RU2843654C1 - Immuno-oncological combination therapy using conjugates based on il-2 and antibodies to egfr - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: group of inventions relates to the field of medicine, in particular to methods and a kit for the treatment of cancer expressing EGFR in a subject in need thereof using an IL-2 conjugate in a combination with an EGFR antibody. IL-2-based conjugate contains the amino acid sequence SEQ ID NO: 3, in which amino acid residue P64 is replaced with a structure of formula (I)

EGFR antibodies include cetuximab, panitumumab, necitumumab or JNJ-61186372 (amivantamab).

EFFECT: inventions provide improving the ADCC response to cancer by enhancing the ADCC function of the EGFR antibody.

23 cl, 8 dwg, 5 tbl, 5 ex

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/044,199, filed June 25, 2020, and U.S. Provisional Patent Application No. 63/196,448, filed June 3, 2021, the disclosures of each of which are hereby incorporated by reference in their entireties.

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 condition. Modulation of different T cell populations can be enhanced by the presence of additional agents or methods in combination therapy.

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

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.

In some cases, interleukin 2 (IL-2) mediated signaling is used to modulate T cell responses and therefore to treat cancer. Accordingly, in one aspect, provided herein are methods of treating cancer in a subject comprising administering an IL-2 conjugate in combination with an anti-EGFR antibody.

ESSENCE OF THE INVENTION

Described herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an IL-2-based conjugate and (b) an anti-EGFR antibody.

Illustrative embodiments include the following.

Embodiment 1. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an IL-2-based conjugate and (b) an antibody to EGFR, wherein the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced by a structure of formula (I):

Where:

Z is _CH2 and Y is

Y is _CH2 and Z is

Z is _CH2 and Y is or

Y is _CH2 and Z is

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

X is an L-amino acid characterized by 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;

wherein the position of the structure of formula (I) in the amino acid sequence of the conjugate based on IL-2 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V6B and L71.

Embodiment 2. The method of embodiment 1, wherein in the IL-2-based conjugate, Z is CH ₂ and Y is

Embodiment 3. The method of embodiment 1, wherein in the IL-2-based conjugate, Y is CH ₂ and Z is

Embodiment 4. The method of embodiment 1, wherein in the IL-2-based conjugate, Z is CH ₂ and Y is

Embodiment 5. The method of embodiment 1, wherein in the IL-2-based conjugate, Y is CH ₂ and Z is

Embodiment 6. The method according to any one of embodiments 1-5, wherein in the IL-2-based conjugate the PEG group has an average molecular weight of about 25 kDa, 30 kDa, or 35 kDa.

Embodiment 7. The method of embodiment 6, wherein in the IL-2-based conjugate the PEG group has an average molecular weight of about 30 kDa.

Embodiment 8. The method according to any one of embodiments 1-7, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2-based conjugate is P64.

Embodiment 9. The method according to embodiment 1, wherein the structure of formula (I) has a structure of formula (IV) or formula (V) or is a mixture of structures of formula (IV) and formula (V):

Where:

W is a PEG group having an average molecular weight of approximately 25 kDa, 30 kDa, or 35 kDa;

q is equal to 1, 2, or 3;

X is an L-amino acid characterized by 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.

Embodiment 10. The method according to embodiment 9, wherein the position of the structure of formula (IV) or formula (V) in the amino acid sequence of the IL-2-based conjugate is P64.

Embodiment 11. The method according to any one of embodiments 1-10, wherein the EGFR antibody is cetuximab.

Embodiment 12. 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-based conjugate and (b) cetuximab, wherein the IL-2-based 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):

Where:

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

q is equal to 1, 2, or 3; and

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

Embodiment 13. The method according to any one of embodiments 1-12, wherein q is equal to 1.

Embodiment 14. The method according to any one of embodiments 1-12, wherein q is 2.

Embodiment 15. The method according to any one of embodiments 1-12, wherein q is 3.

Embodiment 16. The method according to any one of embodiments 1-15, wherein the average molecular weight is a number average molecular weight.

Embodiment 17. The method according to any one of embodiments 1-15, wherein the average molecular weight is a weight average molecular weight.

Embodiment 18. The method according to any one of embodiments 1-17, wherein the IL-2-based conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

Embodiment 19. The method of any one of embodiments 1-18, wherein the IL-2 conjugate is administered to the subject about once every two weeks, about once every three weeks, or about once every 4 weeks.

Embodiment 20. The method of any one of embodiments 1-19, wherein the anti-EGFR antibody is administered to the subject about once a week, about once every two weeks, about once every three weeks, or about once every 4 weeks.

Embodiment 21. The method according to any one of embodiments 1-20, wherein the IL-2-based conjugate is administered to the subject via intravenous administration.

Embodiment 22. The method according to any one of embodiments 1-21, wherein the IL-2-based conjugate and the EGFR antibody are administered separately.

Embodiment 23. The method of embodiment 23, wherein the IL-2 conjugate and the EGFR antibody are administered sequentially.

Embodiment 24. The method according to any one of embodiments 1-23, 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 stability 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 cancer prostate cancer, metastatic castration-resistant prostate cancer, or metastatic castration-resistant prostate cancer with defects in DNA damage response (DDR) mechanisms, 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 disseminating systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma.

Embodiment 25. The method according to any one of embodiments 1-24, comprising administering to the subject about 16 μg/kg of the IL-2 conjugate.

Embodiment 26. The method of any one of embodiments 1-24, comprising administering to the subject about 24 μg/kg of the IL-2 conjugate.

Embodiment 27. The method according to any one of embodiments 1-10 or 12-26, wherein the EGFR antibody is selected from panitumumab (Vectibix), necitumumab (Portrazza), JNJ-61186372 (amivantamab), IMC-C225, ABX-EGF, ICR62, and EMD 55900.

Embodiment 28. An IL-2-based conjugate for use in the method according to any one of embodiments 1-27.

Embodiment 29. Use of an IL-2-based conjugate for the manufacture of a medicinal product for the method according to any one of embodiments 1-27.

BRIEF DESCRIPTION OF DRAWINGS

The novel features of the present invention are particularly set forth in the appended claims. 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 used, and the accompanying drawings, which illustrate the following.

Figures 1A-C show the % cytotoxicity against CAL27 cells co-cultured with human PBMCs from 3 individual donors and varying amounts of IL-2-cetuximab conjugate.

Figure 2A shows the % cytotoxicity against CAL27 cells co-cultured with human PBMC and varying amounts of IL-2-cetuximab conjugate.

Fig. 2B shows the cytotoxicity in % against A431 cells co-cultured with human PBMC and varying amounts of IL-2-cetuximab conjugate.

Fig. 3A shows the cytotoxic effect on A431 cells co-cultured with NK92 cells and treated with varying amounts of IL-2-cetuximab conjugate.

Fig. 3B shows the % cytotoxicity against DLD-1 cells co-cultured with NK92 cells and treated with varying amounts of IL-2-cetuximab conjugate.

Fig. 3C shows the cytotoxicity in % against FaDu cells co-cultured with NK92 cells and treated with varying amounts of IL-2-cetuximab conjugate.

Fig. 3D shows the % cytotoxicity against CAL27 cells co-cultured with NK92 cells and treated with varying amounts of IL-2-cetuximab conjugate.

Fig. 4A shows the number of peripheral CD8+ T _eff cells in the indicated subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after administration of IL-2 conjugate.

Figure 4B shows the change in peripheral CD8+ T _eff cell counts in subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after IL-2 conjugate administration. Data are normalized to pre-treatment CD8+ T _eff cell counts (C1D1).

Fig. 5A shows the number of peripheral NK cells in the indicated subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after administration of IL-2 conjugate.

Figure 5B shows the change in peripheral NK cell counts in subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after IL-2 conjugate administration. Data are normalized to pre-treatment NK cell counts (C1D1).

Fig. 6A shows the number of peripheral CD4+ T _reg cells in the indicated subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after administration of IL-2 conjugate.

Figure 6B shows the change in peripheral CD4+ T _reg cell counts in subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after IL-2 conjugate administration. Data are normalized to pre-treatment CD4+ T _reg cell counts (C1D1).

Fig. 7A shows eosinophil cell counts in subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after administration of IL-2 conjugate.

Figure 7B shows the change in eosinophil cell counts in subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after administration of IL-2 conjugate. Data are normalized to pre-treatment eosinophil cell counts (C1D1).

Fig. 8A shows lymphocyte cell counts in subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after administration of IL-2 conjugate.

Fig. 8B shows the change in lymphocyte cell counts in the indicated subjects treated with 16 μg/kg IL-2 conjugate in combination with cetuximab at the indicated time points after administration of the IL-2 conjugate. Data are normalized to pre-treatment lymphocyte cell counts (C1D1).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, 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. It is to be understood that the foregoing general description and the following detailed description are merely illustrative and explanatory and are not limiting of any claimed subject matter. To the extent that any material incorporated herein by reference does not correspond to the express subject matter of the present invention, the express subject matter shall control. In this application, the use of the singular includes the plural unless specifically indicated otherwise. It should be noted that as used in the specification and the appended claims, the singular forms include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless otherwise indicated. Furthermore, 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.

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

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, except for possible antibody variants, such as those containing mutations that occur naturally or that arise during the production of the monoclonal antibody product, such variants are typically present in small amounts. In contrast to polyclonal antibody products, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody product 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 production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and techniques using transgenic animals containing all or part of the human immunoglobulin loci, such techniques and other illustrative methods for producing monoclonal antibodies being described herein.

As used herein, "nucleotide" refers to a compound comprising 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."

As used in this document, the term "dose-limiting toxicity" (DLT) is defined as an adverse event occurring from day 1 to day 29 (inclusive) +1 of a treatment cycle that is clearly or certainly not related to an external cause and that meets the criteria for DLT presented in Example 5.

As used herein, the term "cetuximab" refers to the chimeric (mouse/human) anti-EGFR antibody marketed by Eli Lilly and Co. under the trade name Erbitux.

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 based conjugates

Interleukin 2 (IL-2) is a type 1 pleiotropic cytokine with a 15.5 kDa α-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 peripheral CD4 ⁺ T cells 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 disclosures of each of which are 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 to 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 physicians to effectively use a patient’s own immune cells to combat diseases such as proliferative diseases (e.g., cancer) and infectious diseases. The effect of IL-2-mediated signaling can be further enhanced by the presence of additional agents or modalities in combination therapy. For example, epidermal growth factor receptor (EGFR) is a cell surface receptor overexpressed in many types of cancer. Activation of EGFR induces cell proliferation and survival, as well as angiogenesis, which leads to tumor growth and metastasis. Cell growth and angiogenesis can be controlled by blocking the binding of EGFR to epidermal growth factor (EGF). Anti-EGFR antibodies bind to the extracellular domain of EGFR and prevent EGF from binding to EGFR, thereby inhibiting the subsequent signaling cascade and resulting in decreased cell growth. Anti-EGFR antibodies may also produce the same effect by competitively inhibiting the binding of transforming growth factor alpha (TGF-α) to EGFR.

Disclosed herein are methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of (a) an IL-2-based conjugate and (b) an EGFR antibody. Some exemplary IL-2 polypeptides and IL-2-based conjugates are provided in Table 1.

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 containing a structure of formula (II) or formula (III). For example, when specified, PEC5kDa refers to a linear polyethyleneglycol chain with an average molecular weight of 5 kilodaltons, capped with a methoxy group. The ratio of regioisomers formed by the click reaction is approximately 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety containing a dibenzocyclooctyne group.

[AzK_L1_PEG]=N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG by DBCO-mediated click chemistry to form a compound containing 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 by the click reaction is approximately 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety containing a dibenzocyclooctyne group.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced with a structure of formula (I):

where: Z is _CH2 and Y is Y is _CH2 and Z is Z is CH ₂ and Y is or Y is _CH2 and Z is

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

q is equal to 1, 2, or 3;

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;

wherein the position of the structure of formula (I) in the amino acid sequence of the conjugate based on IL-2 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71.

In any of the embodiments or variations of formula (I) described herein, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt. In some embodiments, the IL-2 conjugate is a solvate. In some embodiments, the IL-2 conjugate is a hydrate.

In some embodiments or variations of formula (I) described herein, X is an L-amino acid.

In some embodiments of formula (I), Z is CH ₂ and Y is

In some embodiments of formula (I), Y is CH ₂ and Z is In some embodiments of formula (I), Z is CH ₂ and Y is In some embodiments of formula (I), Y is CH ₂ and Z is

In some embodiments of formula (I), q is 1. In some embodiments of formula (I), q is 2. In some embodiments of formula (I), q is 3.

In some embodiments of formula (I), q is 1 and the structure of formula (I) is the structure of formula (Ia):

Where:

Z is _CH2 and Y is

Y is _CH2 and Z is

Z is _CH2 and Y is or

Y is _CH2 and Z is

W is a PEG group having an average molecular weight selected from about 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;

wherein the position of the structure of formula (Ia) in the amino acid sequence of the conjugate based on IL-2 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71.

In some embodiments of formula (Ia), Z is CH ₂ and Y is In some embodiments of formula (Ia), Y is CH ₂ and Z is In some embodiments of formula (Ia), Z is CH ₂ and Y is In some embodiments of formula (Ia), Z is CH ₂ and Y is In some embodiments of formula (Ia), Y is CH ₂ and Z is

In some embodiments, the PEG moiety has an average molecular weight selected from about 5 kDa, 10 kDa, 20 kDa, and 30 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 5 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 10 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 15 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 20 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 25 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 30 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 35 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 40 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 45 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 50 kDa. In some embodiments, the PEG moiety has an average molecular weight of about 60 kDa.

In some embodiments, 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 is related to the positions in SEQ ID NO: 3. In some embodiments, 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 is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is K34, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 refers to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is F41, 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, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from F43, 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, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is K4 2, 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, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate IL-2 is E61, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is P64, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is R37, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is T40, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from E67, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is Y44, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is V68, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is L71, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2-based conjugate refers to the positions in SEQ ID NO: 3.

In some embodiments, the IL-2-based conjugate comprises an 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):

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

q is equal to 1, 2, or 3; 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.

