method of embodiment 462, wherein the one or more grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; second degree atrioventricular block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function test abnormalities; gastrointestinal hemorrhage; hematemesis; hemorrhagic diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; increased alkaline phosphatase; increased blood urea nitrogen (BUN) concentration; hyperuricemia; increased non-protein nitrogen (NPN) concentration; respiratory acidosis; drowsiness; agitation; neuropathy; paranoid reaction; spasm; grand mal seizure; delirium; asthma; pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; pupillary abnormalities; renal function abnormalities; renal failure and acute tubular necrosis.

Embodiment 464. The method of any one of embodiments 1-463, wherein administering an effective amount of the IL-2 conjugate to a group of subjects does not cause one or more grade 4 adverse events in more than 1% of the subjects following administration of the IL-2 conjugate to the subjects.

Embodiment 465. The method of embodiment 464, wherein the one or more grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; second degree atrioventricular block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function test abnormalities; gastrointestinal hemorrhage; hematemesis; hemorrhagic diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; increased alkaline phosphatase; increased blood urea nitrogen (BUN) concentration; hyperuricemia; increased non-protein nitrogen (NPN) concentration; respiratory acidosis; drowsiness; agitation; neuropathy; paranoid reaction; spasm; grand mal seizure; delirium; asthma; pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; pupillary abnormalities; renal function abnormalities; renal failure and acute tubular necrosis.

Embodiment 466. The method of any one of embodiments 1-465, wherein administering an effective amount of an IL-2 conjugate to a group of subjects does not cause one or more adverse events in more than 1% of the subjects after administering the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from duodenal ulcer; intestinal necrosis; myocarditis; supraventricular tachycardia; permanent or transient blindness due to optic neuritis; transient ischemic attack; meningitis; cerebral edema; pericarditis; allergic interstitial nephritis; and tracheoesophageal fistula.

Embodiment 467. The method of any one of embodiments 1-466, wherein administering an effective amount of an IL-2 conjugate to a group of subjects does not cause one or more adverse events in more than 1% of the subjects after administering the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary embolism; stroke; intestinal perforation; liver or kidney failure; severe depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure.

Embodiment 468. The method of any one of embodiments 1-467, wherein administering the IL-2 conjugate to the subject results in an increase in the number of CD8+ T and NK cells in the subject's peripheral blood without an increase in the number of CD4+ regulatory T cells in the subject's peripheral blood.

Embodiment 469. The method according to any one of embodiments 1-468, wherein administering the IL-2 conjugate to the subject results in an increase in the number of CD8+ T and NK cells in the subject's peripheral blood without an increase in the number of eosinophils in the subject's peripheral blood.

Embodiment 470. The method of any one of embodiments 1-469, wherein administering the IL-2 conjugate to the subject results in an increase in the number of intratumoral CD8+ T and NK cells in the subject without an increase in the number of intratumoral CD4+ regulatory T cells in the subject.

Embodiment 471. The method according to any one of embodiments 1-470, wherein administering an effective amount of the IL-2 conjugate to the subject does not require an intensive care unit or skilled cardiopulmonary or critical care physicians.

Embodiment 472. The method of embodiment 471, wherein administering an effective amount of the IL-2 conjugate to the subject does not require an intensive care unit.

Embodiment 473. The method of embodiment 471, wherein administering an effective amount of the IL-2 conjugate to the subject does not require skilled cardiopulmonary or critical care professionals.

Embodiment 474. The method according to any one of embodiments 1-473, wherein the cancer is in the form of a solid tumor.

Embodiment 475. The method according to any one of embodiments 1-473, wherein the cancer is in the form of a tumor of liquid tissue.

Embodiment 476. The method of any one of embodiments 381-475, wherein the IL-2 conjugate is administered to the subject prior to administration to the subject of one or more immune checkpoint inhibitors.

Embodiment 477. The method of any one of embodiments 381-475, wherein one or more immune checkpoint inhibitors are administered to the subject prior to administration of the IL-2 conjugate to the subject.

Embodiment 478. The method of any one of embodiments 381-475, wherein the IL-2 conjugate and one or more immune checkpoint inhibitors are administered to the subject simultaneously.

Embodiment 479. 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: 1, SEQ ID NO: 3, or SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a cysteine covalently linked to a PEG group.

Embodiment 480. The method of embodiment 479, wherein 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.

Embodiment 481. The method of embodiment 479, wherein the PEG group has a molecular weight of 5 kDa.

Embodiment 482. The method of embodiment 479, wherein the PEG group has a molecular weight of 10 kDa.

Embodiment 483. The method of embodiment 479, wherein the PEG group has a molecular weight of 15 kDa.

Embodiment 484. The method of embodiment 479, wherein the PEG group has a molecular weight of 20 kDa.

Embodiment 485. The method of embodiment 479, wherein the PEG group has a molecular weight of 25 kDa.

Embodiment 486. The method of embodiment 479, wherein the PEG group has a molecular weight of 30 kDa.

Embodiment 487. The method of embodiment 479, wherein the PEG group has a molecular weight of 35 kDa.

Embodiment 488. The method of embodiment 479, wherein the PEG group has a molecular weight of 40 kDa.

Embodiment 489. The method of embodiment 479, wherein the PEG group has a molecular weight of 45 kDa.

Embodiment 490. The method of embodiment 479, wherein the PEG group has a molecular weight of 50 kDa.

Embodiment 491. The method of embodiment 479, wherein the PEG group has a molecular weight of 60 kDa.

Embodiment 492. The method according to any one of embodiments 479-491, 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 that is replaced with cysteine is selected from K34, T36, R37, T40, F41, K42, F43, Y44, E60, E61, E67, K63, P64, V68, L71, and Y106.

Embodiment 493. The method according to any one of embodiments 479-491, 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 that is replaced with cysteine is selected from K34, T40, F41, K42, Y44, E60, E61, E67, K63, P64, V68 and L71.

Embodiment 494. The method according to any one of embodiments 479-491, wherein 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 that is replaced with cysteine is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.

Embodiment 495. The method of any one of embodiments 479-494, wherein the one or more additional agents is one or more immune checkpoint inhibitors selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, OX40 agonists, and 4-1BB agonists.

Embodiment 496. The method of embodiment 495, wherein one or more immune checkpoint inhibitors are selected from PD-1 inhibitors.

Embodiment 497. The method of embodiment 496, wherein the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (Macrogenics/Incyte), GLS-010 (Arcus/WuXi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer), and genolimzumab (Apollomics/Genor BioPharma).

Embodiment 498. The method of embodiment 495, wherein one or more immune checkpoint inhibitors are selected from PD-L1 inhibitors.

Embodiment 499. The method of embodiment 498, wherein the one or more PD-L1 inhibitors are selected from atezolizumab, avelumab, durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (CytomX), CS1001 (CStone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics).

Embodiment 500. The method of embodiment 495, wherein one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors.

Embodiment 501. The method of embodiment 500, wherein the one or more CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus).

Embodiment 502. 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 non-lysine residue is replaced with lysine, comprising a linker and a water-soluble polymer.

Embodiment 503. The method of embodiment 502, wherein the water-soluble polymer is a PEG group.

Embodiment 504. The method of embodiments 502 or 503, wherein the one or more additional agents is one or more immune checkpoint inhibitors selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, OX40 agonists, and 4-1BB agonists.

Embodiment 505. The method of embodiment 504, wherein one or more immune checkpoint inhibitors are selected from PD-1 inhibitors.

Embodiment 506. The method of embodiment 505, wherein the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (Macrogenics/Incyte), GLS-010 (Arcus/WuXi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer), and genolimzumab (Apollomics/Genor BioPharma).

Embodiment 507. The method of embodiment 506, wherein one or more immune checkpoint inhibitors are selected from PD-L1 inhibitors.

Embodiment 508. The method of embodiment 507, wherein the PD-L1 inhibitors are selected from atezolizumab, avelumab, durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (CytomX), CS1001 (CStone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics).

Embodiment 509. The method of embodiment 508, wherein one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors.

Embodiment 510. The method of embodiment 509, wherein the one or more CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus).

Embodiment 511. The method of any one of embodiments 1-510, wherein the IL-2 conjugate comprises a PEG moiety covalently linked via a non-releasable bond.

Embodiment 512. The method of any one of embodiments 11-511, wherein the IL-2 conjugate comprises a non-releasable covalently linked PEG group.

Embodiment 513. The method of any one of embodiments 1-512, wherein after administration of the IL-2 conjugate and one or more additional agents, the subject experiences a response as measured by the immune-mediated Response Evaluation Criteria in Solid Tumors (iRECIST).

Embodiment 514. The method of embodiment 513, wherein the response is a complete response, a partial response, or stabilization of the disease.

Embodiment 515. The method of any one of embodiments 1-514, wherein the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration.

Embodiment 516. The method of embodiment 515, wherein the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, or intramuscular administration.

Embodiment 517. The method of embodiment 515, wherein the IL-2 conjugate is administered to the subject via intravenous administration.

Embodiment 518. The method of embodiment 515, wherein the IL-2 conjugate is administered to the subject by subcutaneous administration.

Embodiment 519. The method of embodiment 515, wherein the IL-2 conjugate is administered to the subject by intramuscular administration.

Embodiment 520. 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 having SEQ ID NO: 3, wherein an amino acid other than lysine in the IL-2 conjugate is replaced with a lysine residue, and wherein the lysine residue comprises one or more water-soluble polymers and a covalent linker.

Embodiment 521. The method of embodiment 520, wherein the lysine residue is located in the region K34-Y106 of SEQ ID NO: 3.

Embodiment 522. The method of embodiment 521 wherein the lysine residue is located at K34.

Embodiment 523. The method of embodiment 521 wherein the lysine residue is located at F41.

Embodiment 524. The method of embodiment 521 wherein the lysine residue is located at F43.

Embodiment 525. The method of embodiment 521 wherein the lysine residue is located at K42.

Embodiment 526. The method of embodiment 521 wherein the lysine residue is located at E61.

Embodiment 527. The method of embodiment 521 wherein the lysine residue is located at P64.

Embodiment 528. The method of embodiment 521 wherein the lysine residue is located at R37.

Embodiment 529. The method of embodiment 521 wherein the lysine residue is located at T40.

Embodiment 530. The method of embodiment 521, wherein the lysine residue is located at E67.

Embodiment 531. The method of embodiment 521, wherein the lysine residue is located at Y44.

Embodiment 532. The method of embodiment 521, wherein the lysine residue is located at V68.

Embodiment 533. The method of embodiment 521 wherein the lysine residue is located at L71.

Embodiment 534. 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 interleukin-2 (IL-2) variant wherein an amino acid other than lysine in the amino acid sequence of the IL-2 variant is replaced with an amino acid comprising (a) lysine; (b) a covalent linker, and (3) one or more water-soluble polymers.

Embodiment 535. The method of any one of embodiments 520-534, wherein one or more water-soluble polymers comprise a PEG group.

Embodiment 536. The method of embodiment 535 wherein the PEG group is a branched or linear PEG group.

Embodiment 537. The method of any one of embodiments 520-536, wherein the one or more additional agents is one or more immune checkpoint inhibitors selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, OX40 agonists, and 4-1BB agonists.

Embodiment 538. The method of embodiment 537, wherein one or more immune checkpoint inhibitors are selected from PD-1 inhibitors.

Embodiment 539. The method of embodiment 538, wherein the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (Macrogenics/Incyte), GLS-010 (Arcus/WuXi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer), and genolimzumab (Apollomics/Genor BioPharma).

Embodiment 540. The method of embodiment 539, wherein one or more immune checkpoint inhibitors are selected from PD-L1 inhibitors.

Embodiment 541. The method of embodiment 540, wherein the PD-L1 inhibitors are selected from atezolizumab, avelumab, durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (CytomX), CS1001 (CStone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics).

Embodiment 542. The method of embodiment 541, wherein one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors.

Embodiment 543. The method of embodiment 542, wherein the CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus).

Embodiment 544. The method of any one of embodiments 381-543, wherein the method further comprises administering to the subject a therapeutically effective amount of one or more inhibitors of the vascular endothelial cell growth factor (VEGF) or mammalian target of rapamycin (mTOR) signaling pathway.

Embodiment 545. The method of embodiment 544, wherein the subject is administered one or more VEGF signaling pathway inhibitors.

Embodiment 546. The method of embodiment 545, wherein one or more VEGF signaling pathway inhibitors are selected from the group consisting of vascular endothelial cell growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) and monoclonal antibodies to VEGF.

Embodiment 547. The method of embodiment 546, wherein the one or more VEGF signaling pathway inhibitors are selected from one or more VEGFR TKIs.

Embodiment 548. The method of embodiment 547, wherein the one or more VEGFR TKIs are selected from the group consisting of cabozantinib, axitinib, pazopanib, sunitinib, or sorafenib.

Embodiment 549. The method of embodiment 548, wherein the one or more VEGFR TKIs is cabozantinib.

Embodiment 550. The method of embodiment 548, wherein the one or more VEGFR TKIs is axitinib.

Embodiment 551. The method of embodiment 548, wherein the one or more VEGFR TKIs is pazopanib.

Embodiment 552. The method of embodiment 548, wherein the one or more VEGFR TKIs is sunitinib.

Embodiment 553. The method of embodiment 548, wherein the one or more VEGFR TKIs is sorafenib.

Embodiment 554. The method of embodiment 546, wherein the one or more VEGF signaling pathway inhibitors are selected from one or more monoclonal antibodies to VEGF.

Embodiment 555. The method of embodiment 554, wherein the one or more monoclonal antibodies to VEGF are bevacizumab.

Embodiment 556. The method of embodiment 544, wherein the one or more mTOR inhibitors are selected from the group consisting of rapamycin, everolimus, temsirolimus, ridaforolimus, and deforolimus.

Embodiment 557. The method of embodiment 556, wherein the one or more mTOR inhibitors are rapamycin.

Embodiment 558. The method of embodiment 556, wherein the one or more mTOR inhibitors is everolimus.

Embodiment 559. The method of embodiment 556, wherein the one or more mTOR inhibitors is temsirolimus.

Embodiment 560. The method of embodiment 556, wherein the one or more mTOR inhibitors are ridaforolimus.

Embodiment 561. The method of embodiment 556, wherein the one or more mTOR inhibitors is deforolimus.

Embodiment 562. The method of any one of embodiments 544-561, wherein the cancer in the subject is renal cell carcinoma (RCC).

Embodiment 563. The method of embodiment 562, wherein the one or more VEGFR TKIs are axitinib or cabozantinib.

Embodiment 564. The method of embodiment 562, wherein the one or more VEGFR TKIs is cabozantinib.

Embodiment 565. The method of any one of embodiments 381-543, wherein the one or more additional agents further comprise one or more chemotherapeutic agents.

Embodiment 566. The method of embodiment 565, wherein the one or more chemotherapeutic agents comprises one or more platinum-based chemotherapeutic agents.

Embodiment 567. The method of embodiment 565, wherein the one or more chemotherapeutic agents comprises carboplatin and pemetrexed.

Embodiment 568. The method of embodiment 565, wherein the one or more chemotherapeutic agents comprises carboplatin and nab-paclitaxel.

Embodiment 569. The method of embodiment 565, wherein the one or more chemotherapeutic agents comprises carboplatin and docetaxel.

Embodiment 570. The method of any one of embodiments 565-569, wherein the cancer in the subject is non-small cell lung cancer (NSCLC).

Embodiment 571. The method of any one of embodiments 1-570, wherein the one or more additional agents are one or more chemotherapeutic agents.

Embodiment 572. The method of embodiment 571, wherein the one or more chemotherapeutic agents comprises one or more platinum-based chemotherapeutic agents.

Embodiment 573. The method according to any one of embodiments 1-572, wherein the subject tests positive for human papillomavirus (HPV) prior to administration of the IL-2 conjugate and one or more additional agents.

Embodiment 574. The method of embodiment 572, wherein the cancer in the subject is head and neck squamous cell carcinoma (HNSCC).

Embodiment 575. The method of any one of embodiments 1-572, further comprising testing the subject positive for human papillomavirus (HPV+) followed by administering the IL-2 conjugate and one or more additional agents.

Embodiment 576. 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 PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX):

Formula (VIII),

(IX),

Where:

n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; and

X has the structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 576.1: 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 PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX):

Formula (VIII),

(IX),

Where:

n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 577. The method of embodiments 576 or 576.1, wherein the amino acid residue at E61 in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX), and wherein n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons.

Embodiment 578. The method of embodiment 577, wherein n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons.

Embodiment 579. The method of embodiments 577 or 578, wherein the one or more PD-1 inhibitors are pembrolizumab or nivolumab.

Embodiment 580. The method of embodiment 579, wherein the one or more PD-1 inhibitors is pembrolizumab.

Embodiment 581. The method of embodiment 579, wherein the one or more PD-1 inhibitors is nivolumab.

Embodiment 582. The method of embodiments 576 or 576.1, wherein the amino acid residue in P64 in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX), and wherein n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons.

Embodiment 583. The method of embodiment 582, wherein n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons.

Embodiment 584. The method of embodiments 582 or 583, wherein the one or more PD-1 inhibitors are pembrolizumab or nivolumab.

Embodiment 585. The method of embodiment 584, wherein the one or more PD-1 inhibitors is pembrolizumab.

Embodiment 586. The method of embodiment 584, wherein the one or more PD-1 inhibitors is nivolumab.

Embodiment 587. 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 PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII):

Formula (VI),

(VII),

Where:

n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; and

X has the structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 587.1: 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 PD-1 inhibitors, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII):

Formula (VI),

(VII),

Where:

n is an integer such that the molecular weight of the PEG group is from about 15,000 daltons to about 60,000 daltons; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 588. The method of embodiments 587 or 587.1, wherein the amino acid residue at E61 in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), and wherein n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons.

Embodiment 589. The method of embodiment 588, wherein n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons.

Embodiment 590. The method of embodiments 588 or 589, wherein the one or more PD-1 inhibitors are pembrolizumab or nivolumab.

