KR20230027235A - Immuno-oncology combination therapy using IL-2 conjugates and anti-EGFR antibodies - Google Patents

Abstract

Disclosed herein are methods of treating cancer in a subject in need thereof, comprising administering an IL-2 conjugate in combination with an anti-EGFR antibody.

Description

Immuno-oncology combination therapy using IL-2 conjugates and anti-EGFR antibodies

CROSS REFERENCES OF RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/044,199, filed on June 25, 2020, and U.S. Provisional Application No. 63/196,448, filed on June 3, 2021, the disclosures of each of which The entirety is incorporated herein by reference.

Different T cell populations regulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological self-reactivity, whereas cytotoxic T cells target and destroy infected and/or cancer cells. In some instances, modulation of different T cell populations provides options for treatment of a disease or indication. 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 (which play a role in innate and adaptive immune cell homeostasis). Cytokines are produced by immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, endothelial cells, fibroblasts, and other stromal cells. In some cases, cytokines regulate the balance between humoral and cell-based immune responses.

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

In some instances, interleukin 2 (IL-2) signaling is used to modulate T cell responses and subsequently for treatment of cancer. Thus, in one aspect, provided herein is a method of treating cancer in a subject comprising administering an IL-2 conjugate in combination with an anti-EGFR antibody.

Disclosed herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody. includes

Exemplary embodiments include:

Embodiment 1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody. wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues of the IL-2 conjugate are replaced with the structure of Formula I:

[Formula I]

here,

이거나;Z is CH ₂ and Y is

is;

이거나;Y is CH ₂ and Z is

is;

이거나; 또는Z is CH ₂ and Y is

is; or

이며;Y is CH ₂ and Z is

is;

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

X has the following structure:

를 갖는 L-아미노산이며;

It is an L-amino acid having;

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

X+1 represents the point of attachment to the trailing amino acid residue;

wherein the position of structure 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.

인, 방법.Embodiment 2. according to embodiment 1, wherein in the IL-2 conjugate Z is CH ₂ and Y is

in, how.

인, 방법.Embodiment 3. according to embodiment 1, wherein in the IL-2 conjugate Y is CH ₂ and Z is

in, how.

인, 방법.Embodiment 4. according to embodiment 1, wherein in the IL-2 conjugate Z is CH ₂ and Y is

in, how.

인, 방법.Embodiment 5. according to embodiment 1, wherein in the IL-2 conjugate Y is CH ₂ and Z is

in, how.

Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the PEG groups in the IL-2 conjugate have an average molecular weight of about 25 kDa, 30 kDa, or 35 kDa.

Embodiment 7. The method of Embodiment 6, wherein the PEG groups in the IL-2 conjugate have an average molecular weight of about 30 kDa.

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

Embodiment 9. The method of Embodiment 1 wherein the structure of Formula I has the structure of Formula IV or Formula V, or is a mixture of the structure of Formula IV and the structure of Formula V:

[Formula IV]

[Formula V]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 갖는 L-아미노산이며;

It is an L-amino acid having;

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

X+1 represents the point of attachment to the trailing amino acid residue.

Embodiment 10. The method of Embodiment 9, wherein the position of the structure of Formula IV or Formula V in the amino acid sequence of the IL-2 conjugate is P64.

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

Embodiment 12. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) cetuximab. and the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 50, [AzK_L1_PEG30kD] has the structure of Formula XII or Formula XIII below, or a mixture of the structure of Formula XII and the structure of Formula XIII below In, method:

[Formula XII]

[Formula XIII]

here,

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

q is 1, 2 or 3;

wherein the wavy line indicates a covalent bond to an amino acid residue within SEQ ID NO: 50 that is not replaced.

Embodiment 13. The method of any one of Embodiments 1 to 12, wherein q is 1.

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

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

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

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

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

Embodiment 19. The method of any one of Embodiments 1 to 18, wherein the IL-2 conjugate is administered to the subject about once every 2 weeks, about once about every 3 weeks, or about once every 4 weeks.

Embodiment 20. The method of any one of Embodiments 1 to 19, wherein the anti-EGFR antibody is administered about once about every 1 week, about once about every 2 weeks, about once about every 3 weeks, or about once about every 4 weeks. , method.

Embodiment 21. The method of any one of Embodiments 1 to 20, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

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

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

Embodiment 24. The method according to any one of embodiments 1 to 24, wherein the cancer is renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal giant B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable cancer, microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer, 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 DNA damage response (DDR) defects Metastatic castration-resistant prostate cancer, bladder cancer, ovarian cancer, moderate to low mutation burden tumors, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), low to non-expressing PD-L1 tumors, anatomic primary site of origin A tumor disseminated systemically beyond the liver and CNS, and diffuse large B-cell lymphoma.

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

Embodiment 27. The method of any one of Embodiments 1 to 10 or 12 to 26, wherein the anti-EGFR antibody is Panitumumab (Vectibix), Nesitumumab (Portrazza), JNJ-61186372 (Amivantamab), IMC-C225, ABX -EGF, ICR62, and EMD 55900.

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

Embodiment 29. Use of an IL-2 conjugate for the manufacture of a medicament for the method of any one of Embodiments 1 to 27.

The novel features of the invention are pointed out with particularity 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 exemplary embodiments in which the principles of the present invention are employed and the accompanying drawings.
1A-1C show % cytotoxicity in human PBMCs from three individual donors and CAL27 cells co-cultured with various amounts of IL-2 conjugate and cetuximab.
2A shows % cytotoxicity in CAL27 cells co-cultured with human PBMCs and various amounts of IL-2 conjugate and cetuximab.
2B shows % cytotoxicity in A431 cells co-cultured with human PBMCs and various amounts of IL-2 conjugate and cetuximab.
3A shows the cytotoxic effect on A431 cells co-cultured with NK92 cells and treated with varying amounts of IL-2 conjugate and cetuximab.
3B shows % cytotoxicity for DLD-1 cells co-cultured with NK92 cells and treated with various amounts of IL-2 conjugate and cetuximab.
3C shows % cytotoxicity for FaDu cells co-cultured with NK92 cells and treated with various amounts of IL-2 conjugate and cetuximab.
3D shows % cytotoxicity for CAL27 cells co-cultured with NK92 cells and treated with various amounts of IL-2 conjugate and cetuximab.
4A shows peripheral CD8+ Teff cell counts in indicated subjects treated with 16 μg/kg of IL-2 conjugate in combination with _cetuximab at specific time points after IL-2 conjugate administration.
4B shows changes in peripheral CD8+ _Teff cell counts in indicated subjects treated with 16 μg/kg of IL-2 conjugate in combination with cetuximab at specific time points after IL-2 conjugate administration. Data are normalized to pre-treatment (C1D1) CD8+ T _eff cell counts.
FIG. 5A shows peripheral NK cell counts in indicated subjects treated with 16 μg/kg of IL-2 conjugate in combination with cetuximab at specific time points after IL-2 conjugate administration.
5B shows changes in peripheral NK cell counts in indicated subjects treated with 16 μg/kg of IL-2 conjugate in combination with cetuximab at specific time points after administration of IL-2 conjugate. Data are normalized to pre-treatment (C1D1) NK cell counts.
6A shows peripheral CD4+ T reg cell counts in the indicated subjects treated with 16 μg/kg of the IL-2 conjugate in combination with cetuximab at specific time points after administration of the IL-2 _conjugate .
6B shows changes in peripheral CD4+ T _reg cell counts in the indicated subjects treated with 16 μg/kg of IL-2 conjugate in combination with cetuximab at specific time points after IL-2 conjugate administration. Data are normalized to pre-treatment (C1D1) CD4+ T _reg cell counts.
7A shows eosinophil cell counts in the indicated subjects treated with 16 μg/kg of the IL-2 conjugate in combination with cetuximab at specific time points after administration of the IL-2 conjugate.
7B shows the change in eosinophil cell counts in the indicated subjects treated with 16 μg/kg of the IL-2 conjugate in combination with cetuximab at specific time points after IL-2 conjugate administration. Data are normalized to pre-treatment (C1D1) eosinophil cell counts.
8A shows lymphocyte cell counts in indicated subjects treated with 16 μg/kg of IL-2 conjugate in combination with cetuximab at specific time points after IL-2 conjugate administration.
8B shows changes in lymphocyte cell counts in indicated subjects treated with 16 μg/kg of IL-2 conjugate in combination with cetuximab at specific time points after IL-2 conjugate administration. Data are normalized to pre-treatment (C1D1) lymphocyte cell counts.