Herein and throughout this document, the structure of formula (II) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout this document, the structure of formula (III) 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, q is 1 and the structures of formula (II) and formula (III) are the structures of formula (IIa) and formula (IIIa):

Where:

W is a PEG group having an average molecular weight selected from about 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.

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

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

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

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

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

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

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

In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19. In some embodiments, W in the structure of formula (III) is a PEG moiety having an average molecular weight selected from about 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 about 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 about 5 kDa. In some embodiments, W in the structure of formula (III) is a PEG moiety having an average molecular weight of about 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 selected from about 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 about 5 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 about 10 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 about 15 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 about 20 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 about 25 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 about 30 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 about 35 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 about 40 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 about 45 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, [AzK_PEG] comprises a PEG moiety having an average molecular weight of about 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 about 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 selected from about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa, and the PEG moiety is a methoxy-PEG moiety, a linear methoxy-PEG moiety, or a branched methoxy-PEG moiety.

In some embodiments, the IL-2-based conjugate comprises an amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEC5kDa] has a structure of formula (II) or formula (III) or is a mixture of formula (II) and formula (III):

Where:

W is a PEG group having an average molecular weight of approximately 5 kDa; q is 1, 2, or 3; 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, q is 1 and [AzK_PEG5kDa] has a structure of formula (IIa) or formula (IIIa) or is a mixture of formula (IIa) and formula (IIIa).

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

In some embodiments, [AzK_PEG5kDa] has a structure of formula (II):

In some embodiments, q is 1 and [AzK_PEG5kDa] has the structure of formula (IIa).

In some embodiments, [AzK_PEG5kDa] has a structure of formula (III):

In some embodiments, q is 1 and [AzK_PEG5kDa] has the structure of formula (IIIa).

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

W is a PEG group having an average molecular weight of approximately 30 kDa; q is 1, 2, or 3; 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, q is 1 and [AzK_PEG30kDa] has a structure of formula (IIa) or formula (IIIa) or is a mixture of structures of formula (IIa) and formula (IIIa).

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, [AzK_PEG30kDa] has a structure of formula (II):

In some embodiments, q is 1 and [AzK_PEG30kDa] has the structure of formula (IIa).

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

In some embodiments, q is 1 and [AzK_PEG30kDa] has the structure of formula (IIIa).

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

Where:

W is a PEG group characterized by 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;

q is equal to 1, 2, or 3; 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, q is 1 and [AzK_PEG] is a mixture of structures of formula (IIa) and formula (IIIa).

In some embodiments, 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 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 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, W is a linear or branched PEG group. In some embodiments, W is a linear PEG group. In some embodiments, W is a branched PEG group. In some embodiments, W is a methoxy-PEG group. In some embodiments, the methoxy-PEG group is linear or branched. In some embodiments, the methoxy-PEG group is linear. In some embodiments, the methoxy-PEG group is branched.

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

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

q is 1, 2, or 3; 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, q is 1 and [AzK_PEG5kDa] is a mixture of structures of formula (IIa) and formula (IIIa).

In some embodiments, 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 about 1:1. In some embodiments, 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 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.

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

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

q is 1, 2, or 3; 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, q is 1 and [AzK_PEG30kDa] is a mixture of structures of formula (IIa) and formula (IIIa).

In some embodiments, 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 about 1:1. In some embodiments, 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 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 IL-2 conjugate comprises 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, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, W in the structure of Formula (II) or Formula (III) is a linear PEG group. In some embodiments, W in the structure of Formula (II) or Formula (III) is a branched PEG group. In some embodiments, W in the structure of Formula (II) or Formula (III) is a methoxy-PEG group. In some embodiments, W in the structure of Formula (II) or Formula (III) is a methoxy-PEG group that is linear or branched. In some embodiments, the methoxy-PEG group in the structure of Formula (II) or Formula (III) is linear. In some embodiments, the methoxy-PEG group in the structure of Formula (II) or Formula (III) is branched.

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

W is a PEG group characterized by 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;

q is 1, 2, or 3; 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 of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), q is 1. In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), q is 2. In some embodiments of Formula (IV) and Formula (V), or a mixture of Formula (IV) or Formula (V), q is 3.

Here and throughout this document, the structure of formula (IV) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (V) 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, q is 1 and the structures of formula (IV) and formula (V) are formula (IVa) and formula (Va):

Where:

W is a PEG group having an average molecular weight selected from about 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.

In some embodiments, [AzK_L1_PEG] is a mixture of formula (IV) and formula (V). In some embodiments, [AzK_L1_PEG] is a mixture of formula (IVa) and formula (Va).

In some embodiments, [AzK_L1_PEG] has a structure of formula (IV):

In some embodiments, q is 1 and [AzK_L1_PEG] has the structure of formula (IVa).

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

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

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

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

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

In some embodiments, [AzK_L1_PEG] has a structure of formula (V):

In some embodiments, q is 1 and [AzK_L1_PEG] has the structure of formula (V).

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

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

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

In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43. In some embodiments, W in the structure of formula (V) is a PEG moiety having an average molecular weight selected from about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG moiety having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG moiety having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (V) is a PEG moiety having an average molecular weight of about 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 about 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 about 5 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 about 10 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 about 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 about 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 about 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 about 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 about 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 about 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 about 45 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 about 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 about 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 selected from about 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.

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

W is a PEG group with an average molecular weight of approximately 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, q is 1 and [AzK_L1_PEG5kDa] has a structure of formula (IVa) or formula (Va) or is a mixture of formula (IVa) and formula (Va).

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, [AzK_L1_PEG5kDa] has a structure of formula (IV):

In some embodiments, q is 1 and [AzK_L1_PEG5kDa] has the structure of formula (IVa).

In some embodiments, [AzK_L1_PEG5kDa] has a structure of formula (V):

In some embodiments, q is 1 and [AzK_L1_PEG5kDa] has a structure of formula (Va).

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

W is a PEG group having an average molecular weight of approximately 30 kDa; q is 1, 2, or 3; 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, q is 1 and [AzK_L1_PEG30kDa] has a structure of formula (IVa) or formula (Va) or is a mixture of structures of formula (IVa) and formula (Va).

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

In some embodiments, [AzK_L1_PEG30kDa] has a structure of formula (IV):

In some embodiments, q is 1 and [AzK_L1_PEG30kDa] has the structure of formula (IVa).

In some embodiments, [AzK_L1_PEG30kDa] has a structure of formula (V):

In some embodiments, q is 1 and [AzK_L1_PEG30kDa] has the structure of formula (Va).

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

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

q is equal to 1, 2, or 3; 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 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 about 1:1. In some embodiments, 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 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.

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

W is a PEG group having an average molecular weight of approximately 5 kDa; q is 1, 2, or 3; 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, q is 1 and [AzK_L1_PEG5kDa] is a mixture of structures of formula (IVa) and formula (Va).

In some embodiments, 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 about 1:1. In some embodiments, 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 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.

In some embodiments, the IL-2-based conjugate comprises an 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):

Where:

W is a PEG group having an average molecular weight of approximately 30 kDa; q is 1, 2, or 3; 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, q is 1 and [AzK_L1 PEG30kDa] is a mixture of structures of formula (IVa) and formula (Va).

In some embodiments, 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 about 1:1. In some embodiments, 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 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, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight selected from about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight of about 30 kDa. In some embodiments, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight of about 10 kDa. In some embodiments, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight of about 15 kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG moiety having an average molecular weight of about 20 kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG moiety having an average molecular weight of about 25 kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG moiety having an average molecular weight of about 30 kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG moiety having an average molecular weight of about 35 kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG moiety having an average molecular weight of about 40 kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG moiety having an average molecular weight of about 45 kDa. In some embodiments, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight of about 50 kDa. In some embodiments, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight of about 55 kDa. In some embodiments, W in the structure of formula (IV) or (V) is a PEG moiety having an average molecular weight of about 60 kDa.

In some embodiments, an IL-2 conjugate described herein comprises 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, W in the structure of Formula (IV) or Formula (V) is a linear PEG group. In some embodiments, W in the structure of Formula (IV) or Formula (V) is a branched PEG group. In some embodiments, W in the structure of Formula (IV) or Formula (V) is a methoxy-PEG group. In some embodiments, W in the structure of Formula (IV) or Formula (V) is a methoxy-PEG group that is linear or branched. In some embodiments, the methoxy-PEG group in the structure of Formula (IV) or Formula (V) is linear. In some embodiments, the methoxy-PEG group in the structure of Formula (IV) or Formula (V) is branched.

With respect to the IL-2-based conjugates used in the methods described herein, an exemplary structure of the methoxy-PEG moiety is illustrated in the mPEG-DBCO structures below.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced with a structure of formula (VI) or (VII) or a mixture of (VI) and (VII):

Where:

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

q is equal to 1, 2, or 3; 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 of formula (VI) or formula (VII) or a mixture of formula (VI) or formula (VII), q is 1. In some embodiments of formula (VI) or formula (VII) or a mixture of formula (VI) or formula (VII), q is 2. In some embodiments of formula (VI) or formula (VII) or a mixture of formula (VI) or formula (VII), q is 3.

Here and throughout this document, the structure of formula (VI) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (VII) 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, q is 1 and the structures of formula (VI) and formula (VII) are the structures of formula (VIa) and formula (VIIa):

Where:

n is an integer in the range from about 2 to about 5000; and X has the structure

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

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

In some embodiments, 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. In some embodiments, 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.

In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (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, 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, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (VII) 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 (VI) to the amount of the structure of formula (VII) 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 (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 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 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 (VI) or (VII) or a mixture of (VI) and (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 (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 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 (VI) or (VII), or a mixture of (VI) and (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 (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 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 (VI) or (VII), or a mixture of (VI) and (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 (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

In some embodiments, 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 (VI) or (VII), or a mixture of (VI) and (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, 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. 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 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 (VI) or (VII), or a mixture of (VI) and (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 (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 (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 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 (VI) or (VII), or a mixture of (VI) and (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 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 (VI) or (VII), or a mixture of (VI) and (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.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced with a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX):

Where:

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

q is 1, 2, or 3; 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 of formula (VIII) or formula (IX) or a mixture of formula (VIII) or formula (IX), q is 1. In some embodiments of formula (VIII) or formula (IX) or a mixture of formula (VIII) or formula (IX), q is 2. In some embodiments of formula (VIII) or formula (IX) or a mixture of formula (VIII) or formula (IX), q is 3.

Herein and throughout this document, the structure of formula (VIII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout this document, the structure of formula (IX) 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, q is 1 and the structures of formula (VIII) or formula (IX) are structures of formula (VIIIa) or (IXa):

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, 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. In some embodiments, 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.

In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (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, wherein the position of the structures of formula (VIII), formula (IX), or a mixture thereof in the amino acid sequence of the IL-2 conjugate refers to the positions in 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 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 about 1:1. In some embodiments, 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 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 IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of (VIII) and (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 (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 IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of formula (VIII) or 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 IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of formula (VIII) or 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 IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of formula (VIII) or 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 IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or formula (IX), or a mixture of formula (VIII) or 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.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or formula (IX), or a mixture of 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 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. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or formula (IX), or a mixture of 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 IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or formula (IX), or a mixture of 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.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced with a structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI):

Where:

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

q is equal to 1, 2, or 3; and

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

In some embodiments of formula (X) or formula (XI) or a mixture of formula (X) or formula (XI), q is 1. In some embodiments of formula (X) or formula (XI) or a mixture of formula (X) or formula (XI), q is 2. In some embodiments of formula (X) or formula (XI) or a mixture of formula (X) or formula (XI), q is 3.

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, q is 1 and the structures of formula (X) and formula (XI) are the structures of formula (Xa) and formula (XI a):

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.

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 (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 (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), 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 thereof in the amino acid sequence of the IL-2 conjugate is among the positions of SEQ ID NO: 3. In some embodiments, an IL-2 conjugate is provided 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 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 (X), 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), 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 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 of SEQ ID NO: 3 is position F43. In some embodiments, 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 of SEQ ID NO: 3 is position K42. In some embodiments, 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 of SEQ ID NO: 3 is position E61. In some embodiments, 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 of SEQ ID NO: 3 is position P64. In some embodiments, 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 of SEQ ID NO: 3 is position R37. In some embodiments, 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 of SEQ ID NO: 3 is position T40. In some embodiments, 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 of SEQ ID NO: 3 is position E67. In some embodiments, 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 of SEQ ID NO: 3 is position Y44. In some embodiments, 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 of SEQ ID NO: 3 is position V68. In some embodiments, 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 of SEQ ID NO: 3 is position L71.

In some embodiments, 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 about 1:1. In some embodiments, 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 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 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 (X), 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, E41, 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 (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.

In some embodiments, 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 (X), 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 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 (X), 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 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 (X), 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, 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 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 (X), 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 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 (X), 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 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 (X), 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.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced with a structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII):

Where:

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

q is equal to 1, 2, or 3; and

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

In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) or formula (XIII), q is 1. In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) or formula (XIII), q is 2. In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) or formula (XIII), q is 3.

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, q is 1 and the structures of formula (XII) and formula (XIII) are the structures of formula (XII a) and formula (XIII a):

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.

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 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 position of the structure of Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula (XIII) in the amino acid sequence of the IL-2 conjugate is 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 is a position of SEQ ID NO: 3. In some embodiments, 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 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), 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), 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), 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), 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), 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), 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 P64. In some embodiments, 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 of SEQ ID NO: 3 is position R37. In some embodiments, 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 of SEQ ID NO: 3 is position T40. In some embodiments, 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 of SEQ ID NO: 3 is position E67. In some embodiments, 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 of SEQ ID NO: 3 is position Y44. In some embodiments, 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 of SEQ ID NO: 3 is position V68. In some embodiments, 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 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, 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 (XII), 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 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 (XII), 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 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 (XII), 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 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 (XII), 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 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 (XII), 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 structure of Formula (XII) or 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 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 (XII), 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. In some embodiments, 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 (XII), 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.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2-based conjugate is replaced with a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX):

Where:

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

q is equal to 1, 2, or 3; 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 of formula (VIII) or formula (IX) or a mixture of formula (VIII) or formula (IX), q is 1. In some embodiments of formula (VIII) or formula (IX) or a mixture of formula (VIII) or formula (IX), q is 2. In some embodiments of formula (VIII) or formula (IX) or a mixture of formula (VIII) or formula (IX), q is 3.