Embodiment 591. The method of embodiment 590, wherein the one or more PD-1 inhibitors is pembrolizumab.

Embodiment 592. The method of embodiment 590, wherein the one or more PD-1 inhibitors is nivolumab.

Embodiment 593. The method of embodiments 587 or 587.1, wherein the amino acid residue in P64 in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), and wherein n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons.

Embodiment 594. The method of embodiment 593, wherein n is an integer such that the molecular weight of the PEG group is from about 30,000 daltons.

Embodiment 595. The method of embodiments 593 or 594, wherein the one or more PD-1 inhibitors are pembrolizumab or nivolumab.

Embodiment 596. The method of embodiment 595, wherein the one or more PD-1 inhibitors is pembrolizumab.

Embodiment 597. The method of embodiment 595, wherein the one or more PD-1 inhibitors is nivolumab.

Embodiment 598. The method of any one of embodiments 1-597, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

Embodiment 599. An IL-2 conjugate for use in the method of any one of embodiments 1-598.

Embodiment 600. Use of an IL-2 conjugate for the manufacture of a medicament for treating cancer according to the method of any one of embodiments 1-598.

EXAMPLES

These examples are provided for illustrative purposes only and do not limit the scope of the claims presented herein.

Each of the compounds disclosed in Examples 2-8 utilizes SEQ ID NO: 4 and a [AzK_PEG] moiety, wherein the position of the substituted amino acid in the IL-2 conjugate is related to the positions in SEQ ID NO: 4.

For example, the compound designated as "P65_5kDa" in Tables 3A and 3B was prepared by methods similar to those disclosed in Example 2, wherein a protein having SEQ ID NO: 4 was first prepared in which the proline at position 65 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 10). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 5 kDa to yield a product having SEQ ID NO: 20, comprising formula (II), formula (III), or a mixture of formulas (II) and (III), wherein W is a linear methoxy-PEG group having an average molecular weight of 5 kDa.

In another example, the compound designated as "P65_30kDa" in Tables 3A and 3B , which was used in Example 4, Example 5, Example 6, and Example 11 (also referred to as "IL-2_P65[AzK_PEG30kDa]" in Example 11 and referred to in Example 11 and the drawings as "compound A"), was prepared by first preparing a protein having SEQ ID NO: 4 in which the proline at position 65 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 10). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 30 kDa to obtain a product having SEQ ID NO: 25, comprising formula (II), formula (III) or a mixture of formulas (II) and (III), wherein W is a linear methoxy-PEG group having an average molecular weight of 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which proline at position 65 (P65) is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), and wherein n is an integer such that the PEG group has a molecular weight of approximately 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which proline at position 65 (P65) is replaced by a structure of formula (X) or (XI) or a mixture of (X) and (XI), and wherein n is an integer such that the PEG group has a molecular weight of approximately 30 kDa.

In another example, the compound designated as "E62_5kDa" in Tables 3A and 3B was prepared by first preparing a protein having SEQ ID NO: 4 in which the glutamic acid at position 62 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine AzK (SEQ ID NO: 11). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 5 kDa to yield a product having SEQ ID NO: 21, comprising formula (II), formula (III), or a mixture of formulas (II) and (III), wherein W is a linear methoxy-PEG group having an average molecular weight of 5 kDa.

In another example, the compound designated as "E62_30kDa" in Tables 3A and 3B and also used in Example 4 was prepared by first preparing a protein having SEQ ID NO: 4 in which the glutamic acid at position 62 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 11). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 30 kDa to obtain a product having SEQ ID NO: 26 comprising formula (II), formula (III), or a mixture of formulas (II) and (III), wherein W is a linear methoxy-PEG group having an average molecular weight of 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 (E62) is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), and wherein n is an integer such that the PEG group has a molecular weight of about 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 (E62) is replaced by a structure of formula (X) or (XI) or a mixture of (X) and (XI), and wherein n is an integer such that the PEG group has a molecular weight of about 30 kDa.

In another example, the compound designated "K35_30kDa" and used in Example 8 was prepared by first preparing a protein having SEQ ID NO: 4 in which the lysine at position 35 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 14). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 30 kDa to obtain a product having SEQ ID NO: 29, comprising formula (II), formula (III), or a mixture of formulas (II) and (III), wherein W is a linear methoxy-PEG group having an average molecular weight of 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which lysine at position 35 (K35) is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), and wherein n is an integer such that the PEG group has a molecular weight of about 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which lysine at position 35 (K35) is replaced by a structure of formula (X) or (XI) or a mixture of (X) and (XI), and wherein n is an integer such that the PEG group has a molecular weight of about 30 kDa.

In Examples 9 and 10, the compound "IL-2_P65_[AzK_L1_PEG30kDa]-1" was used, which comprises SEQ ID NO: 50, in which the proline at position 64 is replaced by AzK_L1_PEG30kDa, where AzK_L1_PEG30kDa is defined as a structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a linear mPEG chain of 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kDa]-1 is also defined as a compound comprising SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX), and wherein n is an integer such that the molecular weight of the PEG group is approximately 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kDa]-1 is also defined as a compound comprising SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII), and wherein n is an integer such that the molecular weight of the PEG group is approximately 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kDa]-1 is also mentioned in Examples 12 and the following and in the drawings as "compound B". The compound was prepared by methods similar to those disclosed in Example 2, wherein the protein having SEQ ID NO: 3 was first prepared, in which the proline at position 64 was replaced byN6-((2-azidoethoxy)-carbonyl)-L-lysine AzK (SEQ ID NO: 35). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 30 kDa.

In Example 11, the compound "IL-2_P65[AzK_PEG30kDa]" (also referred to herein as "P65_30kDa") was used, which was as described above.

EXAMPLE 1

Kinase and cytokine receptor dimerization assays

Handling of cells

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

Agonist format

For agonist detection, cells were incubated with sample to induce a response. Sample stock solutions were intermediately diluted to produce 5X sample in assay buffer. Approximately 5 µl of 5X sample was added to cells and incubated at 37°C for 6-16 hours depending on the assay. The carrier medium concentration was 1%.

Signal detection

The assay signal was generated by a single addition of 12.5 or 15 µl (50% v/v) of PathHunter Detector Reagent Cocktail for the agonist and antagonist assays, respectively, followed by incubation for one hour at room temperature. In some assays, activity was detected using a highly sensitive detector reagent (PathHunter Flash kit) to improve assay performance. For these assays, an equal volume of detector reagent (25 or 30 µl) was added to the wells followed by incubation for one hour at room temperature. Microplates were read after signal generation using a PerkinElmer EnVisionTM chemiluminescent signal detection instrument.

Data Analysis

Compound activity was analyzed using the CBIS data analysis package (ChemInnovation, California). For agonist mode assays, percent activity was calculated using the following formula:

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

For antagonism mode assays, the percentage of inhibition was calculated using the following formula:

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

EXAMPLE 2

Cell-Based Screening to Identify PEGylated IL-2 Compounds Without Interaction with IL-2R α

Illustrative IL-2 conjugates K35, F42, K43, E62, and P65 were subjected to functional analysis. IL-2 conjugates were expressed as inclusion bodies inE. coliby means of the methods disclosed herein, expression plasmids encoding a protein with a desired amino acid sequence were obtained, which contained (a) an unnatural base pair comprising a first unnatural nucleotide and a second unnatural nucleotide to ensure the presence of a codon at the desired position into which the unnatural amino acid was insertedN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and the corresponding anticodon in tRNA, (b) a plasmid encoding tRNA, obtained from PylM. mazei, which contains an unnatural nucleotide to ensure the presence of the corresponding anticodon in place of its native sequence, (c) a plasmid encoding pyrrolizyl-tRNA synthetase derived fromM. barkeri(MbPylRS), and (d)N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). A double-stranded oligonucleotide that encodes the amino acid sequence of a desired IL-2 variant comprises an AXC codon, such as at position 34, 37, 40, 41, 42, 43, 44, 61, 64, 68, or 71 of the sequence that encodes a protein having SEQ ID NO: 3, or at position 35, 38, 41, 42, 43, 45, 62, 65, 69, or 72 of the sequence that encodes a protein having SEQ ID NO: 4, wherein X is a non-natural nucleotide as disclosed herein. In some embodiments, the cell further comprises a plasmid, which may be a protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene fromM. mazei, which contains a GYT anticodon corresponding to AXC in place of its native sequence, wherein Y is a non-natural nucleotide disclosed herein and may be the same as or different from the non-natural nucleotide in the codon. X and Y are selected from the non-natural nucleotides dTPT3, dNaM, and dCNMO disclosed herein. The expressed protein was purified and refolded using standard procedures prior to site-specific PEGylation of the AzK-containing IL-2 product using copper-free DBCO-mediated click chemistry to stably covalently attach mPEG moieties to AzK (scheme 1).

Scheme 1. Illustrative synthesis of interleukin variants with AzK_PEG (where n denotes the number of repeating PEG units). The reaction of the AzK fragment with the alkynyl moiety of DBCO can yield a single regioisomeric product or a mixture of regioisomeric products.

IL-2 conjugates were screened for functional activity at DiscoverX (Fremont, CA) using the PathHunter IL-2 Cytokine Receptor Assay. This assay utilizes the recombinant human U2OS cell line, which expresses the IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) subunits, each fused to half of a split β-galactosidase reporter enzyme. Additionally, a second cell line was constructed to express the IL-2Rα subunit. Parallel testing with these two cell lines allows for the assessment of activation of the IL-2αβγ receptor variant as well as the basal βγ complex. IL-2 agonist activity at the IL-2βγ receptor complex stimulates receptor dimerization and restoration of the reporter β-galactosidase, resulting in the formation of a chemiluminescent signal. The assay was performed in agonist mode to determine the EC ₅₀ of each test drug, and comparison of dose-response profiles between IL2Rα-positive and -negative cell types allows the contribution of IL2Rα to the observed activity to be determined.

Table 2 shows the EC50 data for IL-2 receptor agonism in cell-based screening of 10 kDa PEGylated IL-2 conjugates (except where noted)

Table 2 Website βγ,
EC50 (nM) αβγ,
EC50 (nM) βγ/αβγ ratio Native 1.68 0.074 23 Optimal 1.68 1.68 1 K35 6.75 0.15 45 F42 6.09 0.515 12 K43 9.84 0.131 75 E62 3 1.5 2 P65* 23.8 4.44 5 R38 4.16 0.165 25 T41 6.37 0.0489 130 E68 7.70 0.0893 86 Y45* 9.06 0,110 83 V69* 9.99 0.083 121 *denotes 30 kDa PEGylated IL-2 conjugate.

Biochemical interactions of pegylated IL-2 with human IL-2 receptor subunits

Kinetic parameters of interactions of the pegylated IL-2 compound with human IL-2 receptor subunits were measured using surface plasmon resonance (SPR) at Biosensor Tools LLC (Salt Lake City, UT). For these studies, the extracellular domains of IL-2Rα (Sino Biological, #10165-H02H) and β (Sino Biological, #10696-H02H) fused to human IgG1 Fc were captured on the surface of a protein A-coated CM4 sensor chip from Biacore. These surfaces were probed in duplicate in a two-fold dilution series starting with 2 μM native IL-2 (wild-type IL-2; Thermo #PHC0021), P65_30kDa, P65_5kDa, E62_30kDa, or E62_5kDa using a Biacore 2000 SPR instrument. Test samples were injected for 60 s to allow association measurements, followed by buffer (wash) only for 30 s to measure dissociation. Response units (RU, y-axis) were plotted against time (s, x-axis).

To assess the effect of the IL-2 receptor α subunit on IL-2 binding to β, α was captured in approximately 2-fold excess over β. Native IL-2 (wild-type IL-2), P65_30kDa, P65_5kDa, E62_30kDa, or E62_5kDa were coated onto these surfaces in a three-fold dilution series starting at 2.5 μM. Binding data were fitted with a 1:1 interaction model that included a volume shift and the extracted kinetic parameters are summarized in Table 3A and Table 3B . As shown in Table 3A and Table 3B , small molecule PEGs abolished the interaction with IL2Ralpha but had a less pronounced non-specific effect on the interaction with IL2Rbeta.

Table 3A. Kinetic parameters for interactions of IL-2 variants with individual IL-2 receptor subunit surfaces - IL-2 receptor α-subunit surface k _a (M ^-1 ⋅s ^-1 ) k _d (With ^-1 ) K _D (μM) Native IL-2 4.5 ± 0.3 × 10 ⁷ 0.410 ± 0.01 0.009 ± 0.002 P65_30kDa 114 ± 36 × 10 ⁷ 0.018 ± 0.008 158 ± 21 P65_5kDa 797 ± 226 × 10 ⁷ 0.033 ± 0.004 42 ± 7 E62_30kDa 333 ± 88 × 10 ⁷ 0.050 ± 0.01 162 ± 7 E62_5kDa 1010 ± 41 × 10 ⁷ 0.035 ± 0.002 34.4 ± 0.3

Table 3B. Kinetic parameters for interactions of IL-2 variants with individual IL-2 receptor subunit surfaces - IL-2 receptor β-subunit surface k _a (M ^-1 ⋅s ^-1 ) k _d (With ^-1 ) K _D (μM) Native IL-2 1.3 ± 0.2 × 10 ⁶ 0.185 ± 0.009 0.145 ± 0.005 P65_30kDa 1.8 ± 0.2 × 10 ⁵ 0.370 ± 0.01 2.09 ± 0.09 P65_5kDa 9.0 ± 0.4 × 10 ⁵ 0.270 ± 0.01 0.305 ± 0.002 E62_30kDa 1.8 ± 0.4 × 10 ⁵ 0.208 ± 0.006 1.14 ± 0.01 E62_5kDa 6.6 ± 0.8 × 10 ⁵ 0.281 ± 0.004 0.428 ± 0.00

Determination of the immune response profile ex vivo for IL-2 compounds in PBMC samples obtained from the human primary leukocyte removal system (LRS)

To determine how the differential receptor specificities of IL-2 P65_30kDa, K64_30kDa, K43_30kDa, K35_30kDa, and F42_30kDa influence the activation of primary immune cell subsets, concentration-response profiling of lymphocyte activation was performed in LRS-derived human peripheral blood mononuclear cell (PBMC) samples using multicolor flow cytometry. These studies were performed at PrimityBio LLC (Fremont, CA). Fresh LRS-derived samples were treated with native IL-2, IL-2 P65_30kDa, K64_30kDa, K43_30kDa, K35_30kDa, and F42_30kDa in a 5-fold dilution series starting with a highest concentration of 30 μg/mL. Following 45 min incubation, samples were fixed and stained with antibodies to detect the phosphorylated form of the transcription factor STAT5 (pSTAT5), a marker for upstream recruitment and activation of IL-2 receptor signaling complexes, and a panel of surface markers to monitor pSTAT5 formation in specific T cell and natural killer (NK) cell subsets. The staining panel for flow cytometric examination of LRS-derived PBMC samples included markers for effector T cells (Teff: CD3+, CD4+, CD8+, CD127+), NK cells (CD3-, CD16+), and regulatory T cells (Treg: CD3+, CD4+, CD8-, IL-2Rα+, CD127-1).

Flow cytometry data were analyzed for activation of different T and NK cell subsets in a concentration-response fashion, reading pSTAT5 accumulation after treatment with native IL-2. Due to Treg-specific IL-2Rα expression, native IL-2 demonstrated increased pSTAT5 stimulating activity in Tregs compared to CD8 Teff and NK cells. Compared to native compound, pegylated variants demonstrated moderately reduced activity on CD8 T cell and NK cell populations, but showed differential reduction in activity in IL-2Rα-expressing Tregs compared to native IL-2.

IN Table 4EC50 dependence is presented dose-response for pSTAT5-mediated signaling (EC50) in human LRS samples or for CTTL-2 proliferation upon treatment with the indicated IL-2 variant.

Table 4. EC50 dose-response relationships for pSTAT5-mediated signaling (EC50) in human LRS samples or for CTTL-2 proliferation upon treatment with the indicated IL-2 variant Processing NK cells Treg cells CD8+ T cells CD8/Treg ratio CTLL-2 Native IL-2 5150,5 62.5 25703,5 411.3 846 E62_30kDa 12834 37213 66644 1.8 398012 E62_5kDa 5327,5 18146 41552,5 2,3 275590 E62K 10305 11086 64037 5.8 58213 P65_30kDa 15741 40740,5 113638 2.8 677198 P65_5kDa 1920 6324,5 13769,5 2,2 194924 K35_30kDa 14021 358 63023 176.0 N.D. F42_30kDa 16397 36856 107944 2.9 123936 K43_30kDa 9004 4797 50504 10.5 N.D.

N.D.=not defined.

EC50 values (pg/mL) were calculated from dose-response curves obtained from MFI plots.

EXAMPLE 3

PEG and residue substitution promote non-alpha-subunit-related pharmacological properties of variants

To determine how PEG and residue substitution affect the non-alpha-subunit pharmacological properties of IL-2 E62, concentration-response profiling of lymphocyte activation was performed in LRS-derived human peripheral blood mononuclear cell (PBMC) samples using multicolor flow cytometry. These studies were performed at PrimityBio LLC (Fremont, CA). Fresh LRS-derived samples were treated with native IL-2, E62K, or E62_30kDa in a 5-fold dilution series starting with a highest concentration of 30 μg/mL. Following 45 min incubation, samples were fixed and stained with antibodies to detect the phosphorylated form of the transcription factor STAT5 (pSTAT5), a marker for upstream recruitment and activation of IL-2 receptor signaling complexes, and a panel of surface markers to monitor pSTAT5 formation in specific T cell and natural killer (NK) cell subsets. The staining panel for flow cytometric examination of LRS-derived PBMC samples includes markers for CD4, CD4+ central memory, CD4+ effector memory, CD4+ memory T, CD4+ naive T, CD4+ T, CD8, CD8+ central memory, CD8+ effector memory, CD8+ memory T, CD8+ naive T, CD8+ T, NK, and regulatory T.