Justice

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 should be understood that both the above general description and the following detailed description are illustrative and explanatory only and are not limiting of any claimed subject matter. To the extent any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content of this disclosure controls. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in this specification and the appended claims, it should be noted that the singular forms include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Also, the use of the term "comprising" and other forms, such as "comprises", "includes" and "including" is not limiting.

References in the specification to “some embodiments,” “one embodiment,” “one embodiment,” or “another embodiment” do not necessarily indicate that a particular feature, structure, or characteristic described in connection with the embodiment is the invention. It means that it is not included in all embodiments of, but is included in at least some embodiments of the present invention.

As used herein, ranges and amounts can be expressed as “approximate” specific values or ranges. About includes the exact amount. Thus “about 5 μL” means “about 5 μL” and also “5 μL”. Generally, the term “about” includes amounts expected to be within experimental error, such as within 15%, 10%, or 5%.

Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

As used herein, the terms "individual(s)", "subject(s)", and "patient(s)" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. Neither term requires nor is limited to a situation characterized by the supervision of a health care worker (eg, a physician, registered nurse, nurse practitioner, physician's assistant, janitor, or hospice worker).

As used herein, the terms “significantly” or “significantly” with respect to binding affinity refers to a cytokine (such as an IL-2 polypeptide) that is sufficient to affect binding of the cytokine (such as an IL-2 polypeptide) to a target receptor. , IL-2 polypeptide) means a change in binding affinity. In some cases, the term means a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some cases, the terms mean at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 50x, 100x, 500x, 1000x, or more. means change.

In some instances, the terms "significant" or "significantly" in reference to activation of one or more cell populations through a cytokine signaling complex means a change sufficient to activate the cell population. In some cases, a change to activate a cell population is measured as a receptor signaling potency. In this case, an EC50 value may be provided. In other cases, an ED50 value may be provided. In further instances, concentrations or doses of cytokines may be provided.

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

As used herein, the term “non-natural amino acid” refers to an amino acid other than one of the 20 naturally occurring amino acids. Exemplary non-natural 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 is incorporated herein by reference.

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

As used herein, the term "monoclonal antibody(s)" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprised in the population are capable of variant antibodies, e.g., naturally occurring mutations. Except for antibodies comprising or arising during production of a monoclonal antibody preparation, which are identical and/or bind to the same epitope, such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention include hybridoma methods, recombinant DNA methods, phage-display methods, and methods using transgenic animals comprising all human immunoglobulin loci or portions thereof; It can be made by a variety of techniques, including but not limited to, such methods and other exemplary methods of making monoclonal antibodies are described herein.

As used herein, "nucleotide" refers to a compound comprising a nucleoside moiety and a phosphate moiety. Exemplary natural nucleotides include, without limitation, 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), Deoxycity Dean monophosphate (dCMP), and deoxyguanosine monophosphate (dGMP). Exemplary natural deoxyribonucleotides comprising deoxyribose as the sugar moiety include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural ribonucleotides comprising ribose as the 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 a nucleoside or nucleotide that in some cases may contain additional modifications to the sugar portion of the nucleoside or nucleotide (nucleosides and nucleotides are ribosomes or nucleotides). deoxyribo variants). In some cases, "base" is also used to indicate an entire nucleoside or nucleotide (e.g., "base" can be incorporated into DNA by DNA polymerase, or incorporated into RNA by RNA polymerase). has exist). However, the term “base” should not necessarily be construed as indicating an entire nucleoside or nucleotide unless the context requires it. In the chemical structures provided herein of bases or nucleobases, 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 chemical structures provided herein of a base or nucleobase, a wavy line indicates a linkage to a nucleoside or nucleotide at which the sugar moiety of the nucleoside or nucleotide may be further modified. In some embodiments, a wavy line indicates the attachment of a base or nucleobase to a sugar moiety, such as a nucleoside or pentose sugar of a nucleotide. In some embodiments, the pentose sugar is ribose or deoxyribose.

In some embodiments, a nucleobase is generally a heterocyclic base portion of a nucleoside. Nucleobases can be naturally occurring, modified, dissimilar to natural bases, and/or synthesized, for example, by organic synthesis. In certain embodiments, a nucleobase includes any atom or group of atoms within a nucleoside or nucleotide, wherein the atom or group of atoms can interact with bases of other nucleic acids with or without hydrogen bonding. In certain embodiments, the non-natural nucleobase is not derived from a natural nucleobase. It should be noted that unnatural nucleobases do not necessarily have the basic properties, but are referred to as nucleobases for simplicity. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase may be attached to deoxyribose or ribose, while "d" without parentheses indicates that the nucleobase is attached to deoxyribose. indicates being

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 with mimetic bases and/or sugar groups. Nucleosides include nucleosides with any of a variety of substituents. A nucleoside can be a glycosidic compound formed through a glycosidic linkage between a nucleic acid base and a reducing group of a sugar.

An “analog” of a chemical structure, as the term is used herein, refers to a chemical structure that preserves substantial similarity to the parent structure, even though it may not be easily derived synthetically from the parent structure. In some embodiments, nucleotide analogs are unnatural nucleotides. In some embodiments, the nucleoside analog is an unnatural nucleoside. Related chemical structures that are readily derived synthetically from the parent chemical structure are referred to as “derivatives”.

As used herein, a "dose limiting toxicity" (DLT) is defined as an adverse event occurring within ±1 day from Day 1 to Day 29 (inclusive) of a treatment cycle, which is neither apparent nor incontrovertible. It was entirely unrelated to external causes and meets the criteria set forth in Example 5 for DLT.

As used herein, “cetuximab” refers to a chimeric (mouse/human) anti-EGFR antibody sold under the brand name “Erbitux” by Eli Lilly and Co.

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

IL-2 conjugate

Interleukin 2 (IL-2) is a pleiotropic type 1 cytokine whose structure contains a bundle of four α-helices of 15.5 kDa. The precursor form of IL-2 is 153 amino acid residues in length, 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 after antigen stimulation and, to a lesser extent, by CD8+ cells, natural killer (NK) and natural killer T (NKT) cells, activated dendritic cells (DC) and mast cells. do. IL-2 signaling is a specific combination of the 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). occurs through interaction with Interaction of IL-2 with IL-2Rα results in the formation of a “low affinity” IL-2 receptor complex with a K _d of about 10 ⁻⁸ M. Interaction of IL-2 with IL-2Rβ and IL-2Rγ forms an “intermediate affinity” IL-2 receptor complex with a K _d of about 10 ⁻⁹ M. Interaction of IL-2 with all three subunits, IL-2Rα, IL-2Rβ and IL-2Rγ, forms a "high affinity" IL-2 receptor complex with a K _d greater than about 10 ^-11 M .

) T 세포(pTreg 세포)로부터 유도된다. 일부 경우에, Treg 세포는 말초 내용성의 매개체로 간주된다. 실제로 한 연구에서, CD25-고갈 말초 CD4⁺ T 세포의 이전은 누드 마우스에서 다양한 자가면역 질환을 생성하였으며, 반면에 CD4⁺CD25⁺ T 세포의 공동이전은 자가면역의 발달을 억제하였다(문헌[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)], 이들 각각의 개시 내용은 본원에 참고로 포함됨). Treg 세포 집단의 증가는 이펙터 T 세포 증식을 하향조절하고 자가면역 및 T 세포 항종양 반응을 억제한다.In some instances, IL-2 signaling through the “high affinity” IL-2Rαβγ complex regulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T (Treg) cells mediate 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 from the thymus (tTreg cells) or peripherally naïve (tTreg cells).