Herein and throughout this document, the structure of formula (VIII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout this document, the structure of formula (IX) 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, q is 1 and the structures of formula (VIII) or formula (IX) are structures of formula (VIIIa) and formula (IXa):

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.

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 group 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 group is from about 30,000 Daltons. 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 group 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 group is from about 30,000 daltons.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2-based conjugate is replaced with a structure of formula (VIII) or (IX) or a mixture of (VIII) and (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;

q is equal to 1, 2, or 3; 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 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 group 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 group is from about 30,000 Daltons. 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 group 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 group is from about 30,000 daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: I, 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 group has a molecular weight of 25 kDa. In some embodiments, the PEG group has a molecular weight of 30 kDa. In some embodiments, the PEG group has a molecular weight of 35 kDa. In some embodiments, the PEG group has a molecular weight of 40 kDa. In some embodiments, the PEG group has a molecular weight of 45 kDa. In some embodiments, the PEG group has a molecular weight of 50 kDa. In some embodiments, the PEG group has a molecular weight of 60 kDa. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue in the IL-2 conjugate that is replaced with 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

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, in which 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 group.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of 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.

In some embodiments, the IL-2 conjugate comprises an amino acid sequence of SEQ ID NO: 3, wherein an amino acid other than lysine in the amino acid sequence of the IL-2 conjugate 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 group.

In some embodiments, the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced with a structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and 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 IL-2 conjugate comprises 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, m in the compounds of Formula (XIV) and Formula (XV) is 1. In some embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 2. In some embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 3. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 4. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 5. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 6. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 7. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 8. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 9. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 10. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 11. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 12. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 13. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 14. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 15. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 16. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 17. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 18. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 19. In some embodiments, m in the compounds of formula (XIV) and formula (XV) is 20.

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

In some embodiments, the IL-2 conjugate comprises 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 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 IL-2 conjugate comprises 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 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 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 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 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 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 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 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 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 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 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 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 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 compounds of formula (XIV) and (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 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 IL-2 conjugate comprises 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, the position of the structure of formula (XIV), formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-2 conjugate is 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 in SEQ ID NO: 3. In some embodiments, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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.

In some embodiments, 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 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 (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, 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 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 (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.

In some embodiments, 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, 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, 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 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 (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.

In some embodiments, 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, 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.

In some embodiments, 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 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. In some embodiments, 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 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 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 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, 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 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 of 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, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

In some embodiments, 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 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, 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 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.

In some embodiments, 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 of the compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is 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 IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced with a structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and 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 IL-2 conjugate comprises 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 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 1. In some embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 2. In some embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 3. In some embodiments, m in compounds of formula (XVI) and formula (XVII) is 4. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 5. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 6. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 7. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 8. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 9. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 10. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 11. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 12. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 13. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 14. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 15. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 16. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 17. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 18. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 19. In some embodiments, m in the compounds of formula (XVI) and formula (XVII) is 20.

In some embodiments, the IL-2 conjugate comprises 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 7 96, 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 7 96, 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 IL-2 conjugate comprises 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 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 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 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 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 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 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 compounds of Formula (XVI) and Formula (XVII), m is 5 ... 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 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 compounds of Formula (XVI) and Formula (XVII), m is 7 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds of Formula (XVI) and Formula (XVII), m is 8 and n is 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 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 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 compounds of Formula (XVI) and Formula (XVII), m is 11 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the 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 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 IL-2 conjugate comprises 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, the position of the structure of formula (XVI), 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 (XVI), formula (XVII) or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3. In some embodiments, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid

of the IL-2 conjugate sequence of SEQ ID NO: 3 is position K34. In some embodiments, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, the position of the structure of formula (XVI), 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, 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, 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, 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 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 replaced with a structure of Formula (XVI), 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, 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, 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 replaced with a structure of Formula (XVI), 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, 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, 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 replaced with a structure of formula (XVI), 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, 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, 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 replaced with a structure of Formula (XVI), 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, 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, 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 replaced with a structure of formula (XVI), 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, 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, 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 (XVI), 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 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 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 (XVI), 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. In some embodiments, 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 (XVI), 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, 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 replaced with a structure of Formula (XVI), 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 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, 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 replaced with a structure of Formula (XVI), 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 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, 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), formula (XVII), or a mixture. formula (XVI) and formula (XVII), which is replaced by 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 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, 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 replaced with a structure of Formula (XVI), 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 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, the IL-2 conjugate comprises SEQ ID NOs: 1-98. 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 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-based conjugate comprises SEQ ID NO: 1. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 2. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 3. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 4. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 5. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 6. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 7. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 8. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 9. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 10. In some embodiments, the IL-2-based conjugate comprises IL-2 comprises SEQ ID NO: 11. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 12. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 13. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 14. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 15. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 16. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 17. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 18. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 19. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 20. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 21. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 22. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 23. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 24. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 25. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 28. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 24. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 25. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 28. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 29. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 30. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 31. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 32. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 33. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 34. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 35. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 36. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 37. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 38. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 39. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 40. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 41. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 42. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 43. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 44. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 45. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 46. In some embodiments, the conjugate based on IL-2 comprises SEQ ID NO: 47. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 48. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 49. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 50. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 51. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 52. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 53. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 54. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 55. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 56. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 57. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 58. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 59. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 60. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 61. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 62. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 63. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 64. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 65. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 66. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 67. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 68. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 69. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 70. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 71. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 72. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 73. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 74. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 75. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 76. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 77. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 78. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 79. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 80. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 81. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 82. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 83. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 84. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 85. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 86. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 87. In some embodiments, the conjugate based on IL-2 comprises SEQ ID NO: 88. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 89. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 90. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 91. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 92. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 93. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 94. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 95. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 96. In some embodiments, the IL-2-based conjugate comprises SEQ ID NO: 97. In some embodiments, the IL-2-based conjugate contains SEQ ID NO: 98.

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 (XV) or any variation thereof, is inserted into a site containing a non-natural amino acid.

In some embodiments, the IL-2 conjugate is 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 is 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 embodiments, 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 linking 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 _{( OCH2CH2} ) monomers [or ( _CH2CH2O ) _monomers , depending on how the PEG is defined]. As used herein, the number of repeating units is denoted by the subscript "n" in "( _OCH2CH2 ) _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 specific molecular weight of ±10%, or 15%, or 20%, or 25%. For example, the present invention includes IL-2-based 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, approximately 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-based 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, 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 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,

characterized by an average molecular weight of about 5,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear methoxy-PEG group characterized by an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear methoxy-PEG group characterized by 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, 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 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 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, 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,

characterized by an average molecular weight of about 30,000 daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety characterized by an average molecular weight of about 50,000 daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety characterized by 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 specific molecular weight of ±10%, or 15%, or 20%, or 25%. For example, the present invention includes IL-2-based 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.

In any of the embodiments or variations of Formula (I) described herein and the pharmaceutical compositions comprising it, the average molecular weight encompasses both the weight average molecular weight and the number average molecular weight; in other words, for example, a number average molecular weight of 30 kDa and a weight average molecular weight of 30 kDa are both considered a molecular weight of 30 kDa. In some embodiments, the average molecular weight is the weight average molecular weight. In other embodiments, the average molecular weight is the number average molecular weight. In the methods provided herein, it is known that administering an IL-2 conjugate described herein to a subject comprises administering more than one molecule of the IL-2 conjugate; therefore, the use of the term "average" to describe the molecular weight of a PEG moiety refers to the average molecular weight of the PEG moieties of the IL-2 conjugate molecules at the dose administered to the subject.

Conjugation chemistry

Various conjugation reactions are used to conjugate linkers, conjugation moieties, and unnatural amino acids included in the IL-2 polypeptides 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, conjugating moieties, or unnatural amino acids. In some embodiments, the conjugation reactions are mediated by enzymes. In some embodiments, the conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123, the disclosure of which is incorporated herein by reference. In some embodiments, the conjugation reaction used herein is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417, the disclosure of which is incorporated herein by reference.

In some embodiments, the IL-2 conjugates described herein can be prepared by a conjugation reaction comprising a 1,3-dipolar cycloaddition reaction. In some embodiments, the 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, the conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached via a triazole. In some embodiments, the conjugation reaction described herein comprises a reaction of an azide with a strained olefin. In some embodiments, the conjugation reaction described herein comprises a reaction of an azide with a strained alkyne. In some embodiments, the conjugation reaction described herein involves the 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 1, 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.

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, the conjugation reaction described herein comprises the reaction shown in Scheme 2, 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.

In some embodiments, the conjugation reaction described herein comprises the reaction shown in Scheme 3, 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.

In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme 4, 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.

In some embodiments, the conjugation reaction described herein involves a cycloaddition reaction between an azide moiety, such as that contained in a protein containing an amino acid residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as that derived from DBCO, which is a chemical moiety 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 5 and 6.

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 IL-2 polypeptide

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 Expi2 93F™ 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 strains of Pichia pastoris (K. phaffii) yeast such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and a strain of Saccharomyces cerevisiae yeast such as INVScl.

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 7 942.

In some embodiments, the host cell is a prokaryotic host cell.

Illustrative prokaryotic host cells include BL21, Machl™, DH10B™, TOP10, DH5cx, DHlOBac™, 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, pFastBacl, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBac M30b, pFastBac M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 Mil, 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-DEST4 9 delivery vector, pA0815 Pichia vector, pFLD1 Pichia pastoris (K. phaffii) vector, pGAPZ A, B, and C Pichia pastoris (K. phaffii) 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 pYES3/CT yeast vector.

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-MAT 1, 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 the IL-2 polypeptide described herein. In some cases, the cell-free system comprises a mixture of cytoplasmic and/or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some 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 2 018/0051065; 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 IL-2 polypeptide is produced in a ring-rearranged form using either a host expression system or a cell-free system.

Production of IL-2 polypeptide containing unnatural amino acid

The present invention may employ an orthogonal or expanded genetic code in which one or more specific codons present in the nucleotide sequence of the IL-2 polypeptide are designed to encode an unnatural amino acid so that it can be genetically inserted into 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 the 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, a 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, efc al., "Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli," PNAS, 113(38): E5588-5597 (2016), the disclosure of which is incorporated herein by reference. 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 disclosure of which is incorporated herein by reference.

In some cases, unnatural nucleic acids are used, which results in the incorporation of one or more unnatural amino acids into 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-CH ₃ -) 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-D-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, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, 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 which 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, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-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. Many 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 disclosure of each of which is 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; 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 which 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, the disclosures of each of which are incorporated herein by reference). 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'-OCH3 and 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Unnatural nucleic acids may contain 2'-substituted 5'-CH2 (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 analogues 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 reference listed in this subsection 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 )nON[(CH ₂ ) _n CH 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 oxygen atom of the ring 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 a number of U.S. patents that teach the concept of making such modified sugar structures and that detail and describe a number 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 57 00920, the disclosures of each of which are incorporated herein by reference in their entireties.

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-(Ci-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( _CH3 )-O-2' and 4'-CH( _CH2OCH3 )-O-2' and their analogs (see U.S. Patent No. 7,399,845) _; 4'-C( _CH3 )( _CH3 )-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 Nos. 4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461, and 7,399,845; International Publication Nos. WO2004/106356, WO1994/14226, WO2005/021570, WO2007/090071, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. Provisional 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 disclosure of each listed reference in this subsection is hereby incorporated 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 linkage 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 can contain one modification. Unnatural nucleic acids can contain multiple modifications in one of the moieties or between different moieties.

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. 2 4:2318-2323; Schultz et al., (1996) Nucleic Acids Res. 2 4:2966-2973; 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. 2 7:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; US Patent No. 5453496 and Micklefield, 2001, Curr. Med. Chem. 8: 1157-117 9; the disclosures of each of which are incorporated herein by reference.

In some cases, backbone modification involves replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral, or cationic group. Examples of such modifications include the anionic internucleoside linkage; the N3'-P5' phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide-linked DNA; methylene(methylimine) linkages; formatetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholine oligonucleotides; peptide nucleic acids (PNA), and positively charged deoxyribonucleic guanidine (DNG) oligonucleotides (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). The modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, for example, 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 moiety 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 N, O, S and _CH2 constituents. Numerous United States patents disclose the preparation and use of these types of phosphate substitutions and include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. In the case of a substituent for a nucleotide, it should also 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 is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 14 97-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogues to enhance, for example, cellular uptake. Conjugates can be chemically linked to a nucleotide or nucleotide analogue. Such conjugates include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether such as hexyl-E-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, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EM50J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 4 9-54), a phospholipid, such as di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-E-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 adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237) or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.Ther., 1996, 277, 923-937). Numerous U.S. patents disclose the concept of producing such conjugates and include, but are not limited to, U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 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 disclosure of each listed source in this subsection is incorporated herein by reference.

In some cases, unnatural nucleic acids additionally form unnatural 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)TATl; (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, 109:12 005-12 010; the disclosure of each of which is incorporated herein by reference. In some embodiments, the non-natural nucleotides include:

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

where R ₂ is selected from 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, bound to a monophosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate or γ-thiotriphosphate group, or incorporated into RNA or DNA or an RNA analogue or a DNA analogue.

In some embodiments, non-natural nucleotides that can be used to prepare the IL-2-based 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. 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-based conjugates disclosed herein can be derived from

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

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

In some embodiments, non-natural nucleotides that can be used to produce the IL-2-based 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).

In some embodiments, the unnatural amino acid is incorporated into the IL-2-based polypeptide using a synthetic codon containing a unnatural nucleic acid. In some cases, the unnatural amino acid is incorporated into IL-2 using a modified synthetase/tRNA orthogonal pair. Such orthogonal pairs comprise a unnatural synthetase that is capable of loading a unnatural tRNA with the unnatural amino acid while minimizing the loading of a) other endogenous amino acids onto the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs. Such orthogonal pairs comprise tRNAs that are capable of being loaded by the unnatural 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 inserted into the IL-2 polypeptide via an aminoacyl (aaRS or RS)-tRNA synthetase-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 inserted into the IL-2 polypeptide via an Mj-TyrRS/tRNA pair. Exemplary UAAs that can be inserted via an Mj-TyrRS/tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine and 3-iodotyrosine; phenylselenocysteine; p-borophenylalanine and o-nitrobenzyltyrosine.