EXAMPLE 4

PK/PD studies in naive (E3826-U1704) and B16-F10 tumor-bearing (E3826-U1803) C57BL/6 mice

The study plans are summarized in Table 5 and Table 6 , with the dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety. Terminal blood samples were collected via cardiac puncture at the indicated time points. Study E3826-U1704 included 13 time points (0.13, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h) with 3 mice sacrificed at each time point, and Study E3826-U1803 included 9 time points (2, 8, 12, 24, 48, 72, 120, 168, and 240 h) with 4-7 mice sacrificed at each time point. Plasma and blood cells (in both studies) and tumors in study E3826-U1803 were collected for PK and PD analyses.

Bioassay of plasma samples was performed using a certified human IL-2 ELISA assay (Abcam, Cambridge, UK). Aldesleukin, E62_30kDa, P65_30kDa, and internal standard concentrations in plasma-derived samples were determined by ELISA. PK data analysis was performed at NW Solutions (Seattle, WA). PK data were imported into Phoenix WinNonlin v6.4 (Certara/Pharsight, Princeton, NJ) for analysis. Group mean plasma concentration-time data were analyzed by non-compartmental methods using an IV bolus model.

Table 5. PK/PD study #E3826-U1704 - control and test groups in naive C57/BL6 mice Processing Dose*(mg/kg) Path, scheme Moments of time N Control 0 IV, single dose 13 3 Aldesleukin 0.3 IV, single dose 13 3 P65_30kDa 0.3 IV, single dose 13 3 E62_30kDa 0.3 IV, single dose 13 3 *Dose refers to the amount of IL-2 P65_30kDa polypeptide, and the dose was calculated based on the mass of the protein component, excluding the mass of the PEG fragment.

Table 6. PK/PD study #E3826-U1803 - control and test groups for treatment of mice bearing B16F-10 melanoma tumor (with the dose calculated based on the mass of the protein component, excluding the mass of the PEG fragment) Processing Dose (mg/kg) Path, scheme Moment in time N Absent (before dosing) 0 mg/kg Absent 1 6 Control medium carrier 0 mg/kg IV, single dose 9 3 P65_30kDa 1 mg/kg IV, single dose 9 4 P65_30kDa 3 mg/kg IV, single dose 9 4

In study E3826-U1704, both P65_30kDa and E62_30kDa demonstrated superior PK profiles compared to aldesleukin, as summarized in Table 7. Following a single IV bolus dose of aldesleukin, Tmax occurred at 0.03 h post-dose (first post-dose time point), and mean plasma concentrations were measurable for up to 4 h post-dose. Following a single IV bolus dose of P65_30kDa and E62_30kDa, Tmax occurred at 0.03 h post-dose, and mean plasma concentrations were measurable for up to 120 h post-dose (last post-dose time point). In a separate study following IV dosing of E62_5kDa, T _max was observed at 0.133 h post-dose, and mean plasma concentrations were measurable for up to 12 h post-dose.

Exposure based on C _max and AUC _0-t was as follows: P65_30kDa > E62_30kDa >> E62_5kDa > aldesleukin. E62_5kDa with lower molecular weight PEG had a PK profile similar to rIL-2 ( Table 7 ). P65_30kDa had 5.5- and 200-fold greater exposure than aldesleukin based on C max and AUC 0-t, respectively. In addition, P65_30kDa had a 23-fold increase in t1/2 (13.3 h vs. 0.57 h) and approximately 198-fold decrease in CL (6.58 vs. 1300 mL/h/kg) compared to aldesleukin. For both P65_30kDa and E62_30kDa, the volume of distribution (82.4 and 92.3 ml/kg, respectively) was approximately 4.2-4.7-fold reduced compared to aldesleukin and similar to the mouse blood volume (85 ml/kg [Boersen 2013]). This suggests that P65_30kDa and E62_30kDa are distributed primarily into the systemic circulation.

Table 7. PK parameters of P65_30kDa in female C57BL/6 mice Parameter Units of measurement P65_30kDa E62_30kDa E62_5kDa Aldesleukin T _max h. 0,030 0,030 0.133 0,030 C _max ng/ml 4870 4230 936 884 AUC _0-t h.*ng/ml 45600 37100 798 229 R ² 0.992 0.986 0.851 0,900 AUC _INF h.*ng/ml 45600 37100 807 230 t _1/2 h. 13300 14500 2.56 0.573 CL ml/h/kg 6,580 8.07 372 1300 V _ss ml/kg 82.4 92.3 404 390

Note: R ² is the goodness-of-fit parameter for the final phase of each concentration-time profile.

All parameters are given with an accuracy of up to 3 significant digits.

EXAMPLE 5

Pharmacodynamic observations in the peripheral blood compartment

STAT5 phosphorylation and induction of cell proliferation (early molecular marker Ki-67 and cell number) were used as pharmacodynamic endpoints to evaluate the pharmacological profile of P65_30kDa relative to its pharmacokinetic characteristics. The PD marker pSTAT5 showed good correlation with PK for both P65_30kDa and aldesleukin in CD8+ effector T cells (Table 7). Sustained elevation of pSTAT5 was observed in both NK and CD8+ T cells for up to 72 h and up to 24 h in Tregs. pSTAT5 induction returned to baseline levels after only 2 h in aldesleukin-treated mice. STAT5 phosphorylation resulted in proliferative responses (72-120 h) of CD8+ effector T cells and NK cells, but not Tregs. Phenotypic analysis of CD8+ effector T cells revealed a significant expansion of CD44+ memory cells in this population.

Pharmacodynamic observations in the tumor compartment of B16-F10 (E3826-U1803) tumor-bearing C57BL/6 mice

Table 8 shows plasma and tumor drug concentrations following a single 3 mg/kg dose of P65_30kDa in B16-F10 tumor-bearing mice, where the dose was calculated based on the mass of the protein component and did not include the mass of the PEG moiety. The tumor half-life was twice that of plasma (24.4 versus 12.6), indicating that P65_30kDa penetrates and is retained in the tumor. The tails of the curves intersect, indicating that plasma clears the drug faster than the tumor (data not shown). The tumor:plasma AUC ratios were 9.7% and 8.4% for the 1 and 3 mg/kg doses, respectively.

Table 8. PK parameters of P65_30kDa in blood plasma and tumor in female C57BL/6 mice bearing B16-F10 tumor Parameter P65_30kDa (3 mg/kg) Blood plasma Tumor T _max (h) *2.00 8 C _max (ng/ml) 40000 1550 t1/2 (h.) 12.60 24.4 AUC _0-t (hr*ng/ml) 656000 55200 R ² 0.974 0.988 AUC _INF (hr*ng/ml) 656000 55200

MTD study in Balb/c mice E3826-U1802

A dose-ranging study of P65_30kDa was performed in naive female Balb/c mice at Crown Biosciences, Inc. (San Diego, CA). The study design is shown in Table 9 , with dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety. Blood samples were collected from the submandibular vein at 8 time points (0.25, 1, 4, 12, 24, 34, 48, and 72 h). Both plasma and blood cells were collected for PK and PD analyses.

All plasma samples were analyzed for human IL-2 and mouse IL-2, TNF-α, IFNγ, IL-5, and IL-6 cytokines using commercially available ELISA kits.

Table 9. PK/PD and MTD Study #E3826-U1802 - Control and Test Treatment Groups in Naïve Balb/C Mice Processing Dose (mg/kg) Path, scheme Moment in time N Previously uninvolved in experiments 0 mg/kg 0 3 Control medium carrier 0 mg/kg IV, BID x 3 3 3 Aldesleukin 0.01 mg/kg IV, BID x 3 3 3 Aldesleukin 0.03 mg/kg IV, BID x 3 3 3 Aldesleukin 0.1 mg/kg IV, BID x 3 3 3 Aldesleukin 1.0 mg/kg IV, BID x 3 3 3 Aldesleukin 3.0 mg/kg IV, BID x 3 3 3 Aldesleukin 5.0 mg/kg IV, BID x 3 3 3 P65_30kDa 0.01 mg/kg IV, single dose 3 3 P65_30kDa 0.03 mg/kg IV, single dose 3 3 P65_30kDa 0.1 mg/kg IV, single dose 3 3 P65_30kDa 1.0 mg/kg IV, single dose 3 3 P65_30kDa 3.0 mg/kg IV, single dose 3 3 P65_30kDa 5.0 mg/kg IV, single dose 3 3 #P65_30kDa 0.3 mg/kg IV, single dose 8 3 * At all time points except 72 h, blood was collected from the submandibular vein. At 72 h, terminal blood was collected.
# Only the 0.3 mg/kg P65_30kDa dose was used for PK/PD evaluation.

Toxicological observations in an MTD study using Balb/c mice

The major form of toxicity associated with high-dose aldesleukin is vascular leak syndrome and its associated cytokine release syndrome (CRS). To assess the potential for this effect, mice were treated with single IV doses of P65_30kDa in the dose range of 0.01 to 5.0 mg/kg ( Table 9 ), with the dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety. Analyses performed included hematology, histopathology, organ weight, and cytokine analysis. No abnormalities were observed in hematology, histopathology, or body weight compared to vehicle control-treated mice for either P65_30kDa or aldesleukin. Regarding the cytokine analysis, aldesleukin was observed to increase IL-5 levels in plasma starting from 1 mg/kg to 5 mg/kg. In the case of P65_30kDa, a moderate increase in IL-5 levels (but less than aldesleukin) was observed only at 5 mg/kg. A transient increase in IFNγ levels in the systemic circulation was observed for both aldesleukin and P65_30kDa.

EXAMPLE 6

PK/PD in Cynomolgus Monkeys - Study #20157276

The pharmacokinetic and pharmacodynamic profiles of P65_30kDa were evaluated in previously studied cynomolgus macaques following a single 0.3 mg/kg intravenous dose, with the dose calculated based on the mass of the protein component and excluding the mass of the PEG moiety. The study was performed at Charles River Laboratories, Inc. (Reno, NV) and PK data analysis was performed at NW Solutions (Seattle, WA). Blood samples were collected predose and at 15 time points (0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 120, 144, 168, 192, and 240 h postdose). Both plasma and blood cells were collected for PK and PD analyses. Selected time points were used for PK, PD, cell population, and hematology analyses.

All plasma samples were analyzed for human IL-2 (PK assays) using commercially available ELISA kits.

Table 10 shows the PK parameters of P65_30kDa in the cynomolgus macaque.

Table 10 0.3 mg/kg Animal 2699 Animal 2705 Average value ROA Parameter Units of measurement Estimated value IV T _max h. 0,500 0,500 0,500 C _max ng/ml 11000 11400 11200 AUC _0-t h.*ng/ml 121000 120000 121000 t _1/2 h. 13.4 13.9 13.6 CL ml/h/kg 2.47 2.49 2.48 V _ss ml/kg 29.0 32.1 30.5

Following administration of a single IV bolus dose, Tmax was observed at 0.5 h post-dose (first post-dose time point), and mean plasma concentrations were measurable for up to 168 h post-dose (last time point). The t _1/2 and AUC for P65_30kDa were 13.6 h and 121,000 h*ng/mL, respectively.

Hematological parameters in cynomolgus macaques - Study #20157276

For hematological parameters, assessment time points correspond to day -1 pre-dose and days 1, 3, 6, 8, 10, 12, 14, 17, 19, 21 post-dose.

EXAMPLE 7

Determination of the immune response profile ex vivo for illustrative IL-2 compounds in PBMC samples obtained from the human primary leukocyte removal system (LRS)

To determine how the different receptor specificities of exemplary IL-2 compounds affect the activation of primary immune cell subsets, concentration-response profiling of lymphocyte activation was performed in human LRS-derived peripheral blood mononuclear cell (PBMC) samples using multicolor flow cytometry. The conjugates in Table 12 were synthesized by modifying SEQ ID NO: 1. These studies were performed at PrimityBio LLC (Fremont, CA). Primary lymphocytes derived from human LRS samples were treated with a dilution series of exemplary IL-2 compounds and quantified based on pSTAT5-mediated signaling in each lymphocyte cell type using the panel shown in Table 11.

Table 11. Designations indicating cell populations Marker Cell population CD3 T cells CD4 Th cells CD8 Effector T cells CD45RA "Non-trained" T cells CD56 NK cells CD14/19 Monocytes/B cells CD25 Tregs or "trained" T cells CD127 Non-Treg cells CD62L Memory T cell versus effector memory T cell pSTAT5 (Y694) Activation marker

Flow cytometry data were analyzed for the activation of different T and NK cell subsets in a concentration-response fashion, measuring pSTAT5 accumulation following treatment with the IL-2 K9_30kDa exemplary variant.

Table 12 shows the EC50 dose-response relationship for pSTAT5-mediated signaling (EC50) in human LRS samples or for CTTL-2 proliferation upon treatment with the indicated IL-2 variant.

Table 12. EC50 dose-response relationships for pSTAT5-mediated signaling (EC50) in human LRS samples or for CTTL-2 proliferation upon treatment with the indicated IL-2 variant Compound NK cells CD8+ T cells Treg cells CD8+/Ratio
Treg Fold increase in EC50 in Tregs compared to native IL-2 Proliferation of CTLL-2 Native IL-2 4586 31024 75 414 1 455.8 K9_30kDa 169578 1100679 2217 496 30 504 H16_30kDa 2545257 12070108 34976 345 466 80755 L19_30kDa 6756768 22436430 93205 241 1243 3510 D20_30kDa 2643930 9505217 1129455 8 15059 689939 M23_30kDa 143620 539824 1030 524 14 1102 N26_30kDa 258531 1188859 2459 483 33 2594 N88_30kDa 3298113 11111537 323201 34 4309 66606 E100_30kDa 35088 195823 483 405 6 1676 N119_30kDa 34010 143380 535 268 11 1215 T123_30kDa 33396 152928 269 569 6 255 Q126_30kDa 3676807 19722480 29454 670 393 3584 S127_30kDa 20210 92190 150 615 3 123 T131_30kDa 24207 132922 258 515 3 641 N88R/D109_30kDa 2780819 12503386 175805 71 3663 59577 V91K 20537 102255 142 720 3 99.5 N88R 2312847 15025734 11082 1356 148 363

*Changes in Treg activity compared to native IL-2 (wild-type IL-2) were analyzed in each individual experiment.

EC50 values (pg/mL) were calculated from dose-response curves obtained from MFI plots.

EXAMPLE 8

PK study in C57BL/6 mice

The experimental details are summarized in Table 13 , with the dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety.

Table 13 Group Number of animals Test/control drug (dose) Route, mode of administration of the dose End point(s) 1 9 Native IL-2 (wild type) (3.0 mg/kg) Concentration: 0.6 mg/ml IV, single dose on day 0 at T=0 at 5 ml/kg Blood collection at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after dose administration 2 9 K35_30kDa (3.0 mg/kg) Concentration: 0.6 mg/ml IV, single dose on day 0 at T=0 at 5 ml/kg 3 9 K35_30kDa (0.3 mg/kg) Concentration: 0.6 mg/ml IV, single dose on day 0 at T=0 at 5 ml/kg Additional 6 N/A N/A Collection of blank samples (outside the time frame) Total 33

The pharmacokinetic properties of an exemplary pegylated IL-2 K35_30kDa compound were evaluated at two dose levels. Lyophilized test drug was reconstituted in PBS and nine male C57BL/6 mice were dosed at 0.3 and 3 mg/kg via intravenous injection into the tail vein for each dose group (see collection details below). Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-dose. The Abcam hIL-2 ELISA kit (ab100566), which is not cross-reactive with native mouse IL-2, was used to detect and quantify test drugs. To correct for ELISA-specific differences in sensitivity between native and PEGylated compounds, native IL-2 and K35_30kDa test drug calibration curves were generated using test drug dilution buffer and the data were analyzed against the corresponding calibration curves. The data plotted represent the mean and SEM of the three individual samples (biological replicates) described above, and the PK parameters for the K35_30kDa test drugs were extracted and summarized in Table 14 .

Table 14 Analyte Unit of measurement Dose 0.3 mg/kg 3 mg/kg Estimated value T _max h. 0,250 0,250 C _max ng/ml 6080 57700 AUC _0-t h.*/ng/ml 38500 425000 R ² 0.994 0.947 IL-2 K35-mPEG30kDa AUC _½extrap % 35.3 37.4 AUC _0-∞ h.*ng/ml 59600 679000 t _1/2 h. 18.2 19.5 C _max /D kg*ng/ml/mg 20300 19200 AUC _0-t /D h.*ng/ml 128000 142000

EXAMPLE 9

Determination of human IL-2R-alpha and IL-2R-beta binding characteristics

A study was performed to determine the binding characteristics of an exemplary IL-2 conjugate IL-2_P65[AzK_L1_PEG30kDa]-1 to human IL-2R-alpha and IL-2R-beta. The IL-2_P65[AzK_L1_PEG30kDa]-1 compound was as previously described and comprised SEQ ID NO: 50 in which the proline at position 64 was replaced with AzK_L1_PEG30kDa, where AzK_L1_PEG30kDa is defined as a structure of formula (IV) or formula (V) or a mixture of formula (IV) and formula (V) and a linear mPEG chain of 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kDa]-1 is also defined as a compound comprising SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX), and wherein n is an integer such that the molecular weight of the PEG group is approximately 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kDa]-1 is also defined as a compound comprising SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII), and wherein n is an integer such that the molecular weight of the PEG group is approximately 30 kDa.