) from T cells (pTreg cells). In some instances, Treg cells are considered mediators of peripheral tolerance. Indeed, in one study, transfer of CD25-depleted peripheral CD4 ⁺ T cells produced various autoimmune diseases in nude mice, whereas cotransfer of CD4 ⁺ CD25 ⁺ T cells inhibited 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)], their respective disclosures. are incorporated herein by reference). An increase in the Treg cell population downregulates effector T cell proliferation and suppresses autoimmune and T cell antitumor responses.

IL-2 signaling through the "intermediate affinity" IL-2Rβγ complex regulates the activation and proliferation of CD8 ⁺ effector T (Teff) cells, NK cells and NKT cells. CD8 ⁺ Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, Tcon or killer T cells) are damaged cells, cancer cells and pathogen-infected cells. It is a T lymphocyte that recognizes and kills. NK and NKT cells are a type of lymphocyte that, similar to CD8 ⁺ Teff cells, target cancer cells and pathogen-infected cells.

In some instances, IL-2 signaling is used to modulate T cell responses and subsequently for treatment of cancer. For example, IL-2 is administered in high dose form to induce expansion of the Teff cell population for cancer treatment. However, high doses of IL-2 additionally result in concomitant Treg cell stimulation that attenuates the antitumor immune response. High doses of IL-2 also induce toxic adverse events mediated by the involvement of IL-2R alpha chain expressing cells in the vasculature, including type 2 innate immune cells (ILC-2), eosinophils and endothelial cells. This results in eosinophilia, capillary leak and vascular leak syndrome (VLS).

Adoptive cell therapy allows doctors to effectively utilize a patient's own immune cells to fight diseases such as proliferative diseases (eg cancer) and infectious diseases. The effect of IL-2 signaling may be further enhanced by the presence of additional agents or methods in combination therapy. For example, epidermal growth factor receptor (EGFR) is a cell surface receptor that is overexpressed in many types of cancer. Activation of EGFR promotes cell proliferation and survival and angiogenesis, resulting in tumor growth and metastasis. Cell growth and angiogenesis can be regulated 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 downstream signaling cascades and reducing cell growth. Anti-EGFR antibodies can also cause the same effect by competitively inhibiting the binding of transforming growth factor alpha (TGF-α) to EGFR.

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

[Table 1]

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

[AzK_PEG] = N6-((2-azidoethoxy)-carbonyl)-L-lysine is stably added to PEG via DBCO-mediated click chemistry to form a compound containing the structure of Formula II or Formula III Conjugated. For example, where specified, PEG5kD refers to a linear polyethylene glycol chain with an average molecular weight of 5 kDa capped with methoxy groups. The ratio of regioisomers resulting from the click reaction is about 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety comprising a dibenzocyclooctyne group.

[AzK_L1_PEG] = N6-((2-azidoethoxy)-carbonyl)-L-lysine is stably conjugated to PEG via DBCO-mediated click chemistry to form a compound comprising the structure of Formula IV or Formula V Gated. For example, where specified, PEG5kD refers to a linear polyethylene glycol chain with an average molecular weight of 5 kDa capped with methoxy groups. The ratio of regioisomers resulting from the click reaction is about 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety comprising a dibenzocyclooctyne group.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with the structure of Formula I:

[Formula I]

here,

이거나;Z is CH ₂ and Y is

is;

이거나;Y is CH ₂ and Z is

is;

이거나; 또는Z is CH ₂ and Y is

is; or

이며;Y is CH ₂ and Z is

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 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue;

The position of structure 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.

In any embodiment or variation 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.

이다.In some embodiments of Formula I, Z is CH ₂ and Y is

am.

이다. 화학식 I의 일부 실시 형태에서, Z는 CH₂이고 Y는

이다. 화학식 I의 일부 실시 형태에서, Y는 CH₂이고 Z는

이다.In some embodiments of Formula I, Y is CH ₂ and Z is

am. In some embodiments of Formula I, Z is CH ₂ and Y is

am. In some embodiments of Formula I, Y is CH ₂ and Z is

am.

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:

[Formula Ia]

here,

이거나;Z is CH ₂ and Y is

is;

이거나;Y is CH ₂ and Z is

is;

이거나; 또는Z is CH ₂ and Y is

is; or

이며;Y is CH ₂ and Z is

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;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue;

The position of the structure (Ia) 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.

이다. 화학식 Ia의 일부 실시 형태에서, Y는 CH₂이고 Z는

이다. 화학식 Ia의 일부 실시 형태에서, Z는 CH₂이고 Y는

이다. 화학식 Ia의 일부 실시 형태에서, Z는 CH₂이고 Y는

이다. 화학식 Ia의 일부 실시 형태에서, Y는 CH₂이고 Z는

이다.In some embodiments of Formula Ia, Z is CH ₂ and Y is

am. In some embodiments of Formula Ia, Y is CH ₂ and Z is

am. In some embodiments of Formula Ia, Z is CH ₂ and Y is

am. In some embodiments of Formula Ia, Z is CH ₂ and Y is

am. In some embodiments of Formula Ia, Y is CH ₂ and Z is

am.

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

In some embodiments, 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 relative to the position 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 position in SEQ ID NO:3. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is K34, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is F41, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, 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 is a position in SEQ ID NO:3 related to In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is K42, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is E61, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is P64, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate is relative to the position in SEQ ID NO:3. do. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is R37, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is T40, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, 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 a position in SEQ ID NO:3 related to In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is Y44, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is V68, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do. In some embodiments, the structure of Formula I in the amino acid sequence of the IL-10 conjugate is L71, wherein the position of the structure of Formula I in the amino acid sequence of the IL-10 conjugate correlates with the position in SEQ ID NO:3. do.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] has the structure of Formula II or Formula III, or a combination of Formula II and Formula III: has:

[Formula II]

[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 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

As used herein and throughout, structures of Formula II include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of Formula III include 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 structures of Formula IIa and Formula IIIa:

[Formula IIa]

[Formula IIIa]

here,

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;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, [AzK_PEG] is a combination of Formula II and Formula III. In some embodiments, [AzK_PEG] is a combination of Formula IIa and Formula IIIa.

In some embodiments, [AzK_PEG] has the structure of Formula II. In some embodiments, [AzK_PEG] has the structure of Formula IIa.

In some embodiments, [AzK_PEG] has the structure of Formula III. In some embodiments, [AzK_PEG] has the 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 structure II is a PEG group 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 structure II is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure II is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure II is a PEG group 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 structure II is a PEG group 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 structure II is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure II is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure II is a PEG group 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 structure II is a PEG group 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 structure II is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure II is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure II is a PEG group 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 structure II is a PEG group 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 structure II is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure II is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure II is a PEG group 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 structure II is a PEG group 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 structure II is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure II is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure II is a PEG group having an average molecular weight of about 30 kDa.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kD] is a structure of Formula II or Formula III below, or a mixture of Formula II and Formula III has:

[Formula II]

[Formula III]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_PEG5kD] is a structure of Formula IIa or Formula IIIa, or a mixture of Formula IIa and Formula IIIa.

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

In some embodiments, [AzK_PEG5kD] has the structure of Formula II:

[Formula II]

.

.

In some embodiments, q is 1 and [AzK_PEG5kD] has the structure of Formula IIa.

In some embodiments, [AzK_PEG5kD] has the structure of Formula III:

[Formula III]

.

.

In some embodiments, q is 1 and [AzK_PEG5kD] has the structure of Formula IIIa.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kD] has the structure of Formula II or Formula III, or the structure of Formula II: and a structure of Formula III:

[Formula II]

[Formula III]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_PEG30kD] has the structure of Formula IIa or Formula IIIa, or a mixture of the structure 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_PEG30kD] has the structure of Formula II:

[Formula II]

.

.

In some embodiments, q is 1 and [AzK_PEG30kD] has the structure of Formula IIa.

In some embodiments, the method uses an IL-2 conjugate in which [AzK_PEG30kD] has the structure of Formula III:

[Formula III]

In some embodiments, q is 1 and [AzK_PEG30kD] has the structure of Formula IIIa.

In some embodiments, [AzK_PEG] is a mixture of the structures of Formula II and Formula III:

[Formula II]

[Formula III]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_PEG] is a mixture of a structure of Formula IIa and a structure of Formula IIIa.