In some cases, the unnatural amino acid is incorporated into the IL-2 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 inserted into the IL-2 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 Methanesarcina barkeri, Methanosarcina mazei, or Methanesarcina acetivorans. Exemplary UAAs that can be inserted 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 N6-{(2-azidoethoxy)-carbonyl)-L-lysine (AzK), using M. barker! pyrrolizyl-tRNA synthetase (MbPylRS). 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 the IL-2 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), PainNTT1, 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 may be used are disclosed in Zhang et al., Nature 2011, 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 disclosure of each of which is 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 _tRNA CUA pairs, E. coli LeuRS (Ec-Leu)/B. stearothermophilus tRNA _CUA 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, described in Feldman et al., J Am Chem Soc, 2018 14 0:1447-1454 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 the 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 as E. coli, comprising (a) a PfcNTT2 nucleotide triphosphate transporter (including a truncated variant in which the first 65 amino acid residues of the full-length protein are removed), (b) a plasmid comprising a double-stranded oligonucleotide that encodes an IL-2 variant having a desired amino acid sequence and that comprises 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 a non-natural amino acid, such as JV6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), will be inserted, (c) a plasmid encoding a tRNA derived from M. mazei that comprises a non-natural nucleotide to provide a recognizable anticodon (for the IL-2 variant codon) in place of its native sequence, and (oh!) a plasmid encoding pyrrolizyl-tRNA synthetase derived from M. barker! (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 as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, a double-stranded oligonucleotide that encodes an amino acid sequence of a desired IL-2 variant comprises an ACS codon, such as at position 3, 4, 37, 40, 41, 42, 43, 44, 61, 64, 6B 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 may be a protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene from M. mazei that comprises a GYT anticodon corresponding to ACS 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 comprising the non-natural base pair in the double-stranded oligonucleotide can 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 In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise 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 In some embodiments, the M. mazei tRNA may comprise an anticodon comprising a non-natural nucleotide that recognizes a codon comprising a non-natural nucleotide of the mRNA. The anticodon in the M. mazei tRNA may comprise 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 variants

mRNA contains an unnatural 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

contains an unnatural 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

an unnatural 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 the desired average molecular weight 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 the desired average molecular weight 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 which are incorporated herein by reference.

Alternatively, IL-2 polypeptides containing unnatural amino acid(s) are produced by introducing into a host cell the nucleic acid constructs described herein that contain a tRNA and aminoacyl-tRNA synthetase and contain 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 a novel codon and anticodon, (b) ribonucleoside triphosphates required for transcription of mRNA corresponding to (i) the cytokine sequence containing the codon and (ii) an 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) inserted into the polypeptide chain corresponding to the unnatural codon. For example, one or more unnatural amino acids are inserted into the IL-2 polypeptide. Alternatively, two or more unnatural amino acids may be inserted into the IL-2 polypeptide at two or more sites in the protein.

Once the IL-2 polypeptide with the incorporated non-natural amino acid(s) has been produced in the host cell, it can be recovered from the host cell using various techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. The IL-2 polypeptide can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, or any other suitable technique known to those skilled 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.

Antibodies to EGFR

The cancer treatment methods described herein include administering an anti-EGFR antibody in combination with the IL-2 conjugates described herein.

In some embodiments, the anti-EGFR antibody is an inhibitory antibody. In some embodiments, the anti-EGFR inhibitory antibody is selected from cetuximab (Erbitux), panitumumab (Vectibix), necitumumab (Portrazza), JNJ-61186372 (amivantamab), IMC-C225, ABX-EGF, ICR62, and EMD 55900. In some embodiments, the anti-EGFR inhibitory antibody is cetuximab (Erbitux). In some embodiments, the anti-EGFR inhibitory antibody is panitumumab (Vectibix). In some embodiments, the anti-EGFR inhibitory antibody is necitumumab (Portra). In some embodiments, the anti-EGFR inhibitory antibody is JNJ-61186372 (amivantamab). In some embodiments, the EGFR inhibitory antibody is IMC-C225. In some embodiments, the EGFR inhibitory antibody is ABX-EGF. In some embodiments, the EGFR inhibitory antibody is ICR62. In some embodiments, the EGFR inhibitory antibody is EMD 55900.

Treatment methods

In one aspect, provided herein are methods of treating 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) an anti-EGFR antibody.

Also provided is an IL-2 conjugate described herein for use in the method disclosed herein for treating cancer in a subject in need thereof.

In a further aspect, there is provided the use of the IL-2 conjugate described herein for the manufacture of a medicament for the method disclosed herein for treating cancer in a subject in need thereof.

Types of cancer

In some embodiments, 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 stability 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) mechanisms, 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 disseminating systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma.

In some embodiments, 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 some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is Merkel cell carcinoma (MCC). In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC).

In some embodiments, the cancer is in the form of a solid tumor. In some embodiments, the cancer is a late-stage solid tumor or a metastatic solid tumor. In some embodiments, the cancer is in the form of a liquid tissue tumor.

Introduction

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, the duration of treatment is no more than 24 months, such as 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, or 24 months. In some embodiments, the duration of treatment is further increased to no more than 24 months.

In some embodiments, the IL-2 conjugate is administered to the subject prior to administration of the anti-EGFR antibody to the subject. In some embodiments, the anti-EGFR antibody is administered to the subject prior to administration of the IL-2 conjugate to the subject. In some embodiments, the IL-2 conjugate and the anti-EGFR antibody are administered to the subject simultaneously. In some embodiments, the IL-2 conjugate is administered to the subject separately from administration of the anti-EGFR antibody. In some embodiments, the IL-2 conjugate and the anti-EGFR antibody are administered to the subject sequentially. In some embodiments, the IL-2 conjugate and the anti-EGFR antibody are administered to the subject on the same day. In some embodiments, the IL-2 conjugate and the anti-EGFR antibody are administered to the subject on different days.

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, an effective amount of an IL-2 conjugate is administered about once every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, an effective amount of an IL-2 conjugate is administered about once every 14, 15, 16, 17, 18, 19, 20, or 21 days.

In some embodiments, the amount of said agent that corresponds to such an amount varies depending on factors such as the specific 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 specific 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 IL-2 conjugate is administered at a dose of about 8 μg/kg to 24 μg/kg. In some embodiments, the IL-2 conjugate is administered at a dose of about 8 μg/kg. In some embodiments, the IL-2 conjugate is administered at a dose of about 16 μg/kg. In some embodiments, the IL-2 conjugate is administered at a dose of about 24 μg/kg. In any of these embodiments, the IL-2 conjugate is administered at a dose described herein once every 3 weeks.

In some embodiments, the anti-EGFR antibody may be administered at a dose and using a dosing regimen that has been determined to be safe and effective for the antibody, alone or in combination with an IL-2 conjugate. In some embodiments, the anti-EGFR antibody is administered by intravenous infusion. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once per 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, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once a week. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every two weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every three weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 4 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 5 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 6 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 7 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 8 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 9 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 10 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 11 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 12 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 13 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 14 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 15 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 16 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 17 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 18 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 19 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 20 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 21 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 22 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 23 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need thereof once every 24 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered about once every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, cetuximab is administered at a loading dose of about 100 mg/ ^m2 to about 500 mg/ ^m2 by intravenous infusion. In any of the embodiments described herein, the loading dose of cetuximab is given in mg/ ^m2 of body surface area of the subject. In some embodiments, cetuximab is administered at a loading dose of about 100 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 150 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 200 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 250 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 300 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 350 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 400 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 450 mg/ ^m2 by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 500 mg/ ^m2 by intravenous infusion. In some embodiments, an initial dose of cetuximab is administered at a loading dose of about 400 mg/ ^m2 by intravenous infusion, and all subsequent doses of cetuximab are administered at a loading dose of about 250 mg/ ^m2 by intravenous infusion. In any of such embodiments, the cetuximab is infused over about 30-240 minutes. In some embodiments, the cetuximab is infused over about 30 minutes. In some embodiments, the cetuximab is infused over about 60 minutes. In some embodiments, the cetuximab is infused over about 90 minutes. In some embodiments, the cetuximab infusion is performed over about 120 minutes. In some embodiments, the cetuximab infusion is performed over about 150 minutes. In some embodiments, the cetuximab infusion is performed over about 180 minutes. In some embodiments, the cetuximab infusion is performed over about 210 minutes. In some embodiments, the cetuximab infusion is performed over about 240 minutes. In any of these embodiments, cetuximab is administered at an infusion rate of from about 1 mg/min to about 10 mg/min, such as 1 mg/min, 2 mg/min, 3 mg/min, 4 mg/min, 5 mg/min, 6 mg/min, 7 mg/min, 8 mg/min, 9 mg/min, or 10 mg/min. In some embodiments, the first dose of cetuximab is administered at a higher loading dose than subsequent doses of cetuximab. In some embodiments, the infusion time of the first dose of cetuximab is longer than the infusion time of subsequent doses of cetuximab. In some embodiments, cetuximab is administered at the dose described herein once every 3 weeks. In some embodiments, cetuximab is administered at the dose described herein once every 2 weeks. In some embodiments, cetuximab is administered at the dose described herein once a week.

Additional means/medications for premedication

In some embodiments, any of the methods described herein further comprises administering an antihistamine. In some embodiments, the antihistamine is cetirizine. In some embodiments, the antihistamine is promethazine. In some embodiments, the antihistamine is dexchlorpheniramine. In some embodiments, the antihistamine is diphenhydramine. In some embodiments, diphenhydramine is administered intravenously at a dose of about 25 to 50 mg.

In some embodiments, any of the methods described herein further comprises administering a pain reliever, such as acetaminophen. In some embodiments, acetaminophen is administered orally at a dose of about 650 to 1000 mg.

In some embodiments, any of the methods described herein further comprises administering a serotonin 5-HT ₃ receptor antagonist. In some embodiments, the serotonin 5-HT ₃ receptor antagonist is granisetron. In some embodiments, the serotonin 5-HT ₃ receptor antagonist is dolasetron. In some embodiments, the serotonin 5-HT ₃ receptor antagonist is tropisetron. In some embodiments, the serotonin 5-HT ₃ receptor antagonist is palonosetron. In some embodiments, the serotonin 5-HT ₃ receptor antagonist is ondansetron. In some embodiments, ondansetron is administered intravenously at a dose of from about 8 mg to 0.15 mg/kg.

In some embodiments, any of the methods described herein further comprises administering an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine), an analgesic (such as acetaminophen), and/or a serotonin 5-HT ₃ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, the method further comprises administering an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine) and an analgesic (such as acetaminophen). In some embodiments, the method further comprises administering an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine) and a serotonin 5-HT ₃ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, the method further comprises administering a pain reliever (such as acetaminophen) and a serotonin 5-HT ₃ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, any of the methods described herein further comprises administering an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine), an analgesic (such as acetaminophen), and a serotonin 5-HT ³ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron).

In some embodiments, any of the methods described herein further comprises administering a premedication drug, such as to prevent or reduce the incidence of acute infusion reactions (IARs) or flu-like symptoms. In some embodiments, the premedication drug is administered prior to administering the IL-2 conjugate and/or the anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication drug is administered prior to administering the IL-2 conjugate. In some embodiments, the premedication drug is administered prior to administering the anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication drug is administered prior to administering the IL-2 conjugate and the anti-EGFR antibody (such as cetuximab).

In some embodiments, the premedication medicament for the IL-2-based conjugate is different from the premedication medicament for the anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication medicament for the IL-2-based conjugate is the same as the premedication medicament for the anti-EGFR antibody (such as cetuximab). In some cases, where the premedication medicament for the IL-2-based conjugate and the anti-EGFR antibody (such as cetuximab) is the same, only one dose of the premedication medicament is administered. In other cases, where the premedication medicament for the IL-2-based conjugate and the anti-EGFR antibody (such as cetuximab) is the same, multiple doses of the premedication medicament are administered. In some embodiments, the premedication drug is administered for all doses of the IL-2 conjugate administered. In some embodiments, the premedication drug is administered for the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of the IL-2 conjugate and is not administered for any subsequent doses of the IL-2 conjugate. In some embodiments, the premedication drug is administered for the first 4 doses of the IL-2 conjugate and is not administered for any subsequent doses of the IL-2 conjugate. In some embodiments, the premedication drug is administered for all doses of the anti-EGFR antibody (such as cetuximab) administered. In some embodiments, the premedication drug is administered for the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of the anti-EGFR antibody (such as cetuximab) and is not administered for any subsequent doses of the anti-EGFR antibody. In some embodiments, the premedication drug is administered for the first dose of the anti-EGFR antibody (such as cetuximab) and is not administered for any subsequent doses of the anti-EGFR antibody.

In some embodiments, any of the methods described herein further comprises administering a premedication drug prior to administering the IL-2 conjugate. In some embodiments, the premedication drug prior to the IL-2 conjugate is an antihistamine such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine. In some embodiments, the antihistamine is diphenhydramine. In some embodiments, diphenhydramine is administered intravenously at a dose of about 25 to 50 mg. In some embodiments, the premedication drug prior to the IL-2 conjugate is a serotonin 5-HT ₃ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, the serotonin 5-HT ₃ receptor antagonist is ondansetron. In some embodiments, ondansetron is administered intravenously at a dose of about 8 mg to 0.15 mg/kg. In some embodiments, the premedication drug prior to the IL-2 conjugate is a pain reliever (such as acetaminophen). In some embodiments, acetaminophen is administered orally at a dose of about 650 mg to 1000 mg.