The compound was prepared by methods similar to those disclosed in Example 2, wherein the protein having SEQ ID NO: 3 was first prepared, in which the proline at position 64 was replaced byN6-((2-azidoethoxy)-carbonyl)-L-lysine AzK (SEQ ID NO: 35). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 30 kDa. Briefly, IL-2 used for bioconjugation was expressed as inclusion bodies inE. coliby means of the methods disclosed herein, using (a) an expression plasmid encoding (i) a protein with a desired amino acid sequence, the gene of which contains a first unnatural base pair to ensure the presence of a codon at the desired position into which the unnatural amino acid was insertedN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and (ii) tRNA, obtained from PylM. mazei, the gene of which contains a second unnatural nucleotide to ensure the presence of the corresponding anticodon instead of its native sequence; (b) a plasmid encoding pyrrolizyl-tRNA synthetase obtained fromM. barkeri(MbPylRS), (c) AzK, and (d) a truncated variant of the nucleotide triphosphate transporter PtNTT2 in which the first 65 amino acid residues of the full-length protein have been deleted. The double-stranded oligonucleotide that encodes the amino acid sequence of the IL-2 variant contained the codon AXC as codon 64 in the sequence that encodes the protein having SEQ ID NO: 3, in which P64 is replaced with the unnatural amino acid described herein. The plasmid encoding the orthogonal tRNA gene fromM. mazei, contained a GYT anticodon corresponding to AXC in place of its native sequence, where Y is a non-natural nucleotide disclosed herein. X and Y are selected from the non-natural nucleotides dTPT3 and dNaM disclosed herein. The expressed protein was extracted from the inclusion bodies and refolded using standard procedures prior to site-specific PEGylation of the AzK-containing IL-2 product using copper-free DBCO-mediated click chemistry to stably covalently attach mPEG (a linear methoxy-PEG group having an average molecular weight of 30 kDa) moieties to AzK (as shown in Scheme S6 above).

Test drug samples binding to IL-2Ralpha. Test drug samples in solution were tested for binding to IL-2Ralpha receptor bearing surfaces. Response data were processed by subtracting signals from a reference surface without the receptor as well as the average of the buffer injections using Scrubber-2 (BioLogic Software Pty Ltd). Responses to a series of rhIL-2 concentrations were globally fitted with a 1:1 interaction model including a mass transfer step. A summary of the binding constants is presented in Table 15 .

Table 15 K _D (nM) IL-2R-alpha IL-2R-beta rhIL-2 11 ± 1 0.7 ± 1 IL-2_P65[AzK_L1_PEG30kDa]-1 N.D. 3.1 ± 0.3

Capture of Fc-tagged IL-2R-beta on a protein-coated CM4 sensor chip. The CM4 sensor chip was inserted into a Biacore 4000 optical biosensor and the instrument was initially treated with HBS-P running buffer three times (HBS-P is 1X HBS-N supplemented with 0.005% Tween-20). Coupling with protein A was performed using standard NHS/EDC coupling conditions. IL-2R-beta Fc was dissolved in water to a concentration of 0.1 mg/mL and then diluted 1/1000 with HBS-P running buffer. IL-2R-beta Fc was injected for different periods of time to generate 2 surfaces with different receptor densities (~750 RU and 1500 RU, data not shown).

Characterization of IL-2R-beta binding samples. Samples of test drug in solution were tested for binding to IL-2R-beta receptor-bearing surfaces. Response data were processed by subtracting signals from a reference surface without the receptor as well as the average of the buffer injections using Scrubber-2 (BioLogic Software Pty Ltd). Responses to rhIL-2 (2-fold dilutions of the highest concentration of 4 μM) and IL-2_P65[AzK_L1_PEG30kDa]-1 (2-fold dilutions of the highest concentration of 8 μM) samples, tested in duplicate, were globally approximated by a 1:1 interaction model including a mass transfer step. A summary of the binding constants is presented in Table 15 .

Results: His-tagged IL-2Ralpha was captured at different densities on a nickel-coated NTA sensor chip in the Biacore SPR biosensor system. Fc-tagged IL-2Rbeta was captured at different densities on a protein A-coated CM4 sensor chip. Response data were fitted with a 1:1 interaction model to determine binding constants for each interaction. Recombinant human IL-2 (rhIL-2) bound to IL-2Ralpha with an affinity of ~11 nM, whereas no binding of IL-2_P65[AzK_L1_PEG30kDa]-1 samples to IL-2Ralpha was detected. RhIL-2 bound IL-2R-beta with an affinity of ~700 nM, and IL-2_P65[AzK_L1_PEG30kDa]-1 bound IL-2R-beta with an affinity of ~3 μM under these assay conditions.

EXAMPLE 10

A study was conducted to determine the activity and differential cell type specificity of IL-2_P65[AzK_L1_PEG30kDa]-1 compared to recombinant human interleukin-2 (hIL-2) in terms of phosphorylated form of the transcription factor STAT5 (pSTAT5)-mediated signaling activity in human primary immune cell types. The IL-2_P65[AzK_L1_PEG30kDa]-1 compound was as previously described and comprised SEQ ID NO: 50 wherein the proline at position 64 was replaced with AzK_L1_PEG30kDa, where AzK_L1_PEG30kDa is defined as a structure of formula (IV) or formula (V) or a mixture of formula (IV) and formula (V) and a linear mPEG chain of 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kDa]-1 is also defined as a compound comprising SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX), and wherein n is an integer such that the molecular weight of the PEG group is approximately 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kDa]-1 is also defined as a compound comprising SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII), and wherein n is an integer such that the molecular weight of the PEG group is approximately 30 kDa. The compound was prepared by methods similar to those disclosed in Example 2, wherein a protein having SEQ ID NO: 3 was first prepared in which the proline at position 64 was replaced by N 6-((2-azidoethoxy)-carbonyl)-L-lysine AzK (SEQ ID NO: 35). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 30 kDa.

Methods for processing human PBMC samples. Stock solutions of IL-2 (control, 1 mg/mL) and IL-2_P65[AzK_L1_PEG30kDa]-1 (“batch 1”: 1.27 mg/mL; “batch 2”: 2.29 mg/mL) were stored as stock solutions frozen at -20°C.

Lot 1 and Lot 2 IL-2_P65[AzK_L1_PEG30kDa]-1 compounds were diluted in PBS and IL-2 was diluted with PBS+0.1% BSA to generate 10X stock solutions. The concentration of the 10X IL-2 stock solution was 5 μg/mL, and the concentration of the GLP-1 and GLP-2 stock solutions ranged from 6 to 300 μg/mL depending on the experiment. The 10X stock solutions were diluted in serial 5-fold dilutions to perform 10-point dose titration. The highest dose of IL-2 was 5 μg/mL, and for the Lot 1 and Lot 2 stock solutions, the dose ranged from 6 to 300 μg/mL depending on the experiment. 10 µL of each stock solution was added to 90 µL of cell samples to achieve a final peak dose of 500 ng/mL for IL-2 and 0.6 to 30 µg/mL for each of Batch 1 and Batch 2.

Stimulation of samples. For stimulation, 10 µl of the adjusted dose presented above was added to 90 µl of blood sample pre-equilibrated to 37°C. Samples were incubated at 37°C for 45 minutes. At the end of the incubation period, red blood cells were lysed and cells were simultaneously fixed as follows:

100 µl of cells were transferred to 900 µl of BD Lysis/Fixation Buffer (Beckton Dickinson, Cat #558049) and immediately vortexed. BD Lysis/Fixation was prepared by diluting the stock solution 1:5 with cell culture water immediately before addition. Samples were incubated for 10 min at room temperature, then centrifuged at 450 x g for 5 min to pellet the cells. The pelleted cells were washed with PBS+0.5% BSA and stored at -37°C until analysis.

Staining protocol

Step 1: Cells were thawed at room temperature. Step 2: Fc receptor blocking reagent (TruStain FcX™) was added. Step 3: Incubate at room temperature for 5 minutes. Step 4: The following antibodies from Table 16 were added.

Table 16. Antibodies for the human panel Fluorophore CD4 BUV737 CD56 BV711 CD16 BV711 CD8 BUV805 CD27 BV786 CD45RA BUV395 CD127 FITC CD25 Biotin

Step 5. Incubate for 20 minutes at room temperature. Step 6. Cells were washed twice with PBS+0.5% BSA. Step 7. Cells were permeabilized by adding 10 volumes of methanol per cell volume. Step 8. Cells were incubated for 10 minutes at 4°C. Step 9. Washed with PBS. Step 10. Cells were washed with PBS containing 0.5% BSA. Step 11. Fc receptor blocking reagent (TruStain FcX™) was added. Step 12. The following post-permeabilization staining panel from Table 17 was added.

Table 17. Staining reagents Fluorophore CD3 PE-Cy7 STAT5 Ax647 Streptavidin BV421 FOXp3 P.E.

Flow cytometry and data analysis. Samples were run on a Becton Dickinson Fortessa and LSR II with five lasers (372 nM, 405 nM, 488 nM, 561 nM, and 640 nM). The instruments were equipped with 20 detectors providing scatter parameters. The instruments were calibrated regularly using Becton Dickinson Cytometer Setup & Tracking beads. 96-well plates containing stained samples were analyzed at less than 8,000 cells/sec using a high-throughput 96-well plate sampler.

The data were exported as .fcs files to a network drive and compensated for fluorophore excess and the .fcs files were annotated. The data contained in the .fcs files were then gated. Cells were first gated on singlets using FSC-A through FSC-H to exclude any aggregates or doublets. Within this gate, cells were gated on mid to high forward scatter (FSC-A) and side scatter (SSC-A) to exclude red blood cells, debris and granulocytes. T cells were then gated as the CD3+ CD56/16 negative population in panel ^3. NK cells were identified as the CD3 negative population with high levels of CD56/16 in panel ^3. T cells were then separated into CD4+ T cells and CD8+ T cells. Treg cells were then gated from CD4+ T cells as the CD25 ^hi x C127 ^lo population.

Statistics and plotting for deriving EC50 values. The median fluorescence intensity (MFI) for each cell population, donor, and compound treatment was calculated from the signal in the phosphorylation channel. Statistics were analyzed using Spotfire. In Spotfire, data were plotted on a logarithmic scale for compound doses and a linear scale for MFI readouts. The data were fitted using a 4-parameter logistic regression equation. The EC50 was calculated as the inflection point of the curve.

Results. Human IL-2 and IL-2_P65[AzK_L1_PEG30kDa]-1 samples were diluted and tested in triplicate against each of three individual donors as described above. The calculated half-maximal effective concentration (EC50) values are listed in Table 19. The results demonstrate that IL-2_P65[AzK_L1_PEG30kDa]-1 is a potent agonist of IL-2 receptor signaling in human lymphocytes. Consistent with previous in vitro binding studies that showed that IL-2_P65[AzK_L1_PEG30kDa]-1 specifically interacts with the IL-2Rβ subunit and not with IL2Rα, it demonstrated a specific reduction in signaling activity in Treg cells, whose activity is dependent on interaction with IL-2Rα, compared with Teff and NK cells, which do not have constitutive expression of high levels of IL-2Rα.

Table 18. Activity characteristics of hIL-2 and IL-2_P65[AzK_L1_PEG30kDa]-1 batches against primary CD8+ T cell, NK cell, and Treg cell subsets from human donors EC50 in human cells (ng/ml) Material CD8+ T cells CD8+ memory T cells NK Treg CD8/Treg ratio Human IL-2 12.4 ± 1.29 11.3 ± 1.63 2.88 ± 1.63 0.027 ± 0.005 460 Party 1* 224 ± 25.3 240 ± 27.3 54.7 ± 6.55 114 ± 20.3 2.0 Party 2* 265 ± 12.4 284 ± 18.3 55.7 ± 8.62 132 ± 24.0 2.0

Data represent mean ± SEM calculated from testing triplicate samples from three independent donors.

*IL-2_P65[AzK_L1_PEG30kDa]-1.

EXAMPLE 11

Use of Compound A in Combination with a Checkpoint Inhibitor in the Treatment of CT-26 Tumor-Bearing Balb/c Mice Compound IL-2_P65[AzK_PEG30kDa] (also referred to herein as "P65_30kDa" and in the drawings as "Compound A") was prepared according to the methods disclosed herein by first preparing the protein of SEQ ID NO: 4, in which the proline at position 65 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 10). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a linear methoxy-PEG group having an average molecular weight of 30 kDa to obtain a product having SEQ ID NO: 25, comprising formula (II), formula (III) or a mixture of formula (II) and formula (III), wherein W is a linear methoxy-PEG group having an average molecular weight of 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which proline at position 65 (P65) is replaced by a structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII), and wherein n is an integer such that the PEG group has a molecular weight of approximately 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which proline at position 65 (P65) is replaced by a structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI), and wherein n is an integer such that the PEG group has a molecular weight of approximately 30 kDa.

Studies of compound A as monotherapy and in combination with an anti-PD-1 antibody were performed in female Balb/c mice. For Studies 1 and 2, female Balb/c mice, 6–8 weeks old and weighing an average of 16 g to 21 g, were purchased from Jackson Laboratories (Sacramento, CA). For Study 3, female Balb/c mice, 7–8 weeks old and weighing an average of 18 to 22 g, were purchased for HD Biosciences from Taconic Biosciences. Cryogenically preserved vials of CT-26 colon cancer cells were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). Cells were thawed and cultured according to the manufacturer's protocol. On the day of tumor cell inoculation, cells were washed in serum-free medium, counted, and resuspended in cold serum-free medium at a concentration of 250,000 (studies 1 and 2) or 300,000 (study 3) viable cells per 0.1 ml. CT-26 cells (0.1 ml) were injected subcutaneously into the flanks of individual mice and tumors were allowed to grow.

In Studies 1 and 2, which used a combination of Compound A and an anti-PD-1 antibody, the antibody used was anti-mouse PD-1 (BioXCell; RMP1-14) and the control antibody was an IgG1 isotype antibody (BioXCell; Catalog # BP0089, Lot # 2A3). In Study 3, which used an anti-PD-1 antibody, the antibody used was anti-mouse PD-1 (BioXCell; Catalog # BP0146, RMP1-14, Lot # 695318A1) and the control antibody was an IgG1 isotype antibody (BioXCell; Catalog # BP0089, Lot # 2A3).

Lyophilized compound A was reconstituted as a 10 mg/mL stock solution with 0.1 M acetic acid. It was then further diluted to the working concentration with 1x phosphate-buffered saline (PBS). Compound was reconstituted and diluted 1 hour before dosing animals and kept on ice until dosing. Lyophilized compound was stored at -80°C until use. Vehicle medium was stored at 4°C.

Three separate activities were carried out efficacy studies using CT-26 tumor-bearing Balb/c mice. Study design 1, in which Compound A was evaluated for dose-dependent efficacy as a monotherapy, is presented inTable 19. The designs of studies 2 and 3, which assessed the efficacy of compound A in combination with an anti-PD-1 antibody, are presented intable 20 and Table 21respectively. The route of administration of compound A was intravenous (IV). IV dosing of mice was via the tail vein. The antibody was administered intraperitoneally (IP). All agents were administered based on the individual body weight of each animal, obtained immediately prior to each dose. Details of the dosing regimen are described below.

Table 19. Study 1. Control and test treatment groups for CT-26 tumor-bearing mice Means Dose (mg/kg) Path, scheme Number of mice Control medium carrier 0 IV, QWx3 10 Connection A 0.3 IV, QWx3 10 Connection A 0.3 IV, Q2Wx2 10 Connection A 1 IV, QWx3 10 Connection A 1 IV, Q2Wx2 10 Connection A 3 IV, QWx3 10 Connection A 3 IV, Q2Wx2 10

IV=intravenous; QWx3=once a week for a total of 3 doses; Q2Wx2=once every 2 weeks for a total of 2 doses.

Table 20. Study #2. Control and test treatment groups for CT-26 tumor-bearing mice Means Dose (mg/kg) Path, scheme Number of mice Control medium carrier +
IgG isotype control 0+10 IV, QWx3+IP, BIWx3 14 Connection A 3 IV, QWx3 14 Connection A 6 IV, QWx3 14 Antibody to PD-1 10 IP, BIWx3 14 Compound A+antibody to PD-1 6+10 IV, QWx3+IP, BIWx3 14

BIWx3=twice a week for 3 weeks for a total of 6 doses; IP=intraperitoneal; IV=intravenous; QWx3=once a week for a total of 3 doses.

Table 21. Study #3. Control and test treatment groups for CT-26 tumor-bearing mice Means Dose (mg/kg) Path, scheme Number of mice Control medium carrier+
isotypic
IgG control 0+10 IV, QWx3+IP, BIWx3 14 Connection A 1 IV, QWx3 14 Connection A 3 IV, QWx3 14 Connection A 6 IV, QWx3 14 Connection A 9 IV, QWx3 14 Antibody to PD-1 10 IP, BIWx3 14 Connection A+
Anti-PD-1 antibody 1+10 IV, QWx3+IP, BIWx3 14 Connection A+
Anti-PD-1 antibody 3+10 IV, QWx3+IP, BIWx3 14 Connection A+
Anti-PD-1 antibody 6+10 IV, QWx3+IP, BIWx3 14

BIWx3=twice a week for 3 weeks for a total of 6 doses; IP=intraperitoneal; IV=intravenous; QWx3=once a week for a total of 3 doses; Q2Wx2=once every 2 weeks for a total of 2 doses.

In Study 1, CT-26 tumor-bearing mice were treated with vehicle IV once weekly for a total of 3 doses (QWx3) or compound A at 0.3, 1, or 3 mg/kg IV once weekly for a total of 3 doses (QWx3) or once every 2 weeks for a total of 2 doses (Q2Wx2), beginning on day 4 after tumor cell inoculation, when the average tumor volume was ~80 mm ³ .

In Study 2, CT-26 tumor-bearing mice were treated at day 5 post-tumor cell inoculation, when the mean tumor volume was ~80 mm ³ , and were dosed with vehicle vehicle IV QWx3+IgG isotype control IP, or Compound A at 3 or 6 mg/kg IV on a QWx3 dosing schedule, or anti-PD-1 antibody at 10 mg/kg IP, or a combination of Compound A at 6 mg/kg IV QWx3+anti-PD-1 antibody at 10 mg/kg IP. IP antibody dosing was given twice weekly in all cases for 3 weeks for a total of 6 doses (BIWx3).

In Study 3, CT-26 tumor-bearing mice were treated at day 7 post-tumor cell inoculation, when the mean tumor volume was ~70 mm3. They were dosed with vehicle IV QWx3+IgG isotype control IP BIWx3; or Compound A at 1, 3, 6, or 9 mg/kg IV according to the dosing schedule of QWx3, or anti-PD-1 antibody at 10 mg/kg IP BIWx3; or the combination of Compound A at 1, 3, or 6 mg/kg IV QWx3+anti-PD-1 antibody at 10 mg/kg IP BIWx3.