In some embodiments, the ratio of the amount of structure II: the amount of structure III (including the total amount of [AzK_PEG]) in the IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure II: the amount of structure III (including 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 structure II: the amount of structure III (including 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 groups are 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 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kD] is a mixture of structures of Formula II and Formula III:

[Formula II]

[Formula III]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_PEG5kD] is a mixture of structure IIa and structure IIIa.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kD] is a mixture of structures of Formula II and Formula III:

[Formula II]

[Formula III]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_PEG30kD] is a mixture of a structure of Formula IIa and a structure of Formula IIIa.

In some embodiments, the ratio of the amount of structure II: the amount of structure III (including the total amount of [AzK_PEG30kD]) in the IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure II: the amount of structure III (including the total amount of [AzK_PEG30 kD]) in the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure II: the amount of structure III (including the total amount of [AzK_PEG30kD]) 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, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein W is linear or a branched PEG group. In some embodiments, W in structures of Formula II or Formula III is a linear PEG group. In some embodiments, W in structures of Formula II or Formula III is a branched PEG group. In some embodiments, W in structures of Formula II or Formula III is a methoxy PEG group. In some embodiments, W in structures of Formula II or Formula III is a methoxy PEG group that is linear or branched. In some embodiments, the methoxy PEG groups in structures of Formula II or Formula III are linear. In some embodiments, the methoxy PEG groups in structures of Formula II or Formula III are branched.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [AzK_L1_PEG] is a structure of Formula IV or Formula V, or a mixture of Formula IV and Formula V has:

[Formula IV]

[Formula V]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the following amino acid residue.

In some embodiments of Formula IV or Formula V, or mixtures of Formula IV or Formula V, q is 1. In some embodiments of Formula IV or Formula V, or mixtures of Formula IV or Formula V, q is 2. In some embodiments of Formula IV or Formula V, or mixtures of Formula IV or Formula V, q is 3.

As used herein and throughout, structures of Formula IV include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of Formula V include 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 structures of Formula IVa and Formula Va below:

[Formula IVa]

[Formula Va]

here,

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;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing 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 the structure of Formula IV:

[Formula IV]

.

.

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

In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO:40. In some embodiments, W in structure IV is a PEG group 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 structure IV is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure IV is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure IV is a PEG group with 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 structure IV is a PEG group 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 structure IV is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure IV is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure IV is a PEG group with 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 structure IV is a PEG group 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 structure IV is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W is a PEG group with an average molecular weight of about 5 kDa. In some embodiments, W in structure IV is a PEG group with 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 structure IV is a PEG group 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 structure IV is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure IV is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure IV is a PEG group with an average molecular weight of about 30 kDa.

In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO:44. In some embodiments, W in structure IV is a PEG group 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 structure IV is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in structure IV is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure IV is a PEG group with an average molecular weight of about 30 kDa.

In some embodiments, [AzK_L1_PEG] has the structure of Formula V:

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kD] is a structure of Formula IV or Formula V, or a mixture of Formula IV and Formula V has:

[Formula IV]

[Formula V]

here,

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

X has the following structure:

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_L1_PEG5kD] is a structure of Formula IVa or Formula Va, or 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_PEG5kD] has the structure of Formula IV:

[Formula IV]

.

.

In some embodiments, q is 1 and [AzK_L1_PEG5kD] has the structure IVa.

In some embodiments, [AzK_L1_PEG5kD] has the structure of Formula V:

[Formula V]

.

.

In some embodiments, q is 1 and [AzK_L1_PEG5kD] has the structure Va.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kD] has the structure of Formula IV or Formula V, or It is a mixture of structures of Formula V:

[Formula IV]

[Formula V]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_L1_PEG30kD] has the structure of Formula IVa or Formula Va, or a mixture of the structure of Formula IVa and the structure of Formula Va.

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

In some embodiments, [AzK_L1_PEG30kD] has the structure of Formula IV:

[Formula IV]

.

.

In some embodiments, q is 1 and [AzK_L1_PEG30kD] has the structure IVa.

In some embodiments, [AzK_L1_PEG30kD] has the structure of Formula V:

[Formula V]

.

.

In some embodiments, q is 1 and [AzK_L1_PEG30kD] has the structure Va.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [Azk_L1_PEG] is a mixture of structures of Formula IV and Formula V:

[Formula IV]

[Formula V]

here,

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 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, the ratio of the amount of structure IV: the amount of structure V (including 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 structure IV: the amount of structure V (including 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 structure IV: the amount of structure V (including the total amount of [AzK_L1_PEG]) in the IL-2 conjugate is less than 1:1.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kD] is a mixture of structures of Formula IV and Formula V:

[Formula IV]

[Formula V]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_L1_PEG5kD] is a mixture of structure IVa and structure Va.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1 PEG30kD] is a mixture of structures of Formula IV and Formula V:

[Formula IV]

[Formula V]

here,

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

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, q is 1 and [AzK_L1 PEG30kD] is a mixture of structure IVa and structure Va.

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

In some embodiments, W in structure IV or V is a PEG group 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 structure IV or V is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 30 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 10 kDa. In some embodiments, W in structure IV or V is a PEG group having an average molecular weight of about 15 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 20 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 25 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 30 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 35 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 40 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 45 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 50 kDa. In some embodiments, W in structure IV or V is a PEG group with an average molecular weight of about 55 kDa. In some embodiments, W in structure IV or V is a PEG group with 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 II and Formula III, wherein W is a linear or branched PEG group. In some embodiments, W in structures of Formula IV or Formula V is a linear PEG group. In some embodiments, W in structures of Formula IV or Formula V is a branched PEG group. In some embodiments, W in structures of Formula IV or Formula V is a methoxy PEG group. In some embodiments, W in structures of Formula IV or Formula V is a methoxy PEG group that is linear or branched. In some embodiments, the methoxy PEG groups in structures of Formula IV or Formula V are linear. In some embodiments, the methoxy PEG groups in structures of Formula IV or Formula V are branched.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or VII below, or a mixture of Formula VI and Formula VII: :

[Formula VI]

[Formula VII]

here,

n is an integer ranging from about 2 to about 5000;

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments of Formula VI or Formula VII, or mixtures of Formula VI and Formula VII, q is 1. In some embodiments of Formula VI or Formula VII, or mixtures of Formula VI and Formula VII, q is 2. In some embodiments of Formula VI or Formula VII, or mixtures of Formula VI and Formula VII, q is 3.

As used herein and throughout, structures of Formula VI include pharmaceutically acceptable salts, solvates or hydrates thereof. Structures of Formula VII herein and throughout include 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 structures of Formula VIa and Formula VIIa:

[Formula VIa]

[Formula VIIa]

here,

n is an integer ranging from about 2 to about 5000;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, n in the compounds of Formula VI and Formula VII is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900; or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or is from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, n in the compounds of Formula VI and Formula VII is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 159, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

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

In some embodiments, the ratio of the amount of structure VI to the amount of structure VII included in the total amount of IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure VI: amount of structure VII included in the total amount of IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure VI to the amount of structure VII included in the total amount of 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or Formula VII, or a mixture of Formula VI and Formula VII. wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and n is from 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or 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 about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to from about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to is an integer from about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, n in the compounds of Formula VI and Formula VII is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 159, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or VII, or a mixture of Formula VI and Formula VII; , wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and n is from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or It is an integer from about 568 to about 909. In some embodiments, n in the compounds of Formula VI and Formula VII is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, is an integer selected from 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or VII, or a mixture of Formula VI and Formula VII; , wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909 am. In some embodiments, n in compounds of Formula VI and Formula VII is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or VII, or a mixture of Formula VI and Formula VII; , wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in compounds of Formula VI and Formula VII is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is between about 500 and about 1000. In some embodiments, n is between about 550 and 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or VII, or a mixture of Formula VI and Formula VII; , wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in compounds of Formula VI and Formula VII is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is between about 500 and about 1000. In some embodiments, n is between about 550 and about 800. In some embodiments, n is about 681.