In some embodiments, any of the methods described herein further comprises administering a premedication drug prior to administration of the anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication drug prior to the EGFR antibody is an antihistamine such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine. In some embodiments, the antihistamine is diphenhydramine. In some embodiments, the diphenhydramine is administered intravenously at a dose of about 25 to 50 mg. In some embodiments, the premedication drug prior to cetuximab is a serotonin 5-HT ₃ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, the serotonin 5-HT ₃ receptor antagonist is ondansetron. In some embodiments, ondansetron is administered intravenously at a dose of about 8 mg to 0.15 mg/kg. In some embodiments, the premedication drug for the EGFR antibody is a pain reliever (such as acetaminophen). In some embodiments, acetaminophen is administered orally at a dose of about 650 mg to 1000 mg.

In some embodiments, any of the methods described herein further comprises administering a first dose of a premedication drug prior to administration of the IL-2-based conjugate and a second dose of a premedication drug prior to administration of the anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication drug for the IL-2-based conjugate is the same as the premedication drug for the anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication drug for the IL-2-based conjugate is different from the premedication drug for the anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication drug is an antihistamine such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine. In some embodiments, the antihistamine is diphenhydramine. In some embodiments, diphenhydramine is administered intravenously at a dose of about 25 to 50 mg. In some embodiments, the premedication drug is a serotonin 5-HT ₃ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, the serotonin 5-HT ₃ receptor antagonist is ondansetron. In some embodiments, ondansetron is administered intravenously at a dose of about 8 mg to 0.15 mg/kg. In some embodiments, the premedication drug is a pain reliever (such as acetaminophen). In some embodiments, acetaminophen is administered orally at a dose of about 650 to 1000 mg. In some embodiments, the premedication medicament comprises an antihistamine and a 5-HT ₃ serotonin receptor antagonist. In some embodiments, the premedication medicament comprises an antihistamine and an analgesic. In some embodiments, the premedication medicament comprises a 5-HT ₃ serotonin receptor antagonist and an analgesic. In some embodiments, the premedication medicament comprises an antihistamine, a 5-HT ₃ serotonin receptor antagonist and an analgesic. In some cases, where the premedication medicament for the IL-2-EGFR antibody conjugate (such as cetuximab) is the same (such as diphenhydramine), only one dose of the premedication medicament is administered. In other cases, where the premedication drug for the IL-2-EGFR antibody conjugate (such as cetuximab) is the same, multiple doses of the premedication drug are administered.

In some embodiments of the methods described herein, the sequence of administration is as follows: (i) a premedication drug for an anti-EGFR antibody (such as cetuximab); (ii) an anti-EGFR antibody (such as cetuximab); (iii) a premedication drug for an IL-2 conjugate; and (iv) an IL-2 conjugate. In some embodiments where the premedication drug for the anti-EGFR antibody (such as cetuximab) is the same as the premedication drug for the IL-2 conjugate (such as diphenhydramine), administration of the premedication drug for the IL-2 conjugate may not be involved. In some embodiments, the sequence of administration is as follows: (i) a premedication drug for an IL-2 conjugate; (ii) an IL-2-based conjugate; (iii) a premedication drug for an anti-EGFR antibody (such as cetuximab); and (iv) an anti-EGFR antibody (such as cetuximab). In some embodiments, where the premedication drug for the anti-EGFR antibody (such as cetuximab) is the same as the premedication drug for the IL-2-based conjugate (such as diphenhydramine), administration of the premedication drug for the anti-EGFR antibody may not be provided.

Subject

In some embodiments, administering an effective amount of an IL-2-anti-EGFR antibody conjugate is to an adult. In some embodiments, the adult is a male. In other embodiments, the adult is a female. In some embodiments, the adult is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age. In some embodiments, administering an effective amount of an IL-2-anti-EGFR antibody conjugate is to an infant, child, or adolescent. In some embodiments, the subject is at least 1 month, 2 months, 3 months, 6 months, 9 months, or 12 months old. In some embodiments, the subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 years old.

In some embodiments, the subject has a measurable disease (i.e., cancer) as defined by RECIST v1.1. In some embodiments, the subject has been determined to have a performance status score of 0 or 1 according to the Eastern Cooperative Oncology Group (ECOG) criteria. In some embodiments, the subject has satisfactory cardiovascular function, satisfactory hematologic, hepatic, and renal function as determined by a physician. In some embodiments, it has been determined (e.g., by a physician) that the subject has a life expectancy of 12 weeks or greater.

In some embodiments, the subject has satisfactory cardiovascular function, satisfactory hematological, hepatic, and renal function.

In some embodiments, the subject has a histologically or cytologically confirmed diagnosis of advanced solid tumors and/or metastatic solid tumors with at least one tumor lesion in a location accessible for safe biopsy in accordance with clinical judgment (i.e., as determined by a physician). In some embodiments, the subject has previously received anti-cancer therapy prior to administration of the first dose of the treatment agent. In some embodiments, treatment-related toxicity from the previous anti-cancer therapy has been resolved to an appropriate level.

In some embodiments, the subject is a woman of childbearing potential and uses a medically approved method of contraception during treatment and for at least 3 months after the last dose of the treatment. In some embodiments, the subject is a premenopausal woman who has had a negative pregnancy test result (according to a serum pregnancy test) within 7 days before the first dose of the treatment. In some embodiments, the subject is a woman who is less than 12 months post-menopause and has had a negative pregnancy test result (according to a serum pregnancy test) within 7 days before the first dose of the treatment.

In some embodiments, the subject is a male who is not surgically sterilized and who uses a medically approved method of contraception during the treatment and for at least 3 months after the last dose. In some embodiments, the male does not donate sperm or donate sperm to a sperm bank during the treatment period and for at least 3 months after the last dose of the treatment agent.

In some embodiments, the subject has not received radiation therapy within 14 days prior to the first dose of the treatment agent. In some embodiments, the subject has not received palliative radiation therapy or stereotactic radiosurgery within 7 days prior to the first dose of the treatment agent.

In some embodiments, the subject has not been treated with a systemic anticancer therapy or an experimental anticancer agent for 2 weeks prior to the first dose of the treatment agent. In some embodiments, the subject has not been treated with an immunotherapy or a tyrosine kinase inhibitor therapy for 4 weeks prior to the first dose of the treatment agent.

In some embodiments, the subject has not undergone radical surgery for 30 days prior to the first dose of the treatment. In some embodiments, the subject has undergone radical surgery for more than 30 days prior to the first dose of the treatment and has recovered to at least grade 1 of any adverse effects associated with the procedure. In some embodiments, the subject is not expected to require radical surgery during the course of treatment.

In some embodiments, the subject has no active autoimmune disease requiring systemic treatment for 3 months prior to the first dose of the treatment. In some embodiments, the subject has no history of an autoimmune disease with a severe clinical course that requires systemic administration of steroids or immunosuppressants prior to the first dose of the treatment.

In some embodiments, the subject does not have a primary central nervous system (CNS) disease or leptomeningeal disease. In some embodiments, the subject is known to have CNS metastases, but has received appropriate treatment and is asymptomatic, has no evidence of progression by radiology for at least 8 weeks prior to the first dose of the treatment agent, and has not required steroids or liver enzyme-inducing anticonvulsants for 14 days prior to the first dose of the treatment agent.

In some embodiments, the subject does not have impaired pulmonary function, including pneumonitis, active pneumonitis, interstitial lung disease requiring steroids, idiopathic pulmonary fibrosis, documented pleural effusion, and severe dyspnea at rest or requiring supplemental oxygen therapy, within 6 months prior to the first dose of the treatment agent.

In some embodiments, the subject has not received parenteral antibiotics for 14 days prior to the first dose of the treatment agent.

In some embodiments, the subject does not have a history of allogeneic transplantation or solid organ transplantation. In some embodiments, the subject does not have human immunodeficiency virus (HIV) infection or active hepatitis C infection. In some embodiments, the subject does not have uncontrolled hepatitis B virus (HBV) infection.

In some embodiments, the subject has not had clinically significant bleeding (e.g., gastrointestinal bleeding, intracranial hemorrhage) within 2 weeks prior to the first dose of the treatment. In some embodiments, the subject has not previously been diagnosed with deep vein thrombosis or pulmonary embolism within 3 months prior to the first dose of the treatment.

In some embodiments, the subject does not have a severe or unstable cardiac condition (such as congestive heart failure (New York Heart Association class III or IV), coronary artery bypass grafting or stenting, angioplasty, cardiac ejection fraction below the lower limit of normal, unstable angina, uncontrolled hypertension (e.g., ≥160 mmHg systolic or ≥100 mmHg diastolic), uncontrolled cardiac arrhythmia requiring medical therapy (≥grade 2 according to NCI CTCAE v5.0), or myocardial infarction) within 6 months prior to administration of the first dose of the treatment agent.

In some embodiments, the subject does not have a history of drug-induced corrected prolongation of the QT interval, determined using the Frederick formula (QTcF), of >450 milliseconds (ms) in men or >470 ms in women.

In some embodiments, the subject has no known hypersensitivity or contraindication to any of the IL-2 conjugates disclosed herein, PEG, pegylated drugs, or an anti-EGFR antibody such as, for example, cetuximab.

In some embodiments, the subject does not have an active second malignancy. In some embodiments, the subject does not have a history of malignancy. In some embodiments, the subject had non-melanoma skin cancer or cervical cancer that was surgically removed to provide a cure, prior to administration of the first dose of the treatment agent.

In some embodiments, the subject does not have any serious medical condition (including a pre-existing autoimmune disease or inflammatory disorder), abnormal laboratory values, psychiatric illness, or any other significant or unstable comorbidity that would preclude treatment or make treatment inappropriate.

In some embodiments, the subject is not pregnant or breastfeeding. In some embodiments, the subject is not expected to conceive or father children during the course of treatment and for no more than 3 months after administration of the final dose of the treatment agent.

In some embodiments, the subject does not receive concomitant therapy with any experimental agents, vaccines, or devices during the course of treatment. In some embodiments, the subject receives concomitant therapy with experimental agents, vaccines, or devices during the course of treatment upon approval by a physician.

Effects mediated by administration

In some embodiments, following administration of the IL-2-anti-EGFR conjugate, the subject exhibits a response according to the Response Evaluation Criteria in Solid Tumors to Immunotherapy (iRECIST). In some embodiments, following administration of the IL-2-anti-EGFR conjugate, the subject is assessed for objective response rate (ORR) according to RECIST version 1.1. In some embodiments, following administration of the IL-2-anti-EGFR conjugate, the subject is assessed for duration of response (DOR) according to RECIST version 1.1. In some embodiments, following administration of the IL-2-anti-EGFR conjugate, the subject is assessed for progression-free survival (PFS) according to RECIST version 1.1. In some embodiments, following administration of the IL-2-anti-EGFR conjugate, the subject is assessed for overall survival in accordance with RECIST version 1.1. In some embodiments, following administration of the IL-2-anti-EGFR conjugate, the subject is assessed for time to treatment response (TTR) in accordance with RECIST version 1.1. In some embodiments, following administration of the IL-2-anti-EGFR conjugate, the subject is assessed for disease control rate (DCR) in accordance with RECIST version 1.1. In any of such embodiments, the assessment of the subject is based on a physician's review of radiographic images obtained from the subject.

In some embodiments, treatment is interrupted based on the results of a physician's analysis of radiographic images obtained from the subject.

In some embodiments, the treatment is interrupted based on the physician's analysis of immunophenotyping data from peripheral blood at different time points. In some embodiments, the treatment is interrupted based on the physician's analysis of immunophenotyping data from tumor samples at different time points. In some embodiments, the treatment is interrupted based on the physician's analysis of the presence of antibodies to any of the IL-2 conjugates disclosed herein at different time points. In some embodiments, the treatment is interrupted based on the physician's analysis of the plasma concentration of any of the IL-2 conjugates disclosed herein at different time points. In any of such embodiments, the physician's analysis is based on a corresponding analysis of the parameters in question.

In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause vascular leak syndrome in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause grade 2, grade 3, or grade 4 vascular leak syndrome in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause grade 2 vascular leak syndrome in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause grade 3 vascular leak syndrome in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause grade 4 vascular leak syndrome in the subject.

In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause a loss of vascular tone in the subject.

In some embodiments, administration to a subject of an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause extravasation of proteins and plasma fluid into the extravascular space in the subject.

In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause hypotension or decreased organ perfusion in the subject.

In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause a neutrophil dysfunction in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause a decrease in chemotaxis in the subject.

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

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

In some embodiments, administering to a subject an effective amount of an IL-2-antibody conjugate 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, administering to a subject an effective amount of an IL-2-antibody conjugate does not cause seizures in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-antibody conjugate is not contraindicated in subjects known to have epilepsy.

In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause capillary leak syndrome in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause capillary leak syndrome grade 2, grade 3, or grade 4 in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause capillary leak syndrome grade 2 in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause capillary leak syndrome grade 3 in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR conjugate does not cause capillary leak syndrome grade 4 in the subject.

In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause the subject to experience an acute decrease in mean arterial blood pressure following administration of the IL-2-based conjugate to the subject. In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause the subject to experience hypotension. In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause the subject to experience a systolic blood pressure below 90 mmHg or an acute decrease of 20 mmHg from a baseline systolic pressure.

In some embodiments, administration to a subject of an effective amount of an IL-2-anti-EGFR conjugate does not cause edema or renal or hepatic dysfunction in the subject.

In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR antibody conjugate does not cause eosinophilia in the subject. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR antibody conjugate does not cause the subject to have a peripheral blood eosinophil count greater than 500 per μL. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR antibody conjugate does not cause the subject to have a peripheral blood eosinophil count greater than 500 per μL to 1500 per μL. In some embodiments, administering to a subject an effective amount of an IL-2-anti-EGFR antibody conjugate does not cause the subject to have a peripheral blood eosinophil count greater than 1500 per μL to 5000 per μL. In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody does not cause the subject's peripheral blood eosinophil count to exceed 5,000 per μL. In some embodiments, administering to a subject an effective amount of an IL-2-based conjugate and an anti-EGFR antibody is not contraindicated in subjects on a psychotropic drug regimen.