A summary of all three studies is shown in Table 22. Animals were observed daily for clinical signs. According to IACUC guidelines, animals were humanely euthanized when tumors grew to greater than 2000 ^mm3 in volume, or showed continued deterioration in their condition, or demonstrated obvious signs of severe distress and/or pain.

The survival of each mouse was monitored for over 100 days, after which tumor-free surviving animals from Studies 2 and 3 were enrolled in a follow-up study with restimulation for two cycles 2 months apart. Specifically, tumor-free animals were restimulated with the same tumor cell type (CT-26) inoculated into the contralateral lower flank. Control animals were age-matched, naive mice that were inoculated in parallel with the same number of CT-26 tumor cells into the contralateral lower flank.

Tumor growth was monitored using digital caliper measurements every 3-4 days until the end of the study. Tumor volume was calculated as width ² x length/2, where width is the smallest dimension and length is the largest dimension. Raw tumor volume data are presented in the study reports.

The mean tumor volume data for each group were plotted against time with standard error of the mean (SEM) bars. In addition, individual tumor volume data for the last day before sacrifice were plotted with the mean and SEM bar to examine the distribution of the data.

Statistical analysis of tumor volume data for the last day prior to animal sacrifice was performed using GraphPad Prism v.7.0. Data were analyzed for significance using one-way ANOVA. Pairwise comparisons were performed using Tukey's test procedures (2-tailed). The p-value for each individual comparison is reported.

The percentage of tumor growth inhibition (% TGI) in each treatment group compared to the control group was calculated as follows:

[(control - baseline control) - (treatment - baseline treatment)]/(control - baseline control) x 100%.

The survival of each mouse was recorded and a Kaplan-Meier plot was constructed to show survival by treatment group, and significance was assessed using the log-rank (Cox-Mantel) test. Survival was monitored for over 100 days after the start of treatment in Studies 1, 2, and 3, and for two restimulation cycles in surviving tumor-free mice in Studies 2 and 3. Analyses were performed using GraphPad Prism version 7.0.

Table 22. Inhibition of tumor growth in mice by compound A as monotherapy and in combination with anti-mouse PD-1 antibody Dose of compound A
[antibody dose ^a ]
(mg/kg) % TGI (relative to control vehicle) Study #1 Study #2 Study #3 QWx3 Q2Wx2 QWx3 QWx3 Compound A as a monotherapy 0.3 19 20 - - 1 31 27 - 30 3 51* 45* 56 59*** 6 - - 36* 86*** 9 - - - 85*** Antibody [10] - - 44 ^# 44 ^# Combination therapy ^b 6 + [10] - - 84** 84** ^a BIW doses were administered for 3 weeks (6 doses in total); ^b data for the 1 and 3 mg/kg compound A combination groups are not shown.

% TGI was calculated at day 15 (study 1) and day 17 (studies 2 and 3).

Results represent mean ± SEM.

QWx3=once weekly for a total of 3 doses; Q2Wx2=once every 2 weeks for a total of 2 doses; TGI=tumor growth inhibition. *p < 0.05 versus vehicle control; **p < 0.05 versus monotherapy (compound A or antibody); ***p < 0.001 versus vehicle or isotype control antibody; ^#p < 0.01 versus isotype control antibody.

In Study 1, Compound A was evaluated for dose-dependent efficacy as a monotherapy in female Balb/c mice bearing established subcutaneous colon tumors from CT-26. The study was formally terminated at day 15 post-treatment in accordance with the IACUC humane endpoint, when some tumors in the control group reached a volume greater than 2000 ^mm3 . Figure 1 shows the change in mean tumor volume over time for the QWx3 dose groups of Compound A. Figure 2 shows the tumor volumes at day 15 post-treatment for each animal treated with the QWx3 doses of Compound A. Figure 3 shows the change in mean tumor volume over time for the Q2Wx2 dose groups of Compound A. Figure 4 shows the tumor volumes at day 15 post-treatment for each animal treated with the Q2Wx2 doses of Compound A.

With the QWx3 dosing schedule, Compound A exhibited dose-dependent antitumor activity as a monotherapy, resulting in % TGI compared to vehicle control of 31%, 19%, and 52% for the 0.3, 1, and 3 mg/kg dose groups, respectively. Similarly, with the Q2Wx2 dosing schedule, Compound A exhibited dose-dependent antitumor activity as a monotherapy, resulting in % TGI compared to vehicle control of 20%, 27%, and 45% for the 0.3, 1, and 3 mg/kg dose groups, respectively. However, with both dosing schedules, only the 3 mg/kg dose showed statistical significance (p < 0.05) compared to vehicle control. Both dosing schedules exhibited comparable antitumor activity. Therefore, the QWx3 dosing schedule was chosen for subsequent studies in this mouse model.

In Figs. 1 , 3 , 5 , and 8, black arrows indicate days of Compound A dosing. Data in Figs. 1 and 3 are mean tumor growth curves for QWx3 dosing and Q2Wx2 dosing of Compound A; black arrows indicate days of Compound A dosing. Data in Figs. 2 and 4 represent individual tumor volumes and mean tumor volume ± SEM (10 mice/group) at day 15 post-treatment for QWx3 and Q2Wx2 dosing of Compound A. Data represent individual tumor volumes; mean ± SEM and % TGI compared to vehicle control are also shown.

Data in Fig. 3 represent mean tumor volume ± SEM (10 mice/group) in animals treated with Q2Wx2 Compound A doses. Data in Fig. 4 represent individual and mean tumor volumes at day 15 post-treatment with Q2Wx2 Compound A doses. *p < 0.05 compared to vehicle control at day 15.

Two separate studies (Studies 2 and 3) were conducted in mice bearing CT-26 colon tumors to evaluate Compound A as monotherapy and in combination with an anti-mouse PD-1 checkpoint inhibitor antibody. The dose ranges of Compound A in these studies overlapped, with a wider dose range in Study 3. In both studies, Compound A was administered QWx3 and the antibody was administered at the same dose level BIWx3.

Study 2 assessed the antitumor activity of Compound A alone at 3 and 6 mg/kg (QWx3) in female Balb/c mice bearing established subcutaneous CT-26 colon tumors. Additionally, the antitumor activity of the combination of IV doses of Compound A at 6 mg/kg (QWx3) and anti-PD-1 antibody at 10 mg/kg IP (BIWx3) was assessed. % TGI was calculated at day 15 after the start of treatment, since some tumors in the vehicle control group reached volumes greater than 2000 mm ³ . However, animals in the treatment groups that demonstrated complete tumor regression were monitored by tumor measurements at a frequency of once or twice weekly.

Compound A demonstrated antitumor activity as a single agent, resulting in % TGI compared to vehicle control of 56.3% and 35.6% for the 3 and 6 mg/kg dose groups, respectively. In a combination study, CT-26 tumor-bearing mice were treated IV with Compound A at 6 mg/kg QWx3, IP with anti-PD-1 antibody BIWx3, or the combination at the same dosing schedules, starting 5 days after tumor cell inoculation, when the mean tumor volume was ~80 mm ³ . Figure 5 shows the mean tumor growth curves for mice treated 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. Data in Figure 5 represent mean tumor volume ± SEM (14 mice/group). The upper arrows represent the days of Compound A dosing, and the lower arrows represent the days of anti-PD-1 antibody dosing. The antitumor activity of the combination was significantly increased compared to Compound A or anti-PD-1 antibody alone (p < 0.05). The % TGI data are shown in Fig. 6 and demonstrate significant antitumor effects at day 15 post-treatment in the Compound A and anti-PD-1 antibody combination group compared to the vehicle, Compound A alone, or anti-PD-1 antibody groups (35.6% for the Compound A alone group; 44.1% for the anti-PD-1 alone group; and 74.6% for the Compound A and anti-PD-1 antibody combination group). Data represent individual tumor volumes; mean ± SEM and % TGI compared to vehicle control are also shown. *p < 0.05, **p < 0.01, and ***p <0.01; compared with vehicle control. ^┴ p < 0.05 compared with anti-PD-1 antibody. #p < 0.05 compared with Compound A. The median survival times in the groups are shown in Fig. 7 and were 17, 27, 27.5, and 38 days for the control, Compound A, anti-PD-1 antibody, and Compound A+anti-PD-1 antibody groups, respectively. The median survival time in the combination group was significantly longer than that in the groups treated with either Compound A (p < 0.05) or anti-PD-1 antibody (p < 0.05) as monotherapy. At 98 days post-treatment, only 1 of 14 animals (7%) in each of the Compound A and anti-PD-1 antibody dose groups were tumor-free, whereas 4 of 14 animals (29%) in the combination group were tumor-free. Data in Fig. 7 represent Kaplan-Meier survival curves for treatment groups. *p < 0.05 versus vehicle control. ^┴ p < 0.05 versus anti-PD-1 antibody. #p < 0.05 versus Compound A.

In Study 3, the antitumor activity of Compound A as a single agent was evaluated in female Balb/c mice bearing CT-26 colon SC tumors over a wider dose range (1, 3, 6, and 9 mg/kg) compared to Study 2 using the same IV dosing schedule of QWx3. The data in Fig. 8 represent the mean tumor growth curves for Compound A single agent doses of 1 mg/kg, 3 mg/kg, 6 mg/kg, and 9 mg/kg. Data represent mean tumor volume ± SEM (14 mice/group; except 12 mice/group for 9 mg/kg Compound A). Black arrows indicate the days of compound A dosing. Compound A, dosed alone at 1 mg/kg, 3 mg/kg, 6 mg/kg, and 9 mg/kg, also exhibited dose-dependent antitumor activity, resulting in % TGI compared to vehicle control of 29.8%, 58.8%, 86.2%, and 84.8% for the 1, 3, 6, and 9 mg/kg dose groups, respectively ( Fig. 9 ). % TGI was calculated at day 15 after the start of treatment since some tumors in the vehicle control group reached more than 2000 mm ³ . However, animals in the treatment groups that showed complete tumor regression were monitored by tumor measurements at a frequency of once or twice a week. Data in Fig. 9 represent individual tumor volumes at day 15 after treatment. Data represent individual tumor volumes; Also shown are mean ± SEM and % TGI compared to vehicle control. ***p < 0.01 compared to vehicle control. The lowest dose (1 mg/kg) did not show statistically significant antitumor activity, whereas the remaining 3 dose groups showed statistical significance (p < 0.001) compared to the vehicle treated group. The data also showed that % TGI in the two high dose groups (6 mg/kg and 9 mg/kg) were similar, indicating that maximal antitumor activity was achieved at the 6 mg/kg dose. In the 9 mg/kg dose group, 2 of 14 animals were found dead after losing > 15% of body weight due to treatment.

In the combination phase of the study, Compound A was administered at 1, 3, or 6 mg/kg (QWx3) with anti-PD-1 antibody at 10 mg/kg IP (BIWx3). CT-26 tumor-bearing mice were treated IV with Compound A at 1, 3, 6, or 9 mg/kg QWx3, or IP with anti-PD-1 antibody BIWx3, or the combination at the same dosing schedules, beginning 7 days after tumor inoculation, when the mean tumor volume was ~70 mm3. Of note, in the group receiving 9 mg/kg Compound A monotherapy, two animals were found dead after >15% body weight loss and were not included in the analysis. No additive antitumor activity was observed with the 1 mg/kg Compound A+anti-PD-1 antibody combination based on survival data. Several days after treatment with Compound A, 1 of 14 animals (7%) survived in the anti-PD-1 antibody group, whereas 0 animals survived in the 3 mg/kg monotherapy group. However, in the 3 mg/kg Compound A+anti-PD-1 antibody group, 2 of 14 animals (14%) survived until the days of Compound A administration. As shown in Fig. 10 , the combination of 6 mg/kg Compound A+anti-PD-1 antibody resulted in prolonged survival compared to each monotherapy alone. The median survival times were 21, 35, 24.5, and 49 days for the vehicle control, Compound A (6 mg/kg), anti-PD-1 antibody (10 mg/kg), and Compound A+anti-PD-1 antibody (6 mg/kg Compound A and 10 mg/kg anti-PD-1 antibody) groups, respectively. The median survival time in the combination group was significantly longer than that in the Compound A and anti-PD-1 antibody (p < 0.05) monotherapy groups. Specifically, several days after Compound A treatment, 0 animals in the 6 mg/kg Compound A group were surviving, whereas only 1 of 14 animals (7%) in the anti-PD-1 antibody group were surviving tumor-free. However, 5 of 14 (36%) animals in the combination group were surviving tumor-free (p < 0.05). Data in Fig. 10 represent the Kaplan-Meier survival curves for the treatment groups. *p < 0.05 versus vehicle control. ┴p < 0.05 versus anti-PD-1 antibody. #p < 0.05 versus Compound A.

EXAMPLE 12

Whole blood cytokine release assay

Human whole blood samples from 6 healthy donors were incubated with serial titers of IL-2_P65[AzK_L1_PEG30kDa]-1 (compound B) or IL-2 alone or in combination with PEM (Pembro or pembrolizumab) or NIVO (nivolumab) for 24 h. Cytokines released from whole blood after processing into the supernatant were measured using the U-PLEX Meso Scale Discovery (MSD) six-analyte assay (IFN-γ, IL-4, IL-5, IL-6, IL-8, TNF-α).

Protocol

Blood in heparinized tubes from 6 healthy donors was obtained from the Scripps Research Institute Blood Services (TSRI; San Diego, CA). Specimens were tested on the day of collection; from donors 1–3 on one day and from donors 4–6 on another day.

Human whole blood was incubated with different concentrations of IL-2_P65[AzK_L1_PEG30kDa]-1, as well as 90 μg/mL pembrolizumab or 127 μg/mL nivolumab (expected C _max values of the clinical dose).

Whole blood was first diluted 2-fold with RPMI 1640 medium. A volume of 180 µl of pre-diluted whole blood was seeded into a 96-well tissue culture plate. Then 20 µl of compounds at 10x the final test concentration were added to the assay plate. The test conditions included serial titration of IL-2_P65[AzK_L1_PEG30kDa]-1 (4.5 μg/mL, 1.5 μg/mL, 0.8 μg/mL, 0.45 μg/mL, and 0.2 μg/mL) or IL-2 (0.8 μg/mL, 0.45 μg/mL, 0.2 μg/mL, 0.1 μg/mL, and 0.03 μg/mL) and serial titration of IL-2_P65[AzK_L1_PEG30kDa]-1 or IL-2 in combination with 90 μg/mL PEM or 127 μg/mL NIVO. Additionally, positive and negative controls were included to demonstrate the sensitivity and specificity of the assay: 100 μg/mL Ultra LEAF mouse anti-human CD3 precoated antibody served as positive controls for the assay. 50 μg/mL Ultra LEAF mouse anti-human CD3 precoated IgG1 κ served as an isotype control for the anti-human CD3 antibody mentioned above. IL-2_P65[AzK_L1_PEG30kDa]-1 formulation buffer alone served as a negative control for the assay. No treatment also served as a negative control for the assay.

After adding the compound(s) to the well, the blood was incubated at 37°C. After centrifugation of the samples, the supernatant was collected 24 h after treatment and stored at -80°C until analysis. Human cytokines (IFN-γ, TNF-α, IL-8, IL-6, IL-5, IL-4) released in the assay were quantified using the MSD U-PLEX kit. The lower limit of detection for the assay is listed in the table below. The limit of detection for each plate is reported in the MSD Discovery Workbench software. The limit of detection varies slightly for each plate.

Materials

Table 23. Used materials and sources Material Supplier Catalogue No. Compound Initial solution IL-2_P65[AzK_L1_PEG30kDa]-1, trial series 2, No. 0590-164 Cytovance N/A Buffer for compilation 2.18 mg/ml Buffer for formulation: 10 mM histidine, pH 6.0, 5% sorbitol, 0.01% polysorbate 80 Cytovance N/A N/A N/A IL-2 R&D Systems 202-IL-500 0.1 M acetic acid 1 mg/ml BioLegend Ultra-LEAF™ Purified Human CD3 Antibody (UCHT1) BioLegend 300438 PBS 1 mg/ml Ultra-LEAF™ BioLegend Purified Mouse IgG1, κ Isotype Control (MOPC-21) BioLegend 400166 PBS 1 mg/ml Pembrolizumab (PEM) SelleckChem A200502 PBS 9.97 mg/ml Purified recombinant human IgG4 isotype control antibody Ultra-LEAF™ BioLegend 403702 PBS 1 mg/ml Group 1 of U-PLEX biomarkers (human IFN-γ, TNF-α, IL-10, IL-6, IL-4, IL-5) Meso Scale Discovery (MSD) K15067L-2 N/A N/A RPMI 1640 Gibco™, HEPES Gibco™ 22400089 Falcon™ Polystyrene Microplates, 96-Well, TC Treated Corning 353072

Results

L-2_P65[AzK_L1_PEG30kDa]-1 and IL-2 induced a dose-dependent induction of IFN-γ upon 24-hour treatment, but did not induce significant release of the other 5 cytokines (IL4, IL5, IL8, TNFa, IL6) tested in this assay. In combination with either Pem or Nivo, both IL-2_P65[AzK_L1_PEG30kDa]-1 and IL-2 (R&D) did not significantly alter the profile of cytokines (IFN-γ, IL-4, IL-5, IL-8, TNF-α, IL-6) tested in this assay. in this study. Individual donor data are provided intables 24-32Illustrative graphs for one donor are shown inFig. 11A-11B.