In some embodiments, n in structures of Formula VI and Formula VII is the molecular weight of the PEG moiety between about 1,000 Daltons and about 200,000 Daltons, or between about 2,000 Daltons and about 150,000 Daltons, or between about 3,000 Daltons and about 125,000 Daltons, or about 4,000 daltons to about 100,000 daltons, or about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or about 5,000 daltons to about 60,000 daltons, or about 5,000 daltons to about 50,000 daltons, or about 6,000 daltons to about 50,000 daltons, or about 7,000 daltons to about 50,000 daltons, or about 7,000 daltons daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons , or 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 about 9,500 daltons to about 30,000 daltons, or about 10,000 daltons to about 50,000 daltons, or about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,0 00 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 40,000 daltons, or about 15,000 daltons to about 35,000 daltons, or about 15,000 daltons to about 30,000 daltons, or about 20,000 daltons to about 50,00 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or VII, or a mixture of Formula VI and Formula VII; , where n is the molecular weight of the PEG moiety is about 5,000 Da, about 7,500 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, about 40,000 Da, about 45,000 Da .

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VI or VII, or a mixture of Formula VI and Formula VII; , where n is the molecular weight of the PEG moiety is about 5,000 Da, about 7,500 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, about 40,000 Da, about 45,000 Da , or an integer such that about 50,000 Da.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or IX, or a mixture of Formulas VIII and IX:

[Formula VIII]

[Formula IX]

here,

n is an integer ranging from about 2 to about 5000;

q is 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments of Formula VIII or Formula IX, or mixtures of Formula VIII and Formula IX, q is 1. In some embodiments of Formula VIII or Formula IX, or mixtures of Formula VIII and Formula IX, q is 2. In some embodiments of Formula VIII or Formula IX, or mixtures of Formula VIII and Formula IX, q is 3.

As used herein and throughout, structures of Formula VIII include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of formula (IX) include 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 and Formula IX are structures of Formula VIIIa or Formula IXa:

[Formula VIIIa]

[Formula IXa]

here,

n is an integer ranging from about 2 to about 5000;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments, n in the compounds of Formula VIII and Formula IX is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900; or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136; or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, n in the compounds of Formula VIII and Formula IX is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 159, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

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

In some embodiments, the ratio of the amount of structure VIII to the amount of structure IX included in the total amount of IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure VIII to the amount of structure IX included in the total amount of IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure VIII to the amount of structure IX included in the total amount of 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX. wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and n is from 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or 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 about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, n in the compounds of Formula VIII and Formula IX is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 159, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or IX, or a mixture of Formula VIII and Formula IX; , wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and n is from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or It is an integer from about 568 to about 909. In some embodiments, n in the compounds of Formula VIII and Formula IX is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, is an integer selected from 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX. wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and n is 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 is an integer 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX. wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, 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 between about 500 and about 1000. In some embodiments, n is between about 550 and 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX. wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, 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 between about 500 and about 1000. In some embodiments, n is between about 550 and 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX. where n is the molecular weight of the PEG moiety is from about 1,000 daltons to about 200,000 daltons, or from about 2,000 daltons to about 150,000 daltons, or from about 3,000 daltons to about 125,000 daltons, or from about 4,000 daltons to about 100,000 daltons, or from about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or 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 about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 daltons to about 45,000 daltons, or 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 about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,000 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 15,000 daltons 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 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 in which one or more amino acid residues in the SMS IL-2 conjugate are replaced with a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX. where n is the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons, or about 200,000 daltons. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX. where n is 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 an integer that equals about 50,000 daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with the structure of Formula X or Formula XI below, or a mixture of Formula X and Formula XI: contains:

[Formula X]

[Formula XI]

here,

n is an integer ranging from about 2 to about 5000;

q is 1, 2 or 3;

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 mixtures of Formula X and Formula XI, q is 1. In some embodiments of Formula X or Formula XI, or mixtures of Formula X and Formula XI, q is 2. In some embodiments of Formula X or Formula XI, or mixtures of Formula X and Formula XI, q is 3.

As used herein and throughout, structures of Formula X include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of Formula (XI) include 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 structures of Formula Xa and Formula XIa:

[Formula Xa]

[Formula XIa]

here,

n is an integer ranging from about 2 to about 5000;

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

In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is racemic, enriched in (R), enriched in (S), substantially (R), substantially (S), or ( R) or (S). In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is racemic. In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is enriched in (R). In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is enriched in (S). In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is substantially (R). In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is substantially (S). In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is (R). In some embodiments, the stereochemistry of chiral centers in Formula X and Formula XI is (S).

In an embodiment, n in the compounds of Formula X and Formula XI is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or about 150 to about 2900, or about 125 to about 2900, or about 100 to about 2500, or about 100 to about 2000, or about 100 to about 1900, or about 100 to about 1850, or about 100 to about 1750, or about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 3 41 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, n in compounds of Formula X and Formula XI is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 159, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

In some embodiments, the structure of Formula X, Formula XI, or the position of a mixture of Formula X and Formula XI in the amino acid sequence of the IL-2 conjugate is K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the position of Formula X, structure XI, or mixtures thereof in the amino acid sequence of the IL-2 conjugate is relative to the position in SEQ ID NO:3. In some embodiments, the structure of Formula X, Formula XI, or the position of a mixture of Formula X and Formula XI in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is K34, F41, F43, K42, E61, P64, An IL-2 conjugate selected from 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 at 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 at 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 at 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 at 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 at position E61. In some embodiments, 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 at position P64. In some embodiments, 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 at 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 at 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 at position E67. In some embodiments, 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 at position Y44. In some embodiments, 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 at 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 at position L71.

In some embodiments, the ratio of the amount of structure X to the amount of structure XI included in the total amount of IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure of Formula X to the amount of structure of Formula XI included in the total amount of IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure of Formula X to the amount of structure of Formula XI included in the total amount of 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with Formula X, a structure of Formula XI, or a mixture of Formula X and Formula XI. wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and n is from 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or 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 about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910 , or an integer from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, n in the compounds of Formula VI and Formula VII is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 159, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with Formula X, a structure of Formula XI, or a mixture of Formula X and Formula XI. wherein the amino acid residues in SEQ ID NO: 3 that are replaced are selected from F41, F43, K42, E61, and P64, and n is from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682; or an integer from about 568 to about 909. In some embodiments, n in the compounds of Formula X and Formula XI is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, is an integer selected from 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are replaced with Formula X, a structure of 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 n is 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 is an integer In some embodiments, n in 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with Formula X, a structure of 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 n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in 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 between about 500 and about 1000. In some embodiments, n is between about 550 and 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with Formula X, a structure of 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 n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in 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 between about 500 and about 1000. In some embodiments, n is between about 550 and about 800. In some embodiments, n is about 681.

In some embodiments, n in the structure of Formula X and Formula XI is the molecular weight of the PEG moiety is between about 1,000 Daltons and about 200,000 Daltons, or between about 2,000 and about 150,000 Daltons, or between about 3,000 and about 125,000 Daltons, or about 4,000 daltons to about 100,000 daltons, or about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons , or 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 about 7,000 daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or 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 about 30,000 daltons, or about 10,000 daltons to about 50,000 daltons, or about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or About 15,000 Daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 40,000 daltons, or about 15,000 daltons to about 35,000 daltons, or about 15,000 daltons to about 30,000 daltons, or about 20,000 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with Formula X, a structure of Formula XI, or a mixture of Formula X and Formula XI. where n is the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons, or about 200,000 daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are replaced with Formula X, a structure of Formula XI, or a mixture of Formula X and Formula XI. where n is 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 an integer that equals about 50,000 daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are replaced by a structure of Formula XII or Formula XIII, or a mixture of Formula XII and Formula XIII: contains:

[Formula XII]

[Formula XIII]

here,

n is an integer ranging from about 2 to about 5000;

q is 1, 2 or 3;

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 mixtures of Formula XII and Formula XIII, q is 1. In some embodiments of Formula XII or Formula XIII, or mixtures of Formula XII and Formula XIII, q is 2. In some embodiments of Formula XII or Formula XIII, or mixtures of Formula XII and Formula XIII, q is 3.