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

In some embodiments, administering to a subject an effective amount of an IL-2 conjugate and an anti-EGFR antibody does not cause in the subject one or more grade 4 adverse events following administration of the IL-2 conjugate to the subject. In some embodiments, the grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystoles; 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; bloody 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, administering an effective amount of an IL-2 conjugate and an anti-EGFR antibody 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, the grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystoles; 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 tests abnormal; gastrointestinal hemorrhage; hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; alkaline phosphatase increased; blood urea nitrogen (BUN) increased; hyperuricemia; non-protein nitrogen (NPN) increased; 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, administering an effective amount of an IL-2-based conjugate and an anti-EGFR antibody 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-based 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, administration of an effective amount of an IL-2-EGFR antibody conjugate to a group of subjects does not cause one or more adverse events in more than 1% of subjects after administration, 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, administering an IL-2-anti-EGFR antibody conjugate to a subject results in an increase in the number of peripheral CD8+ T and NK cells in the subject without an increase in the number of peripheral regulatory CD4+ T cells in the subject. In some embodiments, administering an IL-2-anti-EGFR antibody conjugate to a subject results in an increase in the number of peripheral CD8+ T and NK cells in the subject without an increase in the number of peripheral blood eosinophils in the subject. In some embodiments, administering an IL-2-anti-EGFR antibody conjugate to a subject results in an increase in the number of peripheral CD8+ T and NK cells in the subject without an increase in the number of intratumoral CD8+ T and NK cells in the subject and without an increase in the number of intratumoral CD4+ regulatory T cells in the subject.

In some embodiments, administering to a subject an effective amount of an IL-2 conjugate and an anti-EGFR antibody does not require access to an intensive care unit or skilled cardiopulmonary or intensive care physicians. In some embodiments, administering to a subject an effective amount of an IL-2 conjugate does not require access to an intensive care unit or skilled cardiopulmonary or intensive care physicians. In some embodiments, administering to a subject an effective amount of an IL-2 conjugate does not require access to an intensive care unit. In some embodiments, administering to a subject an effective amount of an IL-2 conjugate does not require skilled cardiopulmonary or intensive care physicians.

In some embodiments, administration of a combination therapy comprising an IL-2-based conjugate and an anti-EGFR antibody results in an improved ADCC response to cancer, such as by improving the ADCC function of the anti-EGFR antibody. In some embodiments, administration of a combination therapy comprising an IL-2-based conjugate and an anti-EGFR antibody results in expansion of innate and adaptive immune system cells. In some embodiments, administration of a combination therapy comprising an IL-2-based conjugate and an anti-EGFR antibody results in activation of the immune system within the tumor microenvironment. In some embodiments, administration of an IL-2-based conjugate and an anti-EGFR antibody results in a synergistic improvement in the anti-cancer activity of the combination of the two agents, compared to the anti-cancer activity of each of the two agents alone. In some embodiments, administration of an IL-2-based conjugate results in an increase in the number and quantity of activated NK cells that enhance ADCC stimulated by an anti-EGFR antibody.

Additional funds

In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of one or more chemotherapeutic agents in addition to the anti-EGFR antibody. 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).

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 compartmentalized to accommodate one or more containers, such as vials, tubes, and the like, wherein each of the containers comprises 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 comprises an IL-2 conjugate and an anti-EGFR antibody.

The kit usually contains labels listing the 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 P1. 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-based conjugate and (b) an antibody to EGFR, wherein the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2-based conjugate is replaced by a structure of formula (I):

Where:

Z is _CH2 and Y is

Y is _CH2 and Z is

Z is _CH2 and Y is or

Y is _CH2 and Z is

W is a PEG group characterized by 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;

where 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,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment P2. The method of embodiment P1, wherein in the IL-2 conjugate, Z is _CH2 and Y is

Embodiment P3. The method of embodiment P1, wherein in the IL-2 conjugate, Y is CH2 and Z is

Embodiment P4. The method according to any one of embodiments P1-3, wherein in the IL-2-based conjugate the PEG group has an average molecular weight of 25 kDa, 30 kDa, or 35 kDa.

Embodiment P5. The method according to embodiment 4, wherein in the IL-2-based conjugate the PEG group has an average molecular weight of 30 kDa.

Embodiment P6. The method according to any one of embodiments P1-5, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2-based conjugate is P64.

Embodiment P7. The method according to embodiment P1, wherein the structure of formula (I) has a structure of formula (XII) or formula (XIII) or is a mixture of structures of formula (XII) and formula (XIII):

Where:

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

wavy lines indicate covalent bonds with amino acid residues in SEQ ID NO: 3 that are not replaced,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment P8. The method of embodiment P7, 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 P9. The method of embodiment P8, wherein in the IL-2 conjugate n is an integer such that -( _OCH2CH2 ) _{n -OCH3 has} a molecular weight of about 30 kDa.

Embodiment P10. The method according to any one of embodiments P7-9, wherein the position of the structure of formula (XII) or formula (XIII) in the amino acid sequence of the IL-2-based conjugate is P64.

Embodiment P11. 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-based conjugate and (b) an anti-EGFR antibody, wherein the IL-2-based 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):

Where:

W is a PEG group characterized by 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;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment P12. The method according to embodiment P11, wherein W is a PEG group having an average molecular weight selected from 25 kDa, 30 kDa, or 35 kDa.

Embodiment P13. The method of embodiment P12, wherein W is a PEG group having an average molecular weight of 30 kDa.

Embodiment P14. The method according to any one of embodiments P1-13, wherein the anti-EGFR antibody is cetuximab.

Embodiment P15. 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-based conjugate and (b) cetuximab, wherein the IL-2-based 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):

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 with amino acid residues in SEQ ID NO: 50 that are not replaced,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Implementation option P16. The method according to any one of embodiments P1-15, 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) mechanisms, 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 disseminating systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma.

Embodiment P17. The method of any one of embodiments P1-16, 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 P18. The method of any one of embodiments P1-17, wherein the IL-2 conjugate is administered to the subject via intravenous administration.

EXAMPLES

These examples are provided for illustrative purposes only and do not limit the scope of the claims presented herein.

Example 1. Obtaining a conjugate based on IL-2 IL-2_P65_[AzK_L1_PEG30kDa]-1

IL-2 used for bioconjugation was expressed as inclusion bodies in E. coli by 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 provide a codon at the desired position into which the unnatural amino acid N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) was inserted, and (ii) a tRNA derived from M. mazei Pyl, the gene of which contains a second unnatural nucleotide to provide a corresponding anticodon in place of its native sequence; (b) a plasmid encoding pyrrolizyl-tRNA synthetase derived from M. 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 ACS as codon 64 in the sequence that encodes the protein having SEQ ID NO: 3, in which P64 is replaced with a non-natural amino acid described herein. The plasmid encoding the orthogonal tRNA gene from M. mazei contained the anticodon GYT corresponding to ACS in place of its native sequence, wherein Y is a non-natural nucleotide disclosed herein. X and Y are selected from the non-natural nucleotides dTPT3 and dNaM disclosed herein. Expressed protein was extracted from inclusion bodies and refolded using standard procedures prior to site-specific PEGylation of the AzK-containing IL-2 product using copper-free DBC0-mediated click chemistry to stably covalently attach mPEG (a linear methoxy-PEG group with an average molecular weight of 30 kDa) moieties to AzK (as shown in Scheme 6 above).

The IL-2 conjugate "IL-2_P65_[AzK_L1_PEG3OkDa]-1" comprises SEQ ID NO: 50, wherein the proline at position 64 is replaced with AzK_L1_PEG30kDa, wherein 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 IL-2 conjugate "IL-2 P65 [AzK_L1_PEG30kDa]-1" is also defined as a compound comprising SEQ ID NO: 3, wherein the proline residue at position 64 (P64) 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 group is approximately 30 kDa. The IL-2-based conjugate "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), wherein n is an integer such that the molecular weight of the PEG group is approximately 30 kDa.

Example 2. Antibody-dependent cellular cytotoxicity (ADCC) assays using IL-2_P65_[AzK_L1_PEG30kDa]-1 and cetuximab

The effect of IL-2_P65_[AzK_L1_PE630kDa]-1 on the ADCC function of cetuximab was examined using a calcein-acetoxymethyl (calcein-AM; Invitrogen) release assay.

Materials

The following cell lines were used: A431 (a cell line with high EGFR expression), human PBMCs were obtained from healthy donors and enriched using the EasySep Human NK Cell Enrichment Kit (Stemcell).

The following reagents were used: calcein-acetoxymethyl (calcein-AM; Invitrogen), probenecid (Invitrogen), very low IgG fetal bovine serum (Thermofisher), and human IgG1 isotype antibody (Biolegend).

Analysis procedure.

Primary human NK cells were negatively selected from PBMC using the RoboSep™ instrument according to the manufacturer's recommended protocols. Purified NK cells were cultured with IL-2_P65_[AzK_L1_PE630kDa]-1 at varying concentrations (0.1 μg/ml, 0.01 μg/ml, 0.001 μg/ml, and 0 μg/ml) in RPMI 1640 medium supplemented with 1% low-IgG FBS for 18 h at 37°C in a humidity-controlled incubator with 5% CO ₂ . These cultured cells were used as effector cells. EGFR-positive human cancer cell line (A431) was labeled with calcein-AM for 30 min (50 μg was diluted in 25 μl DMSO to make a stock solution, then 10 μl calcein stock in 4 ml RPMI 1640+1% low-IgG FBS+1% probenecid was added to stain 4×10 ⁶ cells), then washed and seeded in 96-well round-bottom plates at a density of 5×10 ³ cells/well. Cetuximab and human IgG1 isotype antibody were added at different concentrations (10 μg/ml to 1 pg/ml) for 30 min to allow opsonization before addition of NK cells. NK cells activated by IL-2_P65_[AzK_L1_PEG30kDa]-1 were harvested and added as effector cells at an E:T ratio of 3:1 (6× ¹⁰⁴ NK cells for 2× ¹⁰⁴ target cells). The plates were then incubated for 1 h at 37°C in a humidity-controlled incubator with 5% _CO2 , and 90 μl of the supernatants were collected and transferred to opaque 96-well microplates for analysis using fluorometry on an Envision 2104 plate reader (excitation: 492 nm; emission: 515 nm).

For maximum release, cells were lysed with 2% Triton X-100. The background fluorescence value of the culture medium was subtracted from the fluorescence values of experimental release (A), spontaneous release of target cells (B), and maximum release of target cells (C).

The percentage cytotoxicity and ADCC values for each plate (in duplicate) were calculated according to the following formulas:

For each experiment, measurements were performed in triplicate using triplicate wells. Each experiment was repeated at least 3 times. Half-maximal effective concentration (EC50) values were calculated by fitting data points to a 4-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA).

Results.

Without activation by IL-2_P65_[AzK_L1_PEG30kDa]-1, cetuximab-treated human NK cells exhibited cytotoxicity against EGFR-expressing cancer cell lines (A431 and A549), but not against EGFR-null (NCI-H69) cells. After activation by IL-2_P65_[AzK_L1_PEG30kDa]-1, cetuximab-treated human NK cells exhibited enhanced cytotoxicity against EGFR-expressing cancer cell lines (A431 and A549).

Example 3. In vitro study of IL-2 and cetuximab conjugate (ADCC assay against PBMC)

The study was conducted to examine the effects

antibody-dependent cellular cytotoxicity (ADCC) of the IL-2 conjugate from Example 1 in combination with cetuximab using co-culture of human PBMC with calcein-labeled cancer cell lines (CAL27 and A431).

CKL27 cells.

Reagents

Biological Assay Buffer: 1% FBS with very low IgG content was added to RPMI without phenol red. Complete Assay Buffer: 450 μM probenecid was added to 45 ml of biological assay buffer for a final concentration of probenecid of 77 μg/ml. Calcein acetoxymethyl ester (calcein-AM): 50 μg in 25 μl DMSO. Calcein-AM Staining Buffer: 10 μl calcein-AM was added to 4 ml of Complete Assay Buffer (final concentration of calcein-AM 5 μg/ml). Triton-X-100 Lysis Buffer: 20 μl Triton-X-100 was added to 4 ml of Complete Assay Buffer (final concentration 0.5%).

Analysis procedure.

On day 1, a 1 in 5 6-point dilution series of IL-2 conjugate was prepared (in PBS). IL-2 conjugate concentrations were 2, 0.4, 0.08, 0.016, 0.0032, and 0 μg/mL. PBMCs were collected by centrifugation at 200 × g for 5 min and resuspended in RPMI without phenol red +10% very low IgG at 20 million cells/mL. Appropriate volumes of these PBMCs were transferred to a 6-well multiwell vial to which a range of IL-2 conjugate dilutions were added. PBMC were mixed well with IL-2 conjugate by pipetting and 50 μl was transferred to round-bottomed 96-well plates using a multichannel pipette (total PBMC per well was 1 million). Six blank wells were left as controls to be added the following day. Plates were incubated overnight in a humidity-controlled incubator at 37°C in the presence of 5% carbon dioxide.

On day 2, CAL27 cells (EGFR-expressing oral epithelial squamous cell carcinoma cell line) were harvested using TrypLE rapid dissociation buffer and recovered by centrifugation at 200 × g for 5 min. Cells were counted and 5 million cells were resuspended in 4 ml of calcein-AM staining buffer and incubated for 30 min at 37°C in the presence of 5% carbon dioxide. Cells were then collected and washed twice in complete assay buffer by centrifugation at 200 × g for 5 min. Cells were counted and resuspended at 0.4 million cells/ml to generate a final target cell count of 20,000/well.

Cetuximab antibody (Eli Lilly & Co.) was diluted to a working concentration of 3X (3, 0.3, 0.03, 0.003 μg/mL) to give final assay concentrations of 1, 0.1, 0.001, 0.0001 μg/mL. Isotype control (hIgG1, Biolegend) was diluted to 3 μg/mL to give a final concentration of 1 μg/mL in complete assay buffer. Equal volumes of stained CAL27 cells at 0.4 million cells/mL were mixed with antibody dilutions or isotype control and incubated for 30 min at 4°C to allow antibody binding. After incubation, 100 μl of the antibody-CAL27 cell mixture was added to 96-well plates containing 50 μl of IL-2-conjugate-treated PBMCs from day 1.