Table 24. Control conditions for IFN-γ and IL-6 levels Processing Concentration, µg/ml IFN-γ IL-6 D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 Buffer for compound B N/A 43.91 8.77 7.75 9.31 268.77 13.03 2.77 0.68 1.31 0.49 5.60 0.73 Without treatment (culture medium) N/A 21.58 5.12 8.35 7.03 288.70 11.78 0.93 0.31 0.99 2.23 4.53 1.28 Isotype control IgG4 50 33,42 29.17 7.86 12.79 262.93 10.33 4.73 0.47 1.50 0.56 8.54 21:30 Mouse IgG1, κ 100 21.75 BDL 7.19 6.44 94.74 132.02 1.64 0.88 0.89 0.71 4.27 1.85 PEM 90 52.78 9.62 8.80 11.71 301.71 10.08 1.09 0.39 0.92 0.65 8.95 1.06 NIVO 127 29.77 9.34 6.61 7.56 256.75 9.51 1.28 0.48 1.09 0.59 3.29 0.62 Coating antibody to CD3 100 13095,85 110278,12 2890,41 12415,49 46221,98 137695,00 279.20 106.57 20.18 66.79 452.34 1414.77

BDL=below detection limit.

Table 25. Control conditions for IL-5 and IL-8 levels Processing Concentration,
mcg/ml IL-8 TNF-α D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 Buffer for compound B N/A 48.07 8.77 39.07 39.28 143.62 30.69 4.17 0.68 1,2 1 5.82 1.04 Without treatment (culture medium) N/A 20.56 8.77 7.84 27.93 78.91 23.45 3.03 0.68 1.24 1.11 5.61 1.25 Isotype control IgG4 50 37.11 12.77 9.58 24.88 91.61 BDL 2.66 1.33 1.82 0.75 6.58 0.91 Mouse IgG1, κ 100 20.37 7.42 7.92 22.37 57.12 24.8 1.42 0.27 0.14 0.69 3.44 BDL PEM 90 22.01 8.48 11.36 23.29 95.5 28.32 2.33 0.68 1.67 0.97 5.88 1.43 NIVO 127 20.59 7.68 7.95 22.61 67.38 19.68 2.35 0.44 1.33 1.21 4.89 BDL Coating antibody to CD3 100 5211,87 1780.7 1346.96 4031.78 5306.3 10117,33 475.92 2519.34 136.1 894.5 1187,77 3780,63

BDL=below detection limit.

Table 26. Control conditions for TNF-α and IL-4 levels Processing Concentration,
mcg/ml IL-5 IL-4 D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 Buffer for compound B N/A 0.71 0.06 0.59 BDL BDL BDL 0.05 0.06 0.08 0.09 BDL BDL Without processing
(culture medium) N/A 0.81 0.14 0.87 BDL BDL BDL 0.11 0.06 0.11 0.05 BDL BDL Isotypic
IgG4 control 50 0.26 0.23 0.64 BDL 0.77 BDL 0.05 0.12 BDL 0.12 BDL BDL Mouse IgG1, κ 100 0.66 0.09 0.56 BDL BDL BDL 0.04 0.14 0.16 BDL BDL 0.09 PEM 90 1.03 0.72 0.79 BDL BDL BDL 0.10 0.03 0.03 0.09 BDL BDL NIVO 127 2.65 1.50 0.91 BDL BDL BDL 0.11 0.11 0.05 0.10 BDL BDL Coating antibody to CD3 100 34.49 308.57 7.64 31.81 137.92 656.77 11.33 47.81 1.33 11.65 18.59 82.57

BDL=below detection limit.

Table 27. Effect of IL-2 treatment on IFN-γ and IL-6 levels Processing Concentration, µg/ml IFN-γ IL-6 D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 IL-2 0.8 115.31 290.42 314.96 81.72 61.31 850.98 22.76 17.26 10.13 19.67 17.12 51.15 IL-2 0.45 105.72 352.81 331.98 85.34 58.16 433,00 18.47 11.07 7.20 12.82 18.02 65.04 IL-2 0.2 104.20 228.19 278.17 104.40 51.97 437.67 15.93 5.48 4.12 6.96 12.56 49.09 IL-2 0,1 95.61 135.05 224.27 68.31 47,44 387.76 13.48 2.66 2.73 4.02 10.94 28.98 IL-2 0.03 64.49 62.13 81.74 45.09 39.50 170.85 7.89 1.07 1.35 2.31 9.18 16.60 IL-2+Pembro 0.8 138.79 274.43 318.86 117.65 51.99 437.11 27.72 17.63 10.91 22.39 32.49 50.58 IL-2+Pembro 0.45 107.58 247.26 354.82 111.66 55.99 430.47 23.19 11.71 7.45 31,52 15.11 55.85 IL-2+Pembro 0.2 112,00 231.40 283.69 76.41 64.28 244.58 20.84 6.54 4.93 7.65 14.72 29.07 IL-2+Pembro 0,1 99.87 151.13 241.42 106.24 50.17 274.30 16.91 3.10 3.44 13.06 13.48 28.87 IL-2+Pembro 0.03 76.19 44,44 87.79 40.85 38.04 131.98 11.01 1.46 1.47 2.10 9.98 15.04 IL-2+Nivo 0.8 112.83 245.07 396.28 67.50 53.02 344.33 24,21 17.31 12.10 19.46 16.29 50.22 IL-2+Nivo 0.45 98.79 262.60 335.91 66.15 47.95 457.22 17.85 13.65 7.85 11.72 13.01 40.96 IL-2+Nivo 0.2 101.09 204.58 260.76 59.56 46.18 238.28 17.54 6.37 4.23 6.38 13.08 30.47 IL-2+Nivo 0,1 100.69 423.63 230.39 92.71 37.74 206.75 2.71 3.10 3.98 9.98 9.74 20.84 IL-2+Nivo 0.03 72.59 40.13 76.36 N.D. 38.22 49.74 8.23 1.22 1.44 29.61 8.51 14.21

Data for each donor (e.g. D1-D6) were normalized to its specific value with a compilation buffer. ND=not determined.

Table 28. Effect of IL-2 treatment on IL-8 and IFN-α levels Processing Concentration, µg/ml IL-8 TNF-α D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 IL-2 0.8 5.88 6.00 4.53 2.28 5.80 25.94 5.97 14.38 13.84 4.88 16.45 24.31 IL-2 0.45 4.67 5.98 4.98 2.02 5.32 36.29 5.04 14.83 13.27 4.48 16.22 37.41 IL-2 0.2 4.34 5.42 4.01 1.88 4.89 27,24 5.10 12.68 11.74 4.18 15,14 26.06 IL-2 0,1 3.80 5.00 3.76 1.60 4.44 17.45 4.60 11.07 10.68 3.16 12.89 16.94 IL-2 0.03 2.70 2.50 1.42 1.38 3.95 11,11 3.52 3.88 4.45 2.55 10.86 12.96 IL-2+Pembro 0.8 7.00 6.95 4.83 2.97 7.84 24.43 6.02 17.35 13.48 6.42 16.66 23.88 IL-2+Pembro 0.45 5.77 6.41 4.43 3.71 5.86 27.94 5.46 17.44 12.60 6.33 15.51 25.44 IL-2+Pembro 0.2 5.37 6.73 3.59 2.24 6.01 15.52 5.20 15.55 11.03 3.54 15.96 17,18 IL-2+Pembro 0,1 5.39 4.85 3.08 2.73 5.47 18.57 4.85 11.49 10.45 5.06 13.80 16.99 IL-2+Pembro 0.03 3.60 2.28 1.52 1.65 4.72 8.60 4.24 4.94 4.19 2.82 11.29 8.91 IL-2+Nivo 0.8 6.66 6.26 4.31 2.02 6.22 26.11 5.71 17.11 13.39 4.52 14.18 26.91 IL-2+Nivo 0.45 5.06 6.42 4.04 1.80 5.28 22.75 5.35 15.39 12.10 4.61 13.60 22.81 IL-2+Nivo 0.2 4.09 4.96 3.16 1.49 4.82 19.00 4.97 11.83 9.17 3.23 12.57 17,17 IL-2+Nivo 0,1 3.80 4.31 2.97 3.04 5.09 12.19 4.33 10.74 7.90 5.14 11.43 11.44 IL-2+Nivo 0.03 2.28 1.88 1.03 2.86 3.65 8.55 3.35 4.04 3.60 5.41 10.15 9.42

Data for each donor (e.g. D1-D6) were normalized to its specific value with a compilation buffer.

Table 29. Effect of IL-2 treatment on IL-5 and IL-4 levels Processing Concentration, µg/ml IL-5 IL-4 D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 IL-2 0.8 9.45 6.04 4.34 0.71 10.86 22,27 0.25 0.21 0.21 0.13 0.85 1.02 IL-2 0.45 10.57 11.00 5.85 BDL 15.40 22.08 0.14 0.34 0.32 0.08 0.68 0.89 IL-2 0.2 14.53 8.79 3.46 0.93 12.29 28.22 0.30 0.42 0.28 0.10 0.77 1.05 IL-2 0,1 5.24 6.79 3.85 0.26 9.57 23.02 0.21 0.32 0.14 0.09 0.75 0.83 IL-2 0.03 7.56 3.73 1.35 0.66 22.70 13.05 0.27 0.18 0.15 0.14 0.74 0.52 IL-2+Pembro 0.8 5.62 8.14 4.45 1.55 14.17 24.07 0.24 0.35 0.33 0.28 0.78 1.15 IL-2+Pembro 0.45 4.54 7.16 3.00 0.93 9.82 21.83 0.16 0.39 0.27 0.26 0.70 1.20 IL-2+Pembro 0.2 8.95 11.58 6.50 0.56 12.53 15.88 0.31 0.31 0.47 0.12 0.82 0.76 IL-2+Pembro 0,1 7.42 7.03 1.90 BDL 8.53 23.41 0.21 0.27 0.36 0.12 0.64 0.76 IL-2+Pembro 0.03 5.87 2.86 2.05 0.42 14.25 8.53 0.17 BDL 0.13 0.08 0.82 0.51 IL-2+Nivo 0.8 7.16 7.98 4.18 1.38 20.46 20.83 0.22 0.25 0.44 0.14 1.00 1.21 IL-2+Nivo 0.45 7.14 7.05 4.22 BDL 11.55 18.52 0.21 0.31 0.28 0.17 0.54 0.88 IL-2+Nivo 0.2 7.32 7.45 4.18 1.97 12.97 20.73 0.21 0.34 0.30 0.25 0.74 0.75 IL-2+Nivo 0,1 8.67 5.37 3.43 3.04 10.46 16.09 0.26 0.21 0.18 0.14 0.61 0.62 IL-2+Nivo 0.03 3.77 2.02 1.37 BDL 11.82 12.10 0.17 BDL 0.11 0.19 0.74 0.63

BDL=below detection limit.

Table 30. Effect of Compound B (IL-2_P65[AzK_L1_PEG30kDa]-1) Treatment on IFN-γ and IL-6 Levels Processing Concentration
(mcg/
ml) IFN-γ IL-6 D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 Connection B 4.5 213.24 135.44 233.77 75.99 36.04 611.22 39.77 0.56 2.49 4.94 10.61 38.95 Connection B 1.5 93.02 96.56 195.89 49.83 31 122.98 11.82 0.84 1.82 1.87 8.33 16.01 Connection B 0.8 66.82 53.46 119.28 44.78 26.93 92.69 9.38 0.43 1.45 1.71 7.56 11.61 Connection B 0.45 49.07 35.17 85.18 33.58 21.99 74.08 7.87 0.74 1.06 1.29 6.06 3.41 Connection B 0.2 32.02 12.82 32.15 27.63 14.39 21.95 4.50 BDL 0.91 1.11 3.84 1.09 B+Pembro connection 4.5 102.42 164.69 263.89 68.16 38.09 275.34 15.12 1.79 3.26 3.03 10.86 36,39 B+Pembro connection 1.5 80.56 82.28 161.63 49.21 35.01 98.36 11.90 1.29 1.96 3.01 11.07 9.88 B+Pembro connection 0.8 74.09 43.39 130.28 38.2 29.55 62.73 12.28 0.82 1.71 1.31 9.23 6.49 B+Pembro connection 0.45 46.77 27.81 86.44 31.51 19.78 37.93 7.53 0.46 1.17 1.52 6.70 1.78 B+Pembro connection 0.2 29.43 8.62 25.97 20.27 14.52 17.75 5.59 BDL 0.74 0.93 5.04 0.86 B+Nivo connection 4.5 91.59 126.64 269.98 59.66 38.57 901,52 11.56 1.33 4.23 2.83 9.24 34.57 B+Nivo connection 1.5 82.94 71.26 185.79 50.31 30.37 210.47 10.75 1.04 1.97 1.40 7.14 11.44 B+Nivo connection 0.8 59.41 67.83 124.17 30.37 24,23 41.88 7.60 7.56 1.56 0.87 6.38 2.36 B+Nivo connection 0.45 51.93 21.19 84.35 26.44 18.76 30.17 7.73 0.48 1.17 1.33 4.39 2.01 B+Nivo connection 0.2 32.55 7 25.07 16.26 12.1 18.32 4.57 BDL 0.71 1.06 2.59 0.84

"Compound B" is IL-2_P65[AzK_L1_PEG30kDa]-1. Data for each donor (e.g. D1-D6) was normalized to its specific value with compilation buffer. BDL=below detection limit.

Table 31. Effect of Compound B (IL-2_P65[AzK_L1_PEG30kDa]-1) Treatment on IL-8 and TNF-α Levels Processing Concentration
(mcg/ml) IL-8 TNF- α D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 Connection B 4.5 7.67 4.09 2.99 2.18 4.24 20.12 6.23 11.39 10.42 4.96 12.33 26.03 Connection B 1.5 2.83 3.87 3.09 1.66 3.64 11.46 4.43 8.33 8.80 3.30 10.85 13.94 Connection B 0.8 3.08 2.86 2.20 1.36 3.30 6.84 4.16 6.48 6.68 2.92 9.94 8.02 Connection B 0.45 2.21 2.61 1.43 1.42 2.69 3.50 3.56 5.28 4.56 3.12 8.20 5.30 Connection B 0.2 1.48 1.60 0.71 1.27 2.22 1.30 2.64 3.10 2.84 2.24 6.23 2.00 B+Pembro connection 4.5 4.19 4.64 2.61 2.13 4.65 21.72 5.10 11,12 8.91 4.49 12.42 20.50 B+Pembro connection 1.5 4.10 3.46 2.79 2.66 4.45 15.57 4.87 8.68 8.24 3.80 12.07 15.39 B+Pembro connection 0.8 3.36 2.80 2.10 1.53 4.13 5.51 4.41 6.88 6.21 3.15 10.56 6.04 B+Pembro connection 0.45 2.35 2.19 1.58 1.69 3.21 2.09 3.51 4.09 4.89 2.52 8.33 3.46 B+Pembro connection 0.2 1.66 1.41 0.62 1.20 2.46 1.23 2.97 2.59 2.41 2.69 6.18 2.09 B+Nivo connection 4.5 3.97 4.16 3.22 1.63 4.05 16.97 5.36 9.71 9.57 3.83 12.26 21.81 B+Nivo connection 1.5 2.86 2.62 2.16 1.42 3.70 6.78 4.84 6.45 7.11 3.15 10.53 9.76 B+Nivo connection 0.8 2.97 2.55 1.97 1.19 3.56 1.99 3.95 5.33 5.87 2.87 8.88 3.17 B+Nivo connection 0.45 2.15 1.72 1.19 1.10 2.53 2.05 3.66 3.88 3.94 2.86 7.22 3.38 B+Nivo connection 0.2 1.45 1.30 0.59 0.91 1.86 0.90 2.67 2.35 2.21 1.88 4.85 2.31

"Compound B" is IL-2_P65[AzK_L1_PEG30kDa]-1. Data for each donor (e.g. D1-D6) were normalized to its specific value with the composing buffer.

Table 32. Effect of Compound B (IL-2_P65[AzK_L1_PEG30kDa]-1) Treatment on IL-5 and IL-4 Levels Processing Concentration
(mcg/ml) IL-5 IL-4 D1 D2 D3 D4 D5 D6 D1 D2 D3 D4 D5 D6 Connection B 4.5 6.39 6.87 1.39 0.91 8.54 31.80 0.18 0.12 0.18 0.21 0.44 1.07 Connection B 1.5 3.73 5.22 1.57 1.02 8.15 12.23 0.20 0.13 0.16 0.14 0.60 0.43 Connection B 0.8 8.67 2.03 1.51 BDL 3.16 4.73 0.20 BDL BDL 0.06 0.20 0.25 Connection B 0.45 3.63 1.17 1.03 BDL 1.26 5.18 0.08 0.14 0.10 0.11 0.29 0.25 Connection B 0.2 0.66 BDL BDL BDL 0.31 0.27 0.10 0.08 0.12 0.07 0.04 BDL B+Pembro connection 4.5 7.22 8.02 2.77 0.74 9.13 27.48 0.26 0.30 0.20 0.14 0.59 1.10 B+Pembro connection 1.5 3.91 4.10 1.36 BDL 7.45 5.54 0.21 0.13 0.16 0.09 0.59 0.41 B+Pembro connection 0.8 4.74 6.45 0.92 BDL 5.71 2.69 0.18 0.10 0.11 0.11 0.48 0.15 B+Pembro connection 0.45 2.54 0.59 0.82 BDL 2.69 BDL 0.17 0.08 BDL 0.07 0.30 0.08 B+Pembro connection 0.2 0.84 BDL 0.71 BDL 0.33 0.35 0.14 BDL BDL 0.09 0.03 BDL B+Nivo connection 4.5 4.76 5.65 3.54 0.20 9.21 20.27 0.23 0.27 0.24 0.15 0.74 1.00 B+Nivo connection 1.5 3.66 2.11 1.41 0.36 9.57 5.42 0.20 0.09 0.17 0.16 0.69 0.30 B+Nivo connection 0.8 3.20 BDL 0.90 BDL 3.96 1.10 0.21 BDL 0.14 0.09 0.34 0.07 B+Nivo connection 0.45 5.11 BDL 1.03 0.11 2.08 1.06 0.16 BDL 0.13 0.08 0.20 0.07 B+Nivo connection 0.2 2.83 0.60 0.73 BDL 1.97 BDL 0.10 0.14 BDL 0.05 0.07 BDL

"Compound B" is IL-2_P65[AzK_L1_PEG30kDa]-1. BDL=below detection limit.