As used herein and throughout, structures of Formula (XII) include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of Formula (XIII) include 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 of Formula (XIIa) and Formula (XIIIa):

[Formula XIIa]

[Formula XIIIa]

here,

n is an integer ranging from about 2 to about 5000;

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

In some embodiments, the stereochemistry of chiral centers in Formulas XII and Formulas XIII is racemic, enriched in (R), enriched in (S), substantially (R), substantially (S), or ( R) or (S). In some embodiments, the stereochemistry of the chiral centers in Formula XII and Formula XIII is racemic. In some embodiments, the stereochemistry of chiral centers in Formula XII and Formula XIII is enriched in (R). In some embodiments, the stereochemistry of chiral centers in Formula XII and Formula XIII is enriched in (S). In some embodiments, the stereochemistry of the chiral centers in Formula XII and Formula XIII is substantially (R). In some embodiments, the stereochemistry of chiral centers in Formula XII and Formula XIII is substantially (S). In some embodiments, the stereochemistry of chiral centers in Formulas XII and Formulas XIII is (R). In some embodiments, the stereochemistry of chiral centers in Formula XII and Formula XIII is (S).

In some embodiments, n in the compounds of Formula XII and Formula XIII is from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900; or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or about 100 to about 1650, or about 100 to about 1500, or about 100 to about 1400, or about 100 to about 1300, or about 100 to about 1250, or about 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795, or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136; or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or 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 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, 150, 147, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

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

In some embodiments, the ratio of the amount of structure (XII) to the amount of structure (XIII) included in the total amount of IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure (XII) to the amount of structure (XIII) included in the total amount of IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure (XII) to the amount of structure (XIII) included in the total amount of 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 in which one or more amino acid residues in the IL-2 conjugate are 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 n is from 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or 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 about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, n in the compounds of Formula XII and Formula XIII is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680; 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 150, 147, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XII, Formula XIII, or a mixture of Formula XII and Formula XIII. wherein the amino acid residues in SEQ ID NO: 3 that are replaced are selected from F41, F43, K42, E61, and P64, and n is from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682; or an integer from about 568 to about 909. In some embodiments, n in the compounds of Formula XII and Formula XIII is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, is an integer selected from 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are 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 n is 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 is an integer In some embodiments, n in 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 in which one or more amino acid residues in the IL-2 conjugate are 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 n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in 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 between about 500 and about 1000. In some embodiments, n is between about 550 and 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 in which one or more amino acid residues in the IL-2 conjugate are 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 n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in 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 between about 500 and about 1000. In some embodiments, n is between about 550 and about 800. In some embodiments, n is about 681.

In some embodiments, n in the structure of Formula (XII) or Formula (XIII) is when the molecular weight of the PEG moiety is between about 1,000 daltons and about 200,000 daltons, or between about 2,000 daltons and about 150,000 daltons, or between about 3,000 daltons and about 125,000 daltons, or about 4,000 daltons. daltons to about 100,000 daltons, or about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons , or 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 about 7,000 daltons to about 45,000 daltons, or about 7,000 daltons to about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or 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 about 30,000 daltons, or about 10,000 daltons to about 50,000 daltons, or about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,0 00 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 40,000 daltons, or about 15,000 daltons to about 35,000 daltons, or about 15,000 daltons to about 30,000 daltons, or about 20,000 daltons to about 50,00 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XII, Formula XIII, or a mixture of Formula XII and Formula XIII. where n is the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons, or about 200,000 daltons. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XII, Formula XIII, or a mixture of Formula XII and Formula XIII. where n is 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 an integer that equals about 50,000 daltons.

In some embodiments, the IL-2 conjugate is of SEQ ID NO: 3 wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX: It contains the amino acid sequence:

[Formula VIII]

[Formula IX]

here,

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 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

In some embodiments of Formula VIII or Formula IX, or mixtures of Formula VIII and Formula IX, q is 1. In some embodiments of Formula VIII or Formula IX, or mixtures of Formula VIII and Formula IX, q is 2. In some embodiments of Formula VIII or Formula IX, or mixtures of Formula VIII and Formula IX, q is 3.

As used herein and throughout, structures of Formula VIII include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of formula (IX) include 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 and Formula IX are structures of Formula VIIIa and Formula IXa:

[Formula VIIIa]

[Formula IXa]

here,

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

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing 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, wherein n is the molecular weight of the PEG group from about 20,000 Daltons to about It is an integer that equals 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is 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, wherein n is the molecular weight of the PEG group from about 20,000 Daltons to about It is an integer that equals 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 30,000 Daltons.

In some embodiments, the IL-2 conjugate is of SEQ ID NO: 3 wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is replaced by a structure of Formula VIII or Formula IX, or a mixture of Formula VIII and Formula IX: It contains the amino acid sequence:

[Formula VIII]

[Formula IX]

here,

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 1, 2 or 3;

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing 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, wherein n is the molecular weight of the PEG group from about 20,000 Daltons to about It is an integer that equals 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is 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, wherein n is the molecular weight of the PEG group from about 20,000 Daltons to about It is an integer that equals 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 30,000 Daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4 in which one or more amino acid residues in the IL-2 conjugate are replaced with a cysteine covalently linked to a PEG group. . In some embodiments, the PEG group has a molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa. In some embodiments, the PEG group has a molecular weight of 5 kDa. In some embodiments, the PEG group has a molecular weight of 10 kDa. In some embodiments, the PEG group has a molecular weight of 15 kDa. In some embodiments, the PEG group has a molecular weight of 20 kDa. In some embodiments, the PEG group has a molecular weight of 25 kDa. In some embodiments, the PEG group has a molecular weight of 30 kDa. In some embodiments, the PEG group has a molecular weight of 35 kDa. In some embodiments, the PEG group has a molecular weight of 40 kDa. In some embodiments, the PEG group has a molecular weight of 45 kDa. In some embodiments, the PEG group has a molecular weight of 50 kDa. In some embodiments, the PEG group has a molecular weight of 60 kDa. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein one or more amino acid residues in the IL-2 conjugate replaced with cysteine are 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, wherein one or more amino acid residues in the IL-2 conjugate replaced with cysteine are 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, wherein one or more amino acid residues in the IL-2 conjugate replaced with cysteine are K35, T37, R38, T41, F42, K43, F44 , Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more non-lysine residues are replaced with a 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 conjugate comprises a PEG group covalently linked through a non-releasable linkage. In some embodiments, the IL-2 conjugate includes a non-dissociable, covalently linked PEG group.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein non-lysine amino acids in the IL-2 conjugate are replaced with lysine residues, and the lysine residues are covalently linked with one or more water soluble polymers. contains linkers. 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, a 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 the amino acid sequence of SEQ ID NO: 3, wherein the non-lysine amino acids in the amino acid sequence of the IL-2 conjugate are replaced with amino acids comprising: (a) lysine; (b) a covalent linker; and (3) and one or more water soluble polymers. In some embodiments, the one or more water soluble polymers include PEG groups.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are replaced by a structure of Formula XIV or Formula XV, or a mixture of Formula XIV and Formula XV: contains:

[Formula XIV]

[Formula XV]

here,

m is an integer from 0 to 20;

p is an integer from 0 to 20;

n is an integer ranging from about 2 to about 5000;

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

As used herein and throughout, structures of Formula (XIV) include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of Formula (XV) include 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 stereochemistry of chiral centers in Formula (XIV) and Formula (XV) is racemic, enriched in (R), enriched in (S), substantially (R), substantially (S), or ( R) or (S). In some embodiments, the stereochemistry of chiral centers in Formula (XIV) and Formula (XV) is racemic. In some embodiments, the stereochemistry of chiral centers in Formula (XIV) and Formula (XV) is enriched in (R). In some embodiments, the stereochemistry of chiral centers in Formula (XIV) and Formula (XV) is enriched in (S). In some embodiments, the stereochemistry of chiral centers in Formula (XIV) and Formula (XV) is substantially (R). In some embodiments, the stereochemistry of chiral centers in Formula (XIV) and Formula (XV) is substantially (S). In some embodiments, the stereochemistry of chiral centers in Formula XIV and Formula XV is (R). In some embodiments, the stereochemistry of chiral centers 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 0 to 20, or 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments, m in 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 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 is 1 in compounds of Formula XIV and Formula XV. In some embodiments, p is 2 in the compounds of Formula XIV and Formula XV. In some embodiments, p is 3 in the compounds of Formula XIV and Formula XV. In some embodiments, p is 4 in the compounds of Formula XIV and Formula XV. In some embodiments, p is 5 in the compounds of Formula XIV and Formula XV. In some embodiments, p is 6 in the compounds of Formula XIV and Formula XV. In some embodiments, p is 7 in the compounds of Formula XIV and Formula XV. In some embodiments, p in the compounds of Formula XIV and Formula XV is 8. In some embodiments, p is 9 in the compounds of Formula XIV and Formula XV. 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 is 14 in the compounds of Formula XIV and Formula XV. In some embodiments, p 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, p is 17 in the compounds of Formula XIV and Formula XV. In some embodiments, p is 18 in the compounds of Formula XIV and Formula XV. In some embodiments, p is 19 in the compounds of Formula XIV and Formula XV. 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 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 about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.