Control wells without PBMC but with 50 μl calcein-AM stained CAL27 cells were treated with complete assay buffer (background signal) or stained CAL27 were treated with 50 μl Triton-X-100 (for maximum signal after cell lysis), both prepared in a final volume of no more than 150 μl with complete assay buffer, in triplicate. The plates were centrifuged for 1 min at 200 × g and then incubated for 60 min at 37°C in the presence of 5% carbon dioxide. After incubation, the plates were centrifuged again briefly, then 90 μl of the supernatant was transferred to new black clear-bottomed plates and the fluorescent signal was read on an Envision 2104 plate reader (excitation: 492 nm; emission: 515 nm).

Cytotoxicity was calculated using the following formula:

where A is the fluorescence value for treated cells; B is the background level from target cells alone; and C is the maximum release value obtained from treatment with Triton-X-100.

Data represent % cytotoxicity of human PBMC treated with IL-2 conjugate against target cancer cells in the presence of cetuximab. The mean of technical replicates in percentage was transformed into proportion. The analysis was performed using a two-way generalized mixed-effects estimating model (GLMM) with factors for IL-2 conjugate, cetuximab and their interaction, with random donor effects, treating proportion as a pseudobinomial variable. Post hoc analysis (using Dunnett-Hsu correction) was then performed comparing IL-2 conjugate-treated groups with the control group. Statistical analyses were performed using SAS (1) version 9.4 software. A probability of less than 5% (p<0.05) was considered statistically significant.

Results.

At cetuximab dose levels of 1, 0.1, and 0.01 mg/mL, the IL-2 conjugate enhanced the ADCC function of cetuximab on EGFR-expressing CAL27 cells (p<0.05) at concentrations of 0.08, 0.4, and 2 mg/mL (Fig. 1A-C). At the 0.001 mg/mL cetuximab dose level, the IL-2 conjugate enhanced the ADCC function of cetuximab on EGFR-expressing CAL27 cells (p<0.05) at concentrations of 0.4 and 2 mg/mL. Figure 2A further shows the enhancement of ADCC function of cetuximab against EGFR-expressing CAL27 cells (PBMC to CAL27 ratio 50:1).

Fixed effects tests using GLMM model indicate that the factors IL-2 conjugate, cetuximab and their interaction have a significant effect on cytotoxicity, i.e. the differences between IL-2 conjugate groups vary significantly depending on the different concentrations of cetuximab. Pairwise comparisons indicated a significant difference between the 2 mg/mL IL-2 conjugate group and the control group (p=0.0001) and between the 0.4 mg/mL IL-2 conjugate group and the control group (p=0.0001) at the cetuximab concentration of 0.001 mg/mL. Pairwise comparisons also indicated a significant difference between the 2 mg/mL IL-2 conjugate group and the control group (p<0.0001), between the 0.4 mg/mL IL-2 conjugate group and the control group (p<0.0001), and between the 0.08 mg/mL IL-2 conjugate group and the control group (p=0.0003) at a cetuximab concentration of 0.01 mg/mL. In addition, pairwise comparisons also indicated a significant difference between the 2 mg/mL IL-2 conjugate group and the control group (p<0.0001), between the 0.4 mg/mL IL-2 conjugate group and the control group (p<0.0001), and between the 0.08 mg/mL IL-2 conjugate group and the control group (p<0.0001) at a cetuximab concentration of 0.1 mg/mL. Finally, pairwise comparisons also indicated a significant difference between the 2 mg/mL IL-2 conjugate group and the control group (p<0.0001), between the 0.4 mg/mL IL-2 conjugate group and the control group (p<0.0001), and between the 0.08 mg/mL IL-2 conjugate group and the control group (p<0.0001) at a cetuximab concentration of 1 mg/mL.

The data demonstrate that the IL-2 conjugate enhanced the ADCC function of cetuximab against EGFR-expressing CAL27 cancer cells. No significant differences were observed when the IL-2 conjugate was used in combination with the isotype control.

A431 cells.

The studies were performed using EGFR-expressing A431 epidermoid carcinoma cells according to the procedure described above for CAL27 cells. Figure 2B shows the enhancement of ADCC function of cetuximab on EGFR-expressing A431 cells (PBMC to A431 ratio 50:1). The data demonstrate that the IL-2-based conjugate enhanced the ADCC function of cetuximab on EGFR-expressing A431 cancer cells.

Example 4. ADCC assay using engineered NK-92.CD16 V cell line as effector cells

The effect of IL-2_P65_[AzK_L1_PEG30kDa]-1 on the ADCC function of cetuximab was examined using the calcein-acetoxymethyl (calcein-AM; Invitrogen) release assay.

Materials

NK-92.CD16 V (high affinity variant) (Conkwest Inc., San Diego, CA) was used as the effector cell line. The following cell lines were used as target cells: CAL27, A431, DLD-1, and FaDu.

The following reagents were used: cetuximab antibody (Eli Lilly &Co.); human IgG1 isotype antibody (Biolegend); calcein-acetoxymethyl (calcein-AM; Invitrogen C3100MP), and probenecid (Invitrogen; P36400). The biological assay medium was phenol red-free RPMI with 1% very low IgG fetal bovine serum supplemented with 1% probenecid for complete assay medium. MyeloCult H5100 (Stemcell Cat. No. 05150) supplemented with IL-2 (100 U/mL) and hydrocortisone (Sigma H6909; 10 mL at 50 μM) was used for NK-92.CD16 V cell culture.

Analysis procedure.

The IL-2 supplement was removed from the NK-92.CD16 V cell culture, which was then incubated overnight before the assay. The next day, the cells were seeded in 96-well round-bottom plates (60,000 cells were seeded to give a 3:1 effector to target cell ratio) in the presence of IL-2_P65_[AzK_L1_PE630kDa]-1 at varying concentrations (0.1 μg/ml, 0.01 μg/ml, 0.001 μg/ml, and 0 μg/ml) in RPMI 1640 medium without phenol red supplemented with 1% FBS low in IgG for 18 h at 37°C in a humidity-controlled incubator with 5% CO ₂ . These cells were used as effector cells. The following day, EGFR-positive human cancer cell lines (A431, DLD-1, FaDu, or CAL27) were labeled with calcein-AM for 30 min (50 μg was diluted in 25 μl DMSO to make a stock solution, then 10 μl calcein stock was added to 4 ml RPMI 1640 containing 1% low-IgG FBS and 1% probenecid to stain 5× ¹⁰⁶ cells) and then washed. Cells were split into multiple labeled tubes for incubation with varying concentrations of cetuximab or isotype control. Cetuximab and human IgG1 isotype antibody were added at 3X concentrations (to give final assay concentrations of 10 μg/mL to 1 pg/mL), and labeled target cells and antibody were mixed and allowed to stand for 30 min to allow opsonization. Following this incubation, target cells (20,000) and antibody were added on top of NK-92.CD16 V cells in a volume of 100 μL. The plate was briefly centrifuged for 1 min at 1100 rpm, then incubated at 37°C and 5% _CO2 for 1 h.

After incubation, the plates were again briefly centrifuged as before and 90 µl of supernatant was transferred from each well into black, clear-bottomed plates without shaking the cells. The fluorescent signal was read using an Envision 2104 (excitation: 492 nm; emission: 515 nm).

For maximum release, cells were lysed with 2% Triton X-100. The background fluorescence value of the culture medium was subtracted from the fluorescence values of experimental release (A), spontaneous release of target cells (B), and maximum release of target cells (C).

The percentage cytotoxicity and ADCC values for each plate (in duplicate) were calculated according to the following formulas:

For each experiment, measurements were performed in triplicate using triplicate wells. Each experiment was repeated at least 3 times. Half-maximal effective concentration (EC50) values were calculated by fitting data points to a 4-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA).

Results.

Cytotoxicity data from the ADCC assay using the NK92 cell line are shown in Fig. 3A-D for EGFR-expressing A431 (epidermoid carcinoma) (NK92 to A431 ratio 3:1), DLD-1 (adenocarcinoma, colorectal) (NK92 to DLD-1 ratio 3:1), FaDu (epithelial squamous cell carcinoma) (NK92 to FaDu ratio 3:1), and CAL27 (epithelial squamous cell carcinoma) (NK92 to CAL27 ratio 3:1), cells, respectively. The data demonstrate that the IL-2-based conjugate enhanced the ADCC function of cetuximab on EGFR-expressing cancer cells.

Example 5. Clinical study of combination therapy using a conjugate based on IL-2 and cetuximab.

Overview Monotherapy with the IL-2 conjugate of Example 1 has been shown to induce an increase in peripheral NK cells, which are important effector cells mediating antibody-dependent cellular cytotoxicity (ADCC) of IgG1 antibodies such as cetuximab.

An open-label, multicenter, phase 1/2 study was conducted to evaluate the clinical benefit of the IL-2 conjugate described in Example 1 in combination with cetuximab for the treatment of patients with advanced or metastatic solid tumors.

Participants received IL-2 conjugate (16 or 24 mcg/kg dose) via IV infusion once every 3 weeks. Here and throughout the discussion of this cohort, the weight of drug per kg of subject (e.g., 16 mcg/kg) refers to the weight of IL-2, excluding the weight of PEG and linker. Cetuximab was administered on Day 1 of Cycle 1 as an initial loading dose of 400 mg/ ^m2 given by infusion over 120 minutes (maximum infusion rate 10 mg/min), then 250 mg/ ^m2 given by infusion over 60 minutes (maximum infusion rate 10 mg/min) for all subsequent doses beginning with administration on Day 8 of Cycle 1. Cetuximab was administered on Days 1, 8, and 15 of each 21-day cycle. Infusion times for the IL-2 conjugate were approximately 30 minutes in each case. For each treatment cycle, prior to IL-2 conjugate administration, all participants received IL-2 conjugate premedication to prevent or reduce the incidence of acute infusion reactions (IARs) or influenza-like symptoms 30-60 minutes prior to IL-2 conjugate infusion. IL-2 conjugate premedication was as follows: an oral antipyretic and an antihistamine (H1 blocker). Antiemetics were prescribed as prescribed by the supervising physician. All participants received diphenhydramine premedication (approximately 25-50 mg IV) prior to the first dose of cetuximab. Premedication for subsequent doses of cetuximab was optional based on the supervising physician's assessment. If IL-2 conjugate and cetuximab were administered on the same day, participants who received diphenhydramine as premedication before cetuximab could omit the diphenhydramine premedication before IL-2 conjugate. The dosing sequence was as follows: (i) premedication before cetuximab (30-60 min before the start of cetuximab infusion); (ii) cetuximab; (iii) premedication before IL-2 conjugate (administered 30-60 min before the start of IL-2 conjugate infusion); and (iv) IL-2 conjugate. Treatment was repeated for a total of up to 35 cycles or a maximum duration of 735 days.

The following biomarkers serve as indirect predictors of safety and/or efficacy:

eosinophilia (increased number of eosinophils in the peripheral blood): a cellular indirect marker of IL-2-induced cell (eosinophil) proliferation associated with vascular leak syndrome (VLS);

interleukin 5 (IL-5): a cytokine indirect marker of IL-2-induced activation of type 2 innate lymphoid cells, the release of this chemoattractant leads to eosinophilia and possible VLS;

interleukin 6 (IL-6): a cytokine indirect marker of IL-2-induced cytokine release syndrome (CRS); and

interferon γ (IFN-γ): a cytokine indirect marker of IL-2-induced activation of CD8+ cytotoxic T lymphocytes.

The following biomarkers serve as indirect prognostic factors for antitumor immune activity:

peripheral CD8+ effector cells: a marker of IL-2-induced proliferation of these target cells in the peripheral blood, which, when infiltrated, becomes an indirect marker of the induction of a potentially latent therapeutic response;

peripheral CD8+ memory cells: a marker of IL-2-induced proliferation of such target cells in the peripheral blood, which, when infiltrated, becomes an indirect marker of the induction of a potentially long-term latent therapeutic response and maintenance of the memory cell population;

peripheral NK cells: a marker of IL-2-induced proliferation of such target cells in the peripheral blood, which, when infiltrated, becomes an indirect marker for the induction of a potentially rapid therapeutic response; and

Peripheral CD4+ regulatory cells: a marker of IL-2-induced proliferation of such target cells in peripheral blood, which, when infiltrated, becomes an indirect marker of induction of immunosuppressive TME and compensation of the therapeutic effect based on effectors.

First cohort administered with 16 mcg/kg IL-2 conjugate

Results: The five subjects included two men and three women with a median age of 69 years (range 65–72 years). All subjects had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and had received prior first- to fourth-line systemic therapy. Cancer types were anal cancer (1 subject), colon adenocarcinoma (1 subject), adrenocortical carcinoma (1 subject), squamous cell carcinoma of the lung (1 subject), and small bowel cancer (1 subject). All five subjects had metastatic disease.

Subjects received combination therapy comprising IL-2 conjugate (16 mcg/kg) and cetuximab for 2-7 cycles (2-7 doses of IL-2 conjugate). Two subjects, one with anal cancer and one with metastatic adrenocortical carcinoma, demonstrated progressive disease (PD) after 2 cycles of combination therapy, resulting in interruption of IL-2 conjugate and cetuximab combination therapy. One subject with colon cancer demonstrated disease progression after 5 cycles. Two subjects continue treatment: one with squamous cell lung carcinoma has completed 3 cycles and one with small bowel carcinoma has completed 7 cycles.

Peripheral CD8+ T _eff cell counts were measured (Fig. 4A-B). Some subjects showed prolonged CD8+ expansion compared to baseline (e.g., 2-fold change or greater) 3 weeks after the previous dose.

Peripheral NK cell counts are shown in Fig. 5A-B. Increases in NK cell counts were observed in each subject. Subjects generally demonstrated increased NK cell counts compared to baseline at 8 days and 3 weeks after the previous dose.

The number of peripheral CD4+ T _reg is shown in Fig. 6A-B.

Eosinophil counts were measured (Fig. 7A-B). Values measured did not exceed a four-fold increase and were consistently below the range of 2328-15958 eosinophils/μL in patients with IL-2-induced eosinophilia as reported in Pisani et al., Blood 1991 Sep 15;78(6):1538-44. Lymphocyte counts were also measured (Fig. 8A-B).