EXAMPLE 13

Mixed human allogeneic lymphocyte reaction (MLR) assay

A human allogeneic lymphocyte mixed cell reaction (MLR) assay was used to evaluate the ability of compound B (IL-2_P65[AzK_L1_PEG30kDa]-1) to enhance TCR activation as a monotherapy and in combination with a checkpoint inhibitor (nivolumab or pembrolizumab) as a model of the cytolytic response to tumor cells presenting specific antigens or neoantigens.

Table 33. Materials Material Supplier Catalogue No. Compound Concentration of the initial solution Compound B (IL-2_P65[AzK_L1_PEG30kDa]-1) Synthorx Batch No. 0590-187 Composing buffer (see below) 2 mg/ml Buffer for formulation: 10 mM histidine, pH 6.0, 5% sorbitol, 0.01% polysorbate 80 Cytovance N/A N/A N/A Nivolumab (Nivo) SelleckChem A2002 PBS 5 mg/ml Nivolumab Bristol-Meyers Squibb NDC 0003-3772-11 N/A 10 mg/ml Pembrolizumab Merck & Co. NDC 0006-3026-01 N/A 25 mg/ml Purified recombinant human IgG4 isotype control antibody Ultra-LEAF™ BioLegend 403702 PBS 1 mg/ml EasySep Human CD4+ T Cell Isolation Kit StemCell 17952 N/A N/A EasySep Human Monocyte Isolation Kit StemCell 19359 N/A N/A Human IFN-γ ELISA Kit Abcam Ab46025 N/A N/A

Table 34. Materials, continued Ingredient Supplier Catalogue No. Concentration of the initial solution Working concentration Medium for transdifferentiation into monocytic DC BioWhittaker X-VIVO15 serum-free hematopoietic cell culture medium Lonza 04418Q N/A N/A FBS Corning 35011CV 100% 5% Penicillin-streptomycin Fisher Scientific 15140122 100% 1% Solution of replaceable amino acids MEM Gibco 11140-050 100x 1x Sodium pyruvate Gibco 11360-070 100 mM 1 mM HEPES Gibco 15630080 1 M 10 mM Recombinant human IL-4 protein (/CF) R&D 204-IL-050 1.5e+6 IU/ml 1500 IU/ml Recombinant human GM-CSF protein (/CF) R&D 215-GM-050 1.5e+6 IU/ml 1500 IU/ml MLR co-culture medium RPMI 1640, HEPES medium Gibco 22400089 N/A N/A FBS Corning 35011CV 100% 5% Penicillin-streptomycin Fisher Scientific 15140122 100% 1% Solution of replaceable amino acids MEM Gibco 11140-050 100x 1x Sodium pyruvate Gibco 11360-070 100 mM 1 mM HEPES Gibco 15630080 1 M 10 mM 2-Mercaptoethanol Gibco 21985023 50 mM 50 µM

Peripheral blood mononuclear cells (PBMCs) from two normal healthy donors were used for each assay run. Monocytic DCs were generated by culturing monocytes isolated from PBMCs using a monocyte negative selection kit (StemCell) in vitro for 7 days, which included the addition of 1500 IU/ml IL-4 and 1500 IU/ml GM-CSF. The culture medium was replaced on days 3 and 5. Monocytic DCs were harvested on day 7. On this day, CD4+ T cells were isolated from another donor using a negative selection kit (StemCell). CD4+ T cells (1 × 105) and allogeneic monocytic DCs (1 × 104) were co-cultured in 96-well microtiter plates in the presence of a range of concentrations of compound B (0.005-100 μg/mL) alone or with 5, 50, or 500 ng/mL nivolumab, pembrolizumab, or an IgG isotype control. Each run was performed in triplicate for each treatment condition. After 5 days of co-culture, IFN-γ secretion in culture supernatants was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam; Cat. #Ab46025). IFN-γ levels following exposure to the combination of compound B and pembrolizumab are shown in Fig. 12 . IFN-γ levels following exposure to the combination of compound B and nivolumab are shown in Figs. 13 and 14 . Clinical grade nivolumab was used to generate the data shown in Fig. 13 , and research grade nivolumab (SelleckChem) was used to generate the data shown in Fig. 14 . The results demonstrated that compound B, nivolumab, and pembrolizumab, when administered as monotherapy, induced IFN-γ release in a concentration-dependent manner. Surprisingly, the combination of (a) compound B and nivolumab and (b) compound B and pembrolizumab showed a synergistic effect in the MLR assay. The data shown in Figs. 12 , 13 , and 14 represent the mean of 3 replicates ± SEM from one donor pair.

EXAMPLE 14

Compound B induces Ki67 expression in CD8+ T, NK, and Treg cells, but results in expansion of only CD8+ T and NK cells, but not Treg, in peripheral blood and CT-26 tumors

To evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of compound B, female Balb/c mice (6-8 weeks old, average weight 16-22 g, Jackson Laboratories or Taconic Biosciences) were inoculated subcutaneously in the flank region with CT-26 tumor cells (ATCC). Tumor growth was monitored by measuring tumors three times per week. When tumor volumes reached approximately 150 mm ³ , mice were randomized to control or treatment groups. Following IV administration of a single 3 mg/kg dose of Compound B to CT-26 tumor-bearing mice, terminal blood and tumor samples were collected at day 0 (2 h, 8 h, and 12 h), day 1 (N=3 mice at each time point), days 2, 3, 5, 7, 10, and 12 days post-dose (N=4 mice at each time point). Plasma and tumor samples were analyzed for Compound B using an ELISA assay.

Pharmacokinetic analysis

The tumor was divided into two halves, and one half was weighed and frozen in liquid nitrogen for tumor PK analysis. Frozen tumor samples were homogenized with lysis buffer (1 protease inhibitor tablet (SIGMA, cat# 4693159001) in 10 ml 1x PBS). Each 0.1 g of tissue was mixed with 0.4 ml of lysis buffer. A 5 mm stainless steel bead (Qiagen, cat# 69989) was added to each tissue collection tube before homogenization with a TissueLyser II (Qiagen) at 20 Hz for 20 sec. After homogenization, the tumor lysate was pelleted by centrifugation and the supernatant was collected for compound B PK analysis.

As shown inFig. 15, the tumor exposure of compound B was approximately 4% of the plasma exposure (AUC_0-t 429,000 and 15,900 h•ng/ml for blood plasma and tumor, respectively). t_1/2in the tumor it was almost twice as high as t_1/2in blood plasma (19.9 hours and 9.8 hours in the tumor and blood plasma, respectively), indicating that compound B is distributed in the tumor and persists there for a longer period of time compared to the blood compartment.

Pharmacodynamic analysis

Flow cytometry. For whole blood immune cell phenotyping, blood samples were lysed and fixed immediately after terminal blood collection. Briefly, blood samples were treated with 20 volumes of pre-warmed 1x Lysis/Fixation Buffer (BD Phosflow™, Cat. #558049) according to the manufacturer’s protocol. Prior to antibody staining, cell suspensions were blocked with Fc receptor blocking reagent (TruStain FcX anti-CD16/32 antibody, BioLegend). After blocking, cells were first stained for the following cell surface markers: anti-mouse CD3 conjugated to Ax488 (17A2), anti-mouse CD4 conjugated to Bv786 (RM4-5), anti-mouse CD8a conjugated to Bv711 (53-6.7), anti-mouse CD49b conjugated to Bv421 (DX5), biotinylated anti-mouse CD25 (REA568), and anti-mouse CD335 conjugated to Bv605 (29A1.4). Cells were then permeabilized with 4°C pre-chilled methanol (Fisher Chemical, A412-4) and intracellularly stained with PE-conjugated anti-mouse FoxP3 (FJK-16s), PerCP-eFluor710-conjugated anti-Ki67, Ax647-conjugated anti-Pstat5 (Py694), PEcy7-conjugated anti-CD44 (IM7), and BUV395-streptavidin conjugate for biotin. Samples were read using BD LSRFortessa and analyzed using FlowJo software.

CD8 cells were identified as CD3+CD8a+. Treg cells were identified as CD3+CD4+CD25+FoxP3+. Natural killer cells were gated at CD3-CD335+CD49b+. CD8 memory cells were scored as CD3+CD8+CD44 ^hi .

FACS tumors. Single cell suspensions of mouse tumor samples were generated by mincing tumors into small fragments and digesting them using the MACS Mouse Tumor Dissociation Kit (Miltenyi, 130-096-730) according to the manufacturer's protocol. Viable cells were identified using eFluor 780 Viability Capture Dye (eBioscience, 65-0865-14). Cell suspensions were blocked with anti-mouse CD16/32 antibody (TruStain FcX, BioLegend, Cat# 101319) and stained for cell surface markers. Cells were then fixed and permeabilized with FoxP3/transcription factor fixation/permeabilization and staining reagent (eBioscience, Cat #00-5521-00) and then stained for intracellular markers. The antibodies used for surface antigens were anti-mouse CD45 antibody conjugated with PEcy7 (30-F11), anti-mouse CD3e antibody conjugated with BUV395 (17A2), anti-mouse CD4 antibody conjugated with BV510 (GK1.5), anti-mouse CD8a antibody conjugated with PE-eF610 (53-6.7), anti-mouse CD335 antibody conjugated with BV605 (29A1.4), anti-mouse CD25 antibody conjugated with AF700 (PC61), anti-mouse CD49b antibody conjugated with APC (DX5). Antibodies for intracellular antigens were PE-conjugated anti-FoxP3 (FJK-16s) and AF488-conjugated anti-Ki67 (11F6). The CD8 cell population was identified as CD3+CD8+, and the NK cell population was defined as CD3-CD335+CD49b+. Treg cells were gated on CD3+CD4+CD25+FoxP3+. Events were recorded using BD LSRFortessa and analyzed using FlowJo software.

Results: Blood and tumor samples were analyzed for PD indices in cell subsets (CD8+ T, NK, and Treg cells), including levels of intracellular phosphorylated STAT5 (pSTAT5, a marker of receptor occupancy and early signaling), Ki67 (a marker of cell proliferation), and the number of CD8+ T, NK, and Treg cells. pSTAT5 in different cell types was measured in blood only.

CT-26 tumor-bearing mice treated with 3 mg/kg compound B demonstrated robust induction of pSTAT5 in peripheral blood CD8+ T cell, CD8+ memory T cell, NK cell, and Treg cell populations. The percentage of pSTAT5+ cells among peripheral blood CD8+ T cells ( Fig. 16A ) and memory CD8+ T cells ( Fig. 16B ) peaked at 2 h post-dose, remained elevated until approximately 48 h, and returned to baseline at 72 h. Among NK cells ( Fig. 16C ), the percentage of pSTAT5+ cells gradually increased after dosing, peaked at 48 h, and returned to baseline at 120 h. Induction of pSTAT5+ in Tregs ( Fig. 16D ) followed a pattern similar to that in CD8+ T cells.

Following pSTAT5 induction, Compound B induced significant Ki67 upregulation in all three cell populations (CD8+ T, NK, and Treg cells) to a similar extent from day 1 to day 7 (p < 0.05) before returning to vehicle control levels at day 10 ( Fig. 17A–17F ). As shown in Fig. 17A and Fig. 17B , Ki67 upregulation by Compound B resulted in significant CD8+ T cell proliferative responses from days 3 to 12 (p < 0.05 compared to control). Phenotypic analysis of CD8+ T cells revealed significant expansion of CD44+ memory cells in this population during the same time period. In contrast to CD8+ T cells, Compound B induced maximal NK cell expansion at 3 days post-dose, which remained elevated at day 5 (p < 0.05 vs. control) before returning to vehicle control treatment levels at day 7 ( Fig. 17C and Fig. 17D ). In contrast to CD8+ for both T and NK cells, Compound B induced only a very transient expansion of Treg cells at a significantly reduced level (only 2.5% vs. 15-25% for CD8+ T and NK cells) at day 3 post-dose ( Figs. 17E and 17F ). The expansion of CD8+ T cells and the absence of significant expansion of the CD4+ Treg cell subset resulted in a progressive increase in the CD8+ T/Treg ratio, peaking at day 7 in the 3 mg/kg dose group ( Fig. 17G ).

Analysis of tumor samples revealed that 7 days after treatment with Compound B, both CD8+ T cells and NK cells were significantly expanded in the tumor (p < 0.05 vs. control), and their numbers remained elevated until day 10 ( Fig. 18A-18B ). However, in response to Compound B, the Treg cell population in the tumor did not show significant expansion compared to vehicle over time ( Fig. 18C ) . The expansion of CD8+ T cells and the lack of expansion of the CD4+ Treg subset resulted in a progressive increase in the CD8+ T/Treg ratio, peaking at day 7 in the 3 mg/kg dose group ( Fig. 18D ).

Summary: In CT-26 tumor-bearing mice, Compound B at 3 mg/kg induced pSTAT5 activation in peripheral blood in all immune cell types, including CD8+ T, CD8+ memory, NK, and Treg cells, indicating engagement of the IL-2Rβ/γ receptor complex. Additionally, Compound B induced the proliferation marker Ki-67 in all of these cell types at this dose, but proliferation was observed only in CD8+ T and NK cells. This resulted in a CD8/Treg ratio of approximately 20 at this dose in peripheral blood. Although Compound B exposure in tumors was approximately 4% of exposure in plasma, it persisted in the tumor for twice as long, resulting in a CD8 T/Treg ratio sufficient to demonstrate tumor growth inhibition. At higher doses of 6 and 9 mg/kg, more significant inhibition of tumor growth was observed, resulting in tumor regression.

EXAMPLE 15

Compound B increases intratumoral T cell fraction and TCR diversity in CT-26 mouse tumors

The effect of compound B alone and in combination with anti-PD-1 antibody on the T cell repertoire was studied using CT-26 tumor-bearing mice. Female Balb/c mice (6-8 weeks old, average weight 16-22 g, Jackson Laboratories or Taconic Biosciences) were inoculated subcutaneously in the flank with CT-26 tumor cells (ATCC), and tumor growth was monitored by measuring tumors three times a week. When tumors reached approximately 180 ^mm3 , mice (N=4 for each group) were randomized to the following groups: control (compound B vehicle + isotype control), compound B (single IV dose of 6 mg/kg on day 0), mouse anti-PD-1 antibody (10 mg/kg, two doses on days 0 and 3, IP), or compound B + anti-PD-1 antibody. Blood and tumor samples were collected pre-dose and on days 5, 8, 12, and 16 post-dose and stored at -80°C until analysis. Samples were analyzed for intratumoral T cell fraction and TCR diversity (Adaptive Biotechnologies, Seattle, WA). TCR sequencing of infiltrating T cells was performed using ImmunoSEQ™.

By day 8, CT-26 tumors treated with 6 mg/kg compound B alone or in combination with murine anti-PD-1 antibody exhibited significantly lower TCR repertoire clonality as determined by Dunn’s post hoc test compared with vehicle or anti-PD-1 antibody alone (p=0.005). Clonality was quantified as the degree of mono- or oligoclonal dominance in the repertoire by measuring the shape of the clonal frequency distribution. Clonal diversity was determined by reducing the sample size to a minimum number of templates. Consistent with clonality, TCR diversity showed the opposite trend at days 5 and 8 and was higher in the compound B or compound+anti-PD-1 antibody-treated groups (p<0.05) ( Fig. 19 ). No significant difference was observed in T cell repertoire counts at day 12 or 16 with treatment with compound B or combination with anti-PD-1 antibody.

As shown in Fig. 20 , TCR sequencing also demonstrated that Compound B increased the proportion of tumor infiltrating lymphocytes (TILs) alone or in combination with anti-PD-1 antibody (p < 0.05). Analysis of peripheral blood samples at day 8 revealed that Compound B also significantly reduced (p=0.001) T cell clonality compared to vehicle control, consistent with the observations made in the tumor ( Fig. 21 ).

EXAMPLE 16

Compound B reprograms the CT-26 tumor microenvironment to high T _eff activity , IFN-γ induction , and checkpoint ligand expression

CT-26 tumor samples from the study described in Example 15 above were also profiled by mRNA sequencing (OminiSeq, Buffalo, NY) and analyzed by GeneCentric (Research Triangle Park, NC) to identify cellular and molecular signatures of lymphocyte infiltration and activation. Data are shown as an expression heatmap in CT26 tumor samples at day 8 after treatment with control (vehicle), compound B (6 mg/kg), anti-mouse PD-1 (10 mg/kg), or a combination of compound B and anti-mouse PD-1 (N=10 mice per group). All signatures and individual genes are shown with p-values < 0.05 by KW (except PD-L1, p=0.23). Immune activation induced by treatment with compound B and anti-PD1 antibody are presented as heat maps based on values obtained using median-centered log2 expression values of genes comprising the different immune signatures and of individual genes. Box plots showing the expression levels of individual immune activation signatures or individual genes were also generated. Pairwise comparisons were made between control and treatment groups on days 8 and 12 in these box plots, and p-values are shown when Wilcoxon rank sum test p-values were < 0.05. Heat maps and box plots were generated using R version 3.5.3. Box plots show the lower quartile, median, and upper quartile of expression data. Whiskers in the plots show the full distribution of expression data. The nomenclature shown in Fig. 22 corresponds to human orthologous genes.

The top row of the heat map in Figure 22 shows that 8 days after a single dose of Compound B, CT26 tumors were infiltrated with activated CD8+ effector and memory T cells, as well as CD56 ^dim (cytolytic phenotype) NK cells. The abundance of these cell populations was further increased with the combination with the anti-PD-1 antibody. Mean-centered log2 expression levels ( Figure 23 ) show that Compound B significantly (p < 0.05) increased the numbers of activated CD8+ T cells and CD8+ memory T cells in these tumors compared with pre-dose levels, whereas the combination of Compound B and the anti-PD-1 antibody significantly increased the numbers of CD56 dim NK cells compared with pre-dose or control levels (p < 0.01). As shown in Figure 23 , the heat map of Compound B and the anti-PD-1 antibody significantly increased the numbers of CD8 ⁺ T cells compared with pre-dose or control levels. 22 , Compound B induced several markers of IL-2 response and T cell activation, including the three IL-2 receptor chains CD28, 4-1BB, and CD40. Furthermore, Compound B treatment resulted in increased expression of inhibitory checkpoint receptors PD-1 and CTLA4, as well as PD-1 ligands PD-L1 and PD-L2. Compound B also induced genes signaling IFN-γ release and activation of IFN-γ signaling pathways ( Fig. 24A ).