In some embodiments, 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 and is an integer selected from 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 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 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 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 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 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 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 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 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 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 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 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 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 compounds of Formulas XIV and XV, m is 11, p is 2, n is 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, is an integer selected from 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 11, p is 2, and n is 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 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 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, 12510, 12510 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2159 is an integer selected from 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, the structure of Formula XIV, Formula XV, or the position of a mixture of Formula XIV and Formula XV in the amino acid sequence of the IL-2 conjugate is K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the position of formula XIV, structure of formula XV, or mixture of formula XIV and formula XV in the amino acid sequence of the IL-2 conjugate corresponds to the position in SEQ ID NO:3 related In some embodiments, the structure of Formula XIV, Formula XV, or the position of a mixture of Formula XIV and Formula XV in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is K34, F41, F43, K42, E61, P64, is selected from 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 at 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 at 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 at 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 at 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 at 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 at 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 at 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 at position T40. In some embodiments, 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 at position E67. In some embodiments, 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 at 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 at 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 at position L71. In some embodiments, the ratio of the amount of structure (XIV) to the amount of structure (XV) included in the total amount of IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure (XIV) to the amount of structure (XV) included in the total amount of IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure (XIV) to the amount of structure (XV) included in the total amount of 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 one or more amino acid residues in the IL-2 conjugate are 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 n is from 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or 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 about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to from about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to is an integer from 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 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, 150, 147, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XIV or Formula XV, or a mixture of Formula XIV and Formula XV. wherein the amino acid residues in SEQ ID NO: 3 that are replaced are selected from F41, F43, K42, E61, and P64, and n is from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682; or an integer from about 568 to about 909. In some embodiments, n in the compounds of Formula XIV and Formula XV is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, is an integer selected from 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are 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 n is 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 is an integer 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 one or more amino acid residues in the IL-2 conjugate are 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 n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in 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 between about 500 and about 1000. In some embodiments, n is between about 550 and 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 one or more amino acid residues in the IL-2 conjugate are 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 n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, 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 between about 500 and about 1000. In some embodiments, n is between about 550 and 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 one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XIV or Formula XV, or a mixture of Formula XIV and Formula XV. where n is the molecular weight of the PEG moiety is from about 1,000 daltons to about 200,000 daltons, or from about 2,000 daltons to about 150,000 daltons, or from about 3,000 daltons to about 125,000 daltons, or from about 4,000 daltons to about 100,000 daltons, or from about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or 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 about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 daltons to about 45,000 daltons, or 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 about 10,000 daltons to about 45,000 daltons, or about 1 0,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,000 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000,000 4 daltons 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 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 in which one or more amino acid residues in the SMS IL-2 conjugate are replaced with a structure of Formula XIV or Formula XV, or a mixture of Formula XIV and Formula XV. where n is the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons, or about 200,000 daltons. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XIV or Formula XV, or a mixture of Formula XIV and Formula XV. where n is 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 an integer that equals about 50,000 daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XIV or Formula XV, or a mixture of Formula XIV and Formula XV. wherein the amino acid residues in SEQ ID NO: 3 that are replaced are 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 about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the compounds of Formula XIV and Formula XV, m is 2, p is 2, and n is 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 one or more amino acid residues in the IL-2 conjugate are 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, m is an integer from 1 to 6, p is an integer from 1 to 6, and n is from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the compounds of Formula XIV and Formula XV, m is 2, p is 2, n is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and is an integer selected from 910.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate are 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, m is an integer from 1 to 6, p is an integer from 1 to 6, n is from about 450 to about 800, or from about 454 to about 796; or an integer 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, n is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and is an integer selected from 910. In some embodiments, n is between about 500 and about 1000. In some embodiments, n is between about 550 and 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 one or more amino acid residues in the IL-2 conjugate are 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, m is an integer from 1 to 6, p is an integer from 1 to 6, n is from about 450 to about 800, or from about 454 to about 796; or an integer 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, n is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and is an integer selected from 910. In some embodiments, n is between about 500 and about 1000. In some embodiments, n is between about 550 and 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 in which one or more amino acid residues in the IL-2 conjugate are replaced by a structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII): contains:

[Formula XVI]

[Formula XVII]

here,

m is an integer from 0 to 20;

n is an integer ranging from about 2 to about 5000;

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

As used herein and throughout, structures of Formula (XVI) include pharmaceutically acceptable salts, solvates or hydrates thereof. As used herein and throughout, structures of Formula (XVII) include 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 stereochemistry of chiral centers in Formula XVI and Formula XVII is racemic, enriched in (R), enriched in (S), substantially (R), substantially (S), or ( R) or (S). In some embodiments, the stereochemistry of chiral centers in Formula (XVI) and Formula (XVII) is racemic. In some embodiments, the stereochemistry of chiral centers in Formula (XVI) and Formula (XVII) is enriched in (R). In some embodiments, the stereochemistry of chiral centers in Formula (XVI) and Formula (XVII) is enriched in (S). In some embodiments, the stereochemistry of chiral centers in Formula (XVI) and Formula (XVII) is substantially (R). In some embodiments, the stereochemistry of chiral centers in Formula (XVI) and Formula (XVII) is substantially (S). In some embodiments, the stereochemistry of chiral centers in Formula (XVI) and Formula (XVII) is (R). In some embodiments, the stereochemistry of chiral centers 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 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 is 1 in compounds of Formula XVI and Formula XVII. In some embodiments, m in compounds of Formula XVI and Formula XVII is 2. In some embodiments, m in compounds of Formula XVI and Formula XVII is 3. In some embodiments, m is 4 in compounds of Formula XVI and Formula XVII. In some embodiments, m in compounds of Formula XVI and Formula XVII is 5. In some embodiments, m is 6 in compounds of Formula XVI and Formula XVII. In some embodiments, m in compounds of Formula XVI and Formula XVII is 7. In some embodiments, m in compounds of Formula XVI and Formula XVII is 8. In some embodiments, m in compounds of Formula XVI and Formula XVII is 9. In some embodiments, m in compounds of Formula XVI and Formula XVII is 10. In some embodiments, m in compounds of Formula XVI and Formula XVII is 11. In some embodiments, m in compounds of Formula XVI and Formula XVII is 12. In some embodiments, m in compounds of Formula XVI and Formula XVII is 13. In some embodiments, m in compounds of Formula XVI and Formula XVII is 14. In some embodiments, m in compounds of Formula XVI and Formula XVII is 15. In some embodiments, m in compounds of Formula XVI and Formula XVII is 16. In some embodiments, m in compounds of Formula XVI and Formula XVII is 17. In some embodiments, m in compounds of Formula XVI and Formula XVII is 18. In some embodiments, m in compounds of Formula XVI and Formula XVII is 19. In some embodiments, m in 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 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 about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or About 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein m in the compounds of Formulas XVI and XVII is an integer from 1 to 6, and n is 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682; is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682; is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, It is an integer selected from 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds of Formulas XVI and Formulas XVII, m is 3 and n is 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795; It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, It is an integer selected from 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 and n is 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795; It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795; It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795; It is an integer selected from 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 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795; It is an integer selected from 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 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137 is an integer selected from