Summary of Results and Discussion In all subjects tested, expansion of peripheral CD8+ effector T (Teff) cells, NK cells, and CD4+ T _reg cells was observed after dosing.

An adverse event (AE) was defined as any untoward medical event in a subject participating in a clinical trial who was administered a pharmaceutical product, regardless of causal attribution. Dose-limiting toxicity values were defined as an AE occurring from day 1 to day 29 (inclusive) "+1 day of the treatment cycle that was not clearly or indisputably related to an external cause and that met at least one of the following criteria:

• grade 3 neutropenia (absolute neutrophil count <1000/ ^mm3 >500/ ^mm3 ) lasting ≥7 days, or grade 4 neutropenia of any duration;

• febrile neutropenia grade 3+;

• thrombocytopenia grade 4+ (platelet count <25000/ ^m3 );

• grade 3+ thrombocytopenia (platelet count <50,000-25,000/ ^mm3 ), lasting ≥5 days, or associated with clinically significant bleeding, or requiring platelet transfusion;

• failure to meet the criteria for recovery of absolute neutrophil count to at least 1000 cells/ ^mm3 and platelet count to at least 75,000 cells/ ^mm3 within 10 days;

• any other grade 4+ hematological toxicity lasting ≥5 days;

• ALT or AST grade 3+ in combination with a bilirubin level >2 times the ULN in the absence of cholestasis or another cause such as viral infection or use of other drugs (i.e., Hay's law);

• grade 3 infusion reaction that occurs during premedication; grade 4 infusion reaction;

• Grade 3 vascular leak syndrome is defined as hypotension associated with fluid retention and pulmonary edema;

• anaphylaxis grade 3+;

• hypotension grade 3+;

• Grade 3+ AE that does not resolve to <grade 2 within 7 days after initiation of accepted standard clinical treatment;

• cytokine release syndrome grade 3+.

The following exceptions apply to non-hematological AEs:

• grade 3 fatigue, nausea, vomiting, or diarrhea that resolves to grade ≤2 with optimal clinical management for ≥3 days;

• grade 3 fever (defined as >40°C for ≤24 hours);

• Grade 3 infusion reaction that occurs without premedication; premedication should be used for subsequent doses and if the reaction recurs, it will be considered a DLT;

• Grade 3 arthralgia or rash that resolves to ≤ Grade 2 within 7 days after initiation of accepted standard clinical treatment (eg, systemic corticosteroid therapy).

If a subject had a grade 1 or 2 ALT or AST elevation at baseline that was considered secondary to liver metastases, the grade 3 elevation must also be ≥3 times greater than baseline and last for >7 days.

A serious AE is defined as any AE that results in any of the following outcomes: death; life-threatening AE; emergency hospitalization or prolongation of current hospitalization; permanent or significant disability or significant impairment of ability to perform normal life functions; or congenital anomaly/malformation. Important medical events that may not result in death, are life-threatening, or require hospitalization may be considered serious when, based on appropriate medical assessment, they are likely to endanger the subject and may require medical or surgical intervention to prevent one of the above outcomes. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency department or at home, disturbances of blood homeostasis or muscle spasms that do not result in emergency hospitalization, or development of drug dependence or abuse.

There was no significant increase in IL-5 levels.

There was no cumulative toxicity. There was no target organ toxicity. There was no increase in QTc or other cardiac toxicity. Overall, the IL-2 conjugate was considered well tolerated.

Four of the 5 subjects had at least one treatment-emergent AE (TEAE). Most TEAEs were grade 1-2, one subject had at least one grade 3, and one subject had at least one grade 4 TEAE. Four subjects had treatment-related AEs. These included: one grade 1 infusion reaction; one grade 1 nausea; one grade 1 fatigue; one grade 2 diarrhea; and one grade 4 decrease in lymphocyte count. Two subjects had 3 unrelated SAEs: one dysphagia and spinal cord compression and one pleural effusion. TEAEs did not result in any drug discontinuations, no dose reductions, DLTs, anaphylaxis, or CRS. Treatment-related AEs were resolved with accepted standard care. The TEAEs are detailed in Table 2 and the treatment-related adverse events are summarized in Table 3.

Second cohort with 24 mcg/kg IL-2 conjugate dose

Results: The three subjects were male, with a median age of 71 years (range 65-75 years). All subjects had an Eastern Cooperative Oncology Group (ECOG) performance status of 1. One subject had received prior first-line therapy and the other subject had received prior fourth-line therapy. The cancer types were gastric cancer (1 subject), head and neck squamous cell carcinoma (HNSCC) (1 subject), and colon cancer (1 subject). All subjects had metastatic disease. Subjects received combination therapy consisting of IL-2 conjugate (24 mcg/kg) and cetuximab for 1 cycle (1 dose of IL-2 conjugate). One subject demonstrated progressive disease (PD) after the first cycle of combination treatment, precluding administration of an additional therapeutic dose of the combination treatment comprising an IL-2-based conjugate and cetuximab.

Two subjects experienced at least one TEAE. One subject experienced at least one treatment-related Grade 3 AE, which was Grade 3 chills. No subjects experienced treatment-related SAEs. No DLTs were observed, and there were no discontinuations of treatment due to TEAEs. TEAEs are detailed in Table 4, and treatment-related adverse events are summarized in Table 5.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of ease of understanding, the descriptions and examples are not to be construed as limiting the scope of the present invention. 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. To the extent that any material incorporated herein by reference is inconsistent with the express teachings of the present invention, the express teachings shall control.

--->

SEQUENCE LIST

<110> SYNTHORX, INC.

ABBADESSA, Giovanni et al.

<120> IMMUNOONCLOGICAL COMBINATION THERAPY USING CONJUGATES

BASED ON IL-2 AND EGFR ANTIBODIES

<130> 01183-0090-00PCT

<150> US 63/044,199

<151> 2020-06-25

<160> 98

<170> PatentIn version 3.5

<210> 1

<211> 133

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_feature

<223> IL-2 (Homo sapiens) (mature form)

<400> 1

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 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> 2

<211> 153

<212> PROTEIN

<213> Homo sapiens

<220>

<221> other_feature

<223> IL-2 (homo sapiens) (precursor) NCBI accession number: AAB46883.1

<400> 2

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

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

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 3

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<212> PROTEIN

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<223> Synthetic: aldesleukin

<400> 3

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

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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 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> 4

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_C125S

<400> 4

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 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> 5

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65X

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is an unnatural amino acid

<400> 5

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> 6

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62X

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is an unnatural amino acid

<400> 6

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> 7

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42X

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is an unnatural amino acid

<400> 7

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> 8

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43X

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is an unnatural amino acid

<400> 8

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> 9

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35X

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is an unnatural amino acid

<400> 9

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> 10

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK]

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine

<400> 10

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> 11

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK]

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine

<400> 11

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> 12

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_F42[AzK]

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine

<400> 12

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> 13

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K43[AzK]

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine

<400> 13

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> 14

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_K35[AzK]

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine

<400> 14

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> 15

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_P65[AzK_PEG]

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<400> 15

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> 16

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-2_E62[AzK_PEG]

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<400> 16

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> 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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_PEG5kD]

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_PEG5kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kD]

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_PEG5kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kD]

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG5kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kD]

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_PEG5kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa] N6-((2-azidoethoxy)-carbonyl)

-L-lysine stably conjugated to PEG

<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_PEG30kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_PEG30kDa] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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 is an 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 is an 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 is an 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 is an 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 is an 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 is [AzK]: N6-((2-azidoethoxy)-carbonyl)-L-lysine

<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 is [AzK]: N6-((2-azidoethoxy)-carbonyl)-L-lysine

<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 is [AzK]: N6-((2-azidoethoxy)-carbonyl)-L-lysine

<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 is [AzK]: N6-((2-azidoethoxy)-carbonyl)-L-lysine

<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 is [AzK]: N6-((2-azidoethoxy)-carbonyl)-L-lysine

<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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-2

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-2

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-2

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG5kDa]-2

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-2

<220>

<221> other_feature

<222> (65)..(65)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-2

<220>

<221> other_feature

<222> (62)..(62)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-2

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-2

<220>

<221> other_feature

<222> (43)..(43)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-2

<220>

<221> other_feature

<222> (35)..(35)

<223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with 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_PEG5kDa]-1

<220>

<221> other_feature

<222> (64)..(64)

<223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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] N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (61)..(61)

<223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (41)..(41)

<223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (42)..(42)

<223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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_PEG30kDa]-1

<220>

<221> other_feature

<222> (34)..(34)

<223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-

carbonyl)-L-lysine stably conjugated with PEG

<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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 is an 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 (83)

1. A method for treating an epidermal growth factor receptor (EGFR)-expressing cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2-based conjugate and (b) an EGFR antibody comprising cetuximab, panitumumab, necitumumab, or JNJ-61186372 (amivantamab), wherein the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue P64 is replaced by the structure of formula (I) where Z is _CH2 and Y is Y represents _CH2 and Z represents Z represents CH ₂ and Y represents or Y represents _CH2 and Z represents W is a PEG group having a molecular weight of 25 kDa ±15%, 30 kDa ±15%, or 35 kDa ±15%; q represents 1, 2, or 3; 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. 2. The method according to claim 1, wherein in the IL-2-based conjugate Z is CH ₂ and Y is 3. The method according to claim 1, wherein in the IL-2-based conjugate Y is CH ₂ and Z is 4. The method according to claim 1, wherein in the IL-2-based conjugate Z is CH ₂ and Y is 5. The method according to claim 1, wherein in the IL-2-based conjugate Y is CH ₂ and Z is 6. The method according to any one of claims 1-5, wherein in the IL-2-based conjugate the PEG group has a molecular weight of 30 kDa ± 15%. 7. The method according to claim 1, wherein the structure of formula (I) has a structure of formula (IV) or formula (V) or the IL-2-based conjugate is provided as a mixture of IL-2-based conjugates comprising an IL-2-based conjugate in which the structure of formula (I) has a structure of formula (IV) and an IL-2-based conjugate in which the structure of formula (I) has a structure of formula (V): where W is a PEG group having a molecular weight of 25 kDa ± 15%, 30 kDa ± 15%, or 35 kDa ± 15%; q is equal to 1, 2, or 3; 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. 8. The method according to any one of claims 1-7, wherein the antibody to EGFR is cetuximab. 9. A method of treating an epidermal growth factor receptor (EGFR)-expressing cancer in a subject in need thereof, comprising administering (a) an IL-2-based conjugate and (b) cetuximab, wherein (A) the IL-2-based conjugate comprises the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG30kDa] has the structure of formula (XII) or formula (XIII), or (B) the IL-2-based conjugate is provided as a mixture comprising an IL-2-based conjugate that comprises the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG30kDa] has the structure of formula (XII), and an IL-2-based conjugate that comprises the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG30kDa] has the structure of formula (XIII): where n is an integer such that PEG _having the structure -( _OCH2CH2 ) _n - _OCH3 has a molecular weight of 30 kDa ± 15%; q is equal to 1, 2, or 3; and The wavy lines indicate covalent bonds with the first amino acid residue preceding [AzK_L1_PEG30kDa] and the first amino acid residue following [AzK_L1_PEG30kDa], respectively, in SEQ ID NO: 50. 10. The method according to any of paragraphs 1-9, where q equals 1. 11. The method according to any of paragraphs. 1-9, where q equals 2. 12. The method according to any of paragraphs 1-9, where q equals 3. 13. The method according to any one of claims 1-12, wherein the IL-2-based conjugate is a pharmaceutically acceptable salt, solvate or hydrate. 14. The method of any one of claims 1-13, 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, or once every 6 weeks. 15. The method of any one of claims 1-14, wherein the EGFR antibody is administered to the subject once a week, once every two weeks, once every three weeks, or once every 4 weeks. 16. The method according to any one of claims 1-15, wherein the IL-2-based conjugate is administered to the subject via intravenous administration. 17. The method according to any one of claims 1-16, wherein the IL-2-based conjugate and the EGFR antibody are administered separately. 18. The method according to claim 17, wherein the IL-2-based conjugate and the EGFR antibody are administered sequentially. 19. The method according to any of paragraphs. 1-18, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), squamous cell carcinoma (SCC), 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 prostate cancer, metastatic castration-resistant prostate cancer or metastatic castration-resistant prostate cancer with defects in DNA damage response (DDR) mechanisms, 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 disseminating systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma. 20. The method according to any one of claims 1-19, wherein the IL-2-based conjugate is administered to the subject at a dose of 16 μg/kg ± 15% of the conjugate in the form of IL-2 mass excluding the mass of PEG and linker. 21. The method of any one of claims 1-19, wherein the IL-2 conjugate is administered to the subject at a dose of 24 μg/kg ± 15% of the IL-2 conjugate as the bulk of IL-2 excluding the bulk of PEG and linker. 22. Use of an IL-2-based conjugate for the manufacture of a medicinal product for the method according to any one of claims 1 to 21, wherein the IL-2-based conjugate comprises the amino acid sequence SEQ ID NO: 3, in which the amino acid residue P64 is replaced by a structure of formula (I) where Z is _CH2 and Y is Y represents _CH2 and Z represents Z is _CH2 and Y is or Y represents _CH2 and Z represents W is a PEG group having a molecular weight of 25 kDa ±15%, 30 kDa ±15%, or 35 kDa ±15%; q represents 1, 2, or 3; 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. 23. A kit for the treatment of an epidermal growth factor receptor (EGFR) expressing cancer in a subject in need thereof, comprising: an IL-2 conjugate, an anti-EGFR antibody comprising cetuximab, panitumumab, necitumumab, or JNJ-61186372 (amivantamab), and a label or set of instructions indicating that the IL-2 conjugate is to be administered in combination with said anti-EGFR antibody to treat said cancer in said subject in need thereof, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue P64 is replaced by a structure of formula (I) where Z is _CH2 and Y is Y represents _CH2 and Z represents Z is _CH2 and Y is or Y represents _CH2 and Z represents W represents a PEG group having a molecular weight of 25 kDa ± 15%, 30 kDa ± 15%, or 35 kDa ± 15%; q represents 1, 2, or 3; 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.