To construct a response signature to compound B in CT-26 tumor-bearing mice, a supervised assay was performed to identify differentially expressed genes (FDR < 0/01) between vehicle control-treated and compound B-treated animals at day 8 post-treatment. Gene expression profiles were compared across sets of mice, treated with compound B on day 8 and treated with vehicle control on day 8, using significance analyses. Multiple comparisons were adjusted using the false positive rate (FDR=0.01). Genes were ranked by p-value, adjusted by FDR, and the corresponding fold change is indicated intable 35A change factor greater than one indicates higher gene expression in the compound B-treated group; a fold change of less than one indicates to higher gene expression in the control group.

Table 35. Supervised analysis of expression changes to identify novel marker genes for development of a predictive response signature (PRS) to compound B Gene Original p-value P-value Multiplicity Gene Original p-value P-value Multiplicity F10 1,12E-6 0,000 3.2 NR4A3 9,24E-6 0,011 2.5 IL2RA 1,12E-6 0,008 3.8 BCL2L14 9,24E-6 0,011 6.1 CDH1 2,24E-6 0,008 3.0 DCSTAMP 9,24E-6 0,011 4.1 GZMK 2,24E-6 0,009 4.0 CTLA4 1,20E-5 0,012 3.7 SEPT3 5.88E-6 0,009 5.3 ARHGEF37 1,20E-5 0,012 3.0 F7 5.88E-6 0,011 5.4 STRIP2 1,57E-5 0,015 4.6 PDCD1LG2 5.88E-6 0,011 4.6 FAM169A 2,24E-5 0,015 1.7 ASGR2 5.88E-6 0,011 4.6 CCL22 2.41E-5 0,015 3.8 RAMP3 5.88E-6 0,011 3.1 MEX3B 2.55E-5 0,015 1.6 IL12B 5.88E-6 0,011 6.5 HR 2.58E-5 0,015 4.3 FLT3 8,12E-6 0,011 3.9 TACR1 2,69E-5 0,015 2.7 CCL17 8.96E-6 0,011 7.3

Forty-two of the 43 most differentially expressed genes were used for the PRS prototype for compound B. The GT(ROSA)26 gene was excluded from the PRS prototype because this gene is used as a knockin locus in mouse. Compound B prototype calls were performed and comparisons were made between control, compound B, and anti-PD1 antibody treatment groups. Pairwise PRS comparisons between control and treatment groups for days 8 and 12 were performed and presented as box plots ( Fig. 24A ). P-values obtained by paired Wilcoxon rank sum test are shown (p-value < 0.05). Box plots showing lower quartile, median, and upper quartile of expression data were generated using R version 3.5.3. "Whiskers" on the graphs show the median plus or minus 1.5 times the interquartile range (IQR) or the minimum/maximum expression data if the minimum/maximum is within 1.5 times the IQR.

Forty-two genes were upregulated by Compound B at day 8 compared to control. Twenty-three human orthologs shown in Table 35 were used to generate a prototype response signature. As shown in the underlined text in Table 35 , several genes with known IL-2-related biological properties were identified. For Compound B alone and in combination with mouse anti-PD-1, significant signature calls were observed compared to vehicle-treated mice ( FIG. 24B ).

EXAMPLE 17

Compound B promotes robust memory T cell responses, preventing CT-26 tumor growth in surviving animals stimulated by repeated injection of CT-26 cells

In Example 15, 7 mice were tumor-free at day 100 and included 1 animal each from the Compound B 3 mg/kg, Compound B 6 mg/kg, and anti-PD-1 antibody groups, and 4 animals from the Compound B 6 mg/kg+anti-PD-1 antibody combination group. These 7 mice and 7 naive control mice were inoculated in parallel with the same number of CT-26 tumor cells on day 121 (study #2). As shown in Fig. 25 , none of the 7 mice previously treated with Compound B, anti-PD-1, or the combination of both developed tumors, whereas all control animals grew tumors, indicating the formation of robust memory T cell populations in response to the initial treatments. Two months later, on day 181, the 7 tumor-free treated animals that survived the first rechallenge were again inoculated with CT-26 tumor cells along with 7 control mice. Again, the 7 tumor-free treated animals did not develop tumors, whereas tumors grew in the control animals ( Fig. 25 ). The 7 tumor-free animals survived until day 202, when the study was terminated.

The restimulation experiment was repeated in surviving animals in an additional study (Study 3). A total of 9 surviving tumor-free mice (1 animal in each of the Compound B 9 mg/kg and anti-PD-1 antibody groups, 2 animals in the Compound B 3 mg/kg + anti-PD-1 antibody group, and 5 animals in the Compound B 6 mg/kg + anti-PD-1 antibody group) with complete tumor regression at day 102 were restimulated by subcutaneous inoculation of CT-26 tumor cells. An additional 10 naive control mice were also inoculated in parallel. As shown in Fig. 2B , none of the 9 animals developed tumors, indicating the formation of robust memory T cell populations in response to the initial treatment. In contrast, all 10 control animals developed tumors. Two months later, at day 163, a second boost was administered to the same 9 animals that had survived the first boost; an additional 9 naive animals served as controls. As with the first boost, the 9 surviving animals did not develop tumors, confirming the durability of memory T cell responses after the initial treatments, whereas tumors grew in the control animals ( Fig. 26 ). The 9 tumor-free animals survived until day 184, when the study was terminated.

To determine the ability of Compound B to promote a robust memory T cell response, blood samples were collected from 7 surviving mice from Study 2 60 days after the second restimulation. FACS analysis of the blood samples for memory cell expression revealed that Compound B promoted robust immunological memory against CT-26 tumors, as observed as an overall increase in peripheral blood memory T cells (CD3+), including CD8+ memory T cells ( FIGS. 27A-27B ).

Summary: Analysis of TILs in tumors treated with 6 mg/kg compound B revealed an increase in the T cell repertoire and the number of activated CD8+ T cells (including effector memory T cells) and NK cells, as well as the expression of IFNγ signatures that induce checkpoint ligands. Immune checkpoint-targeted therapies have been widely used and shown to be effective against a growing number of cancers, including metastatic melanoma and renal cell carcinoma (Hodi FS et al., N. Engl. J. Med. (2010) 363(8):711–723; Topalian SL et al., N. Engl. J. Med. (2012) 366(26):2443–2454; Wolchok JD et al., N. Engl. J. Med. (2013) 369(2):122–133; Sharma P et al. Cell (2015) 161(2):205–214; Alsaab HO et al., Frontiers in Pharmacology (2017) 8:561, 1–15; Pardoll DM Nat. Rev. Cancer (2016) 12(4): 252–264). However, complete response rates are still quite low. Checkpoint inhibitors remove the limitations of dysfunctional cytotoxic T lymphocytes and prime them, while cytokine therapies such as IL-2 can activate them and induce their proliferation. In addition, IL-2-based therapeutics can promote the expansion and activation of Fc+ lymphocytes such as NK cells. Thus, checkpoint inhibitors and IL-2 in combination act complementarily to mediate effects on immune responses to improve antitumor responses. In current studies, combination treatment with compound B and anti-PD-1 resulted in a survival advantage over either agent alone by increasing both the diversity and clonality of activated cytotoxic CD8+ T and NK cells. Moreover, tumor-free mice treated with Compound B or the combination had a robust population of memory T cells. This resulted in tumor cell rejection in surviving mice after rechallenge with the same tumor cells on two occasions 2 months apart, with the first rechallenge occurring 100 days after the last treatment with Compound B, anti-PD-1, or the combination.

Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are presented by way of example only. Numerous changes, modifications and substitutions will now be apparent to those skilled in the art without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. It is intended that the following claims define the scope of the present invention and that methods and structures within the scope of the claims and their equivalents are encompassed by them. The disclosures of all patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

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SEQUENCE LIST

<110> SYNTHORX, INC.

<120> TYPES OF IMMUNOONCLOGICAL COMBINATION THERAPY

USING IL-2 CONJUGATES

<130> 01183-0073-00PCT

<150> US 62/887,400

<151> 2019-08-15

<150> US 62/903,187

<151> 2019-09-20

<150> US 62/962,668

<151> 2020-01-17

<160> 98

<170> PatentIn version 3.5

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 17

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_PEG]

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG]

<400> 17

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 18

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_PEG]

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_PEG]

<400> 18

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 19

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_PEG]

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_PEG]

<400> 19

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 20

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_PEG5kDa]

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_PEG5kDa]

<400> 20

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 21

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_PEG5kDa]

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_PEG5kDa]

<400> 21

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 22

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_PEG5kDa]

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG5kDa]

<400> 22

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 23

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_PEG5kDa]

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_PEG5kDa]

<400> 23

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 24

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_PEG5kDa]

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_PEG5kDa]

<400> 24

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 25

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_PEG30kD]

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_PEG30kD]

<400> 25

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 26

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_PEG30kD]

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_PEG30kD]

<400> 26

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 27

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_PEG30kD]

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG30kD]

<400> 27

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 28

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_PEG30kD]

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_PEG30kD]

<400> 28

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 29

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_PEG30kD]

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_PEG30kD]

<400> 29

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 30

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65X-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa represents any unnatural amino acid

<400> 30

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 31

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62X-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa represents any unnatural amino acid

<400> 31

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 32

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42X-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa represents any unnatural amino acid

<400> 32

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 33

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43X-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa represents any unnatural amino acid

<400> 33

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 34

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35X-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa represents any unnatural amino acid

<400> 34

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 35

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa represents [AzK]

<400> 35

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 36

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa represents [AzK]

<400> 36

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 37

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa represents [AzK]

<400> 37

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 38

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa represents [AzK]

<400> 38

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 39

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa represents [AzK]

<400> 39

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 40

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_L1_PEG]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_L1_PEG]

<400> 40

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 41

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_L1_PEG]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_L1_PEG]

<400> 41

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 42

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_L1_PEG]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_L1_PEG]

<400> 42

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 43

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_L1_PEG]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_L1_PEG]

<400> 43

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 44

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_L1_PEG]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_L1_PEG]

<400> 44

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 45

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_L1_PEG5kDa]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 45

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 46

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_ L1_PEG5kD]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 46

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 47

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_ L1_PEG5kD]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 47

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 48

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_ L1_PEG5kD]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 48

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 49

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_ L1_PEG5kD]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 49

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 50

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_ L1_PEG30kD]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 50

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 51

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_ L1_PEG30kD]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 51

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 52

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_ L1_PEG30kD]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 52

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 53

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_ L1_PEG30kD]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 53

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 54

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_ L1_PEG30kD]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 54

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 55

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_L1_PEG]-2

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_L1_PEG]

<400> 55

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 56

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_L1_PEG]-2

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_L1_PEG]

<400> 56

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 57

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_L1_PEG]-2

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_L1_PEG]

<400> 57

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 58

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_L1_PEG]-2

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_L1_PEG]

<400> 58

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 59

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_L1_PEG]-2

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_L1_PEG]

<400> 59

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 60

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_L1_PEG5kDa]-2

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 60

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 61

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_ L1_PEG5kD]-2

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 61

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 62

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_ L1_PEG5kD]-2

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 62

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 63

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_ L1_PEG5kD]-2

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 63

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 64

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_ L1_PEG5kD]-2

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_L1_PEG5kD]

<400> 64

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 65

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_ L1_PEG30kD]-2

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 65

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 66

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_ L1_PEG30kD]-2

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 66

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 67

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_ L1_PEG30kD]-2

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 67

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 68

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_ L1_PEG30kD]-2

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 68

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 69

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_ L1_PEG30kD]-2

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_L1_PEG30kD]

<400> 69

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 70

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_PEG]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_PEG]

<400> 70

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 71

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_PEG]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_PEG]

<400> 71

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 72

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_PEG]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_PEG]

<400> 72

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 73

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_PEG]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG]

<400> 73

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 74

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_PEG]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_PEG]

<400> 74

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 75

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_PEG5kD]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_PEG5kDa]

<400> 75

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 76

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_PEG5kDa]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_PEG5kDa]

<400> 76

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 77

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_PEG5kDa]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_PEG5kDa]

<400> 77

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 78

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_PEG5kDa]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG5kDa]

<400> 78

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 79

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_PEG5kDa]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_PEG5kDa]

<400> 79

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 80

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_PEG30kD]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_PEG30kD]

<400> 80

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 81

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_PEG30kD]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_PEG30kD]

<400> 81

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 82

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK_PEG30kD]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_PEG30kD]

<400> 82

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 83

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK_PEG30kD]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG30kD]

<400> 83

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 84

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK_PEG30kD]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_PEG30kD]

<400> 84

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 85

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F44X

<220>

<221> other_feature

<222> (44)..(44)

<223> Xaa represents any unnatural amino acid

<400> 85

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Xaa Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 86

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F44X-1

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa represents any unnatural amino acid

<400> 86

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Xaa Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 87

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_R38X

<220>

<221> other_feature

<222> (38)..(38)

<223> Xaa represents any unnatural amino acid

<400> 87

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Xaa Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 88

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_R38X-1

<220>

<221> other_feature

<222> (37)..(37)

<223> Xaa represents any unnatural amino acid

<400> 88

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Xaa Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 89

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_T41X

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa represents any unnatural amino acid

<400> 89

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Xaa Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 90

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_T41X-1

<220>

<221> other_feature

<222> (40)..(40)

<223> Xaa represents any unnatural amino acid

<400> 90

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Xaa Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 91

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E68X

<220>

<221> other_feature

<222> (68)..(68)

<223> Xaa represents any unnatural amino acid

<400> 91

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 92

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E68X-1

<220>

<221> other_feature

<222> (67)..(67)

<223> Xaa represents any unnatural amino acid

<400> 92

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Xaa Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 93

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_Y45X

<220>

<221> other_feature

<222> (45)..(45)

<223> Xaa represents any unnatural amino acid

<400> 93

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Xaa Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 94

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_Y45X-1

<220>

<221> other_feature

<222> (44)..(44)

<223> Xaa represents any unnatural amino acid

<400> 94

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Xaa Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 95

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_V69X

<220>

<221> other_feature

<222> (69)..(69)

<223> Xaa represents any unnatural amino acid

<400> 95

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 96

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_V69X-1

<220>

<221> other_feature

<222> (68)..(68)

<223> Xaa represents any unnatural amino acid

<400> 96

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Xaa Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 97

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_L72X

<220>

<221> other_feature

<222> (72)..(72)

<223> Xaa represents any unnatural amino acid

<400> 97

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 98

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_L72X-1

<220>

<221> other_feature

<222> (71)..(71)

<223> Xaa represents any unnatural amino acid

<400> 98

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Xaa Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

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

85 90 95

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

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<---

Claims (27)

1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate and (b) one or more PD-1 inhibitors, wherein: (i) 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, in which [AzK_L1_PEG30kDa] of SEQ ID NO: 50 remains, wherein [AzK_L1_PEG30kDa] has the structure of formula (XII); (ii) 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, in which [AzK_L1_PEG30kDa] of SEQ ID NO: 50 remains, wherein [AzK_L1_PEG30kDa] has the structure of formula (XIII); or (iii) the IL-2 conjugate is administered as a pharmaceutical composition comprising a mixture, wherein the mixture comprises the IL-2 conjugate (i) and the IL-2 conjugate (ii), where formula (XII) and formula (XIII) are shown below: Where: n is an integer such that PEG having the structure -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of approximately 30 kDa; and The wavy lines indicate covalent bonds with the first preceding amino acid residues and the first following amino acid residues, respectively, in SEQ ID NO: 50. 2. The method of claim 1, wherein the one or more PD-1 inhibitors are pembrolizumab, nivolumab, or cemiplimab. 3. The method of claim 2, wherein one or more PD-1 inhibitors are pembrolizumab. 4. The method of claim 2, wherein the one or more PD-1 inhibitors are nivolumab. 5. The method of claim 2, wherein one or more PD-1 inhibitors are cemiplimab. 6. The method according to any of paragraphs. 1-5, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer (CRC), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, esophageal squamous cell carcinoma (ESCC), glioblastoma, mesothelioma, breast cancer, triple-negative breast cancer, prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer or metastatic castration-resistant prostate cancer with defects in DNA damage response (DDR), bladder cancer, ovarian cancer, tumors with intermediate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors that have spread systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma. 7. The method of claim 6, wherein the cancer is selected from non-small cell lung cancer (NSCLC), classical Hodgkin lymphoma (cHL), head and neck squamous cell carcinoma (HNSCC), colon cancer, colorectal cancer (CRC), and melanoma. 8. The method according to claim 6, wherein the cancer is selected from colon cancer. 9. The method of any one of claims 1-8, wherein the IL-2 conjugate is administered to the subject once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. 10. The method according to any one of claims 1-8, wherein the IL-2 conjugate is administered to the subject once a week. 11. The method according to any one of claims 1-8, wherein the IL-2 conjugate is administered to the subject once every two weeks. 12. The method according to any one of claims 1-8, wherein the IL-2 conjugate is administered to the subject once every three weeks. 13. The method according to any one of claims 1-12, wherein the IL-2 conjugate is administered to the subject by intravenous administration. 14. The method according to any one of claims 1-12, wherein the IL-2 conjugate is administered to the subject by subcutaneous administration. 15. The method according to any one of claims 1-14, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate or hydrate. 16. The method of any one of claims 1-15, wherein the IL-2 conjugate is administered at a dose of from about 10 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight. 17. The method according to any one of claims 1-16, wherein the IL-2 conjugate comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 50, in which [AzK_L1_PEG30kDa] of SEQ ID NO: 50 remains. 18. The method according to any one of claims 1-16, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 50.