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 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, 12510, 12510 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2159 is an integer selected from 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, the structure of Formula XVI, Formula XVII, or the position of a mixture of Formula XVI and Formula XVII in the amino acid sequence of the IL-2 conjugate is 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 corresponds to the position in SEQ ID NO:3. related In some embodiments, the structure of Formula XVI, Formula XVII, or the position of a mixture of Formula XVI and Formula XVII in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is K34, F41, F43, K42, E61, P64, is selected from R37, T40, E67, Y44, V68, and L71. In some embodiments, 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 at position K34. In some embodiments, 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 at 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 at position F43. In some embodiments, 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 at 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 at position E61. In some embodiments, 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 at 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 at position R37. In some embodiments, 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 at position T40. In some embodiments, 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 at position E67. In some embodiments, 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 at position Y44. In some embodiments, the structure of Formula XII, or the structure of Formula XIII, or the mixture of Formula XII and Formula XIII in the amino acid sequence of the IL-2 conjugate of SEQ ID NO:3 is at position V68. In some embodiments, 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 at position L71. In some embodiments, the ratio of the amount of structure (XVI) to the amount of structure (XVII) included in the total amount of IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure (XVI) to the amount of structure (XVII) included in the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure (XVI) to the amount of structure (XVII) included in the total amount of the IL-2 conjugate is less than 1:1.

In some embodiments, the IL-2 conjugate comprises one or more amino acid residues in the IL-2 conjugate selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. comprises the amino acid sequence of SEQ ID NO: 3 substituted with a structure of Formula XVI, Formula XVII, or a mixture of Formula XVI and Formula XVII, wherein n is 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 About 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or is an integer from about 114 to about 690, or from about 114 to about 575. In some embodiments, n in compounds of Formulas XVI and Formulas XVII is 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680; 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 159, 147, 147, 147 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2953, 2955, is an integer selected from 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

In some embodiments, the IL-2 conjugate has one or more amino acid residues in the IL-2 conjugate selected from F41, F43, K42, E61, and P64 in the structure of Formula XVI, Formula XVII, or Formula XVI and Formula XVII, wherein n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in the compounds of Formula XVI and Formula XVII is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, is an integer selected from 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate is a sequence in which one or more amino acid residues in the IL-2 conjugate, selected from E61 and P64, are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formula XVI and Formula XVII. No. 3, wherein n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formula XVI and Formula XVII (replacement is E61), 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 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 between about 500 and about 1000. In some embodiments, n is between about 550 and about 800. In some embodiments, n is about 681.

In some embodiments, the IL-2 conjugate is a sequence in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formula XVI and Formula XVII, where the replacement is P64. No. 3, wherein n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments, n in 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 between about 500 and about 1000. In some embodiments, n is between about 550 and 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formulas XVI and Formula XVII. where n is the molecular weight of the PEG moiety is from about 1,000 daltons to about 200,000 daltons, or from about 2,000 daltons to about 150,000 daltons, or from about 3,000 daltons to about 125,000 daltons, or from about 4,000 daltons to about 100,000 daltons, or from about 5,000 daltons to about 100,000 daltons, or about 6,000 daltons to about 90,000 daltons, or about 7,000 daltons to about 80,000 daltons, or about 8,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 70,000 daltons, or about 5,000 daltons to about 65,000 daltons, or 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 about 40,000 daltons, or about 8,000 daltons to about 40,000 daltons, or about 8,500 daltons to about 40,000 daltons, or about 8,500 daltons to about 35,000 daltons, or about 9,000 daltons to about 50,000 daltons, or about 9,000 daltons to about 45,000 daltons, or 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 about 10,000 daltons to about 45,000 daltons, or about 10,000 daltons to about 40,000 daltons, or about 10,000 daltons to about 35,000 daltons, or about 10,000 daltons to about 30,000 daltons, or about 15,000 daltons to about 50,000 daltons, or about 15,000 daltons to about 45,000 daltons, or about 15,000 daltons to about 15,000 daltons 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 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 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formulas XVI and Formula XVII. where n is the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons, about 20,000 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 60,000 daltons, about 70,000 daltons, about 80,000 daltons, about 90,000 daltons, about 100,000 daltons, about 125,000 daltons, about 150,000 daltons, about 175,000 daltons, or about 200,000 daltons. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formulas XVI and Formula XVII. where n is 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 an integer that equals about 50,000 daltons.

In some embodiments, the IL-2 conjugate has one or more amino acid residues in the IL-2 conjugate selected from F41, F43, K42, E61, and P64 in the structure of Formula XVI, Formula XVII, or Formula XVI and Formula XVII, wherein m is an integer from 1 to 6, and n is from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the compounds of Formula XVI and Formula XVII, m is 2 and n is 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, is an integer selected from 1023, 1135, 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate is a sequence in which one or more amino acid residues in the IL-2 conjugate, selected from E61 and P64, are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formula XVI and Formula XVII. 3, wherein m is an integer from 1 to 6, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909 am. 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 am.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which one or more amino acid residues in the IL-2 conjugate are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formula XVI and Formula XVII (replacement is E61, wherein m is an integer from 1 to 6, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909 am. 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 am. In some embodiments, n is between about 500 and about 1000. In some embodiments, n is between about 550 and about 800. In some embodiments, n is about 681.

In some embodiments, the IL-2 conjugate is an amino acid of SEQ ID NO: 3 wherein one or more amino acid residues in the IL-2 conjugate, which is P64, are replaced with a structure of Formula XVI, Formula XVII, or a mixture of Formula XVI and Formula XVII. wherein m is an integer from 1 to 6, and n is an integer from about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of the 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 am. In some embodiments, n is between about 500 and about 1000. In some embodiments, n is between about 550 and 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 the structure of Formula I. In some embodiments, the IL-2 conjugate comprises the structure of Formula II. In some embodiments, the IL-2 conjugate comprises the structure of Formula III. In some embodiments, the IL-2 conjugate comprises the structure of Formula IV. In some embodiments, the IL-2 conjugate comprises the structure of Formula V. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 1. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:2. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:3. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:4. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:5. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:6. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:7. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:8. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:9. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:10. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 11. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:12. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:13. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:14. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:15. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:16. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:17. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 18. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:19. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:20. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:21. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:22. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:23. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:24. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:25. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:28. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:24. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:25. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:28. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:29. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:30. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:31. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:32. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:33. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:34. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:35. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:36. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:37. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:38. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:39. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:40. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:41. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:42. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:43. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:44. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:45. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:46. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:47. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:48. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:49. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:50. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:51. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:52. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:53. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:54. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:55. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:56. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:57. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:58. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:59. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:60. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:61. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:62. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:63. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:64. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:65. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:66. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:67. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:68. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:69. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:70. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:71. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:72. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:73. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:74. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:75. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:76. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:77. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:78. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:79. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:80. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:81. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:82. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:83. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:84. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:85. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:86. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:87. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:88. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:89. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:90. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:91. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:92. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:93. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:94. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:95. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:96. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:97. In some embodiments, the IL-2 conjugate comprises SEQ ID NO:98.

In some embodiments, the IL-2 conjugate comprises amino acids of any one of SEQ ID NOs: 86, 88, 90, 92, 94, 96, and 98. In any of the above embodiments, the structure of Formula I, or any variant thereof, such as Formula II-XV or any variant thereof, is incorporated at the site comprising the non-natural amino acid.

In some embodiments, the IL-2 conjugate is modified at certain amino acid positions. In some cases, modifications are to natural amino acids. In some cases, modifications are to non-natural amino acids. In some instances, isolated and modified IL-2 polypeptides comprising at least one non-natural amino acid are described herein. In some cases, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 3-84.

In some embodiments, the IL-2 conjugate further comprises additional mutations. 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, amino acids are conjugated to additional conjugating moieties to increase serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a natural amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine or tyrosine; or mutated to an unnatural amino acid and then linked to additional conjugating moieties.

In some cases, PEG groups are not limited to a particular structure. In some cases, PEG is linear (e.g., end-capped, such as alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), dendritic ( or star) structures, each with or without one or more degradable linkages. In addition, the internal structure of the water-soluble polymer may be composed of various repeating patterns, and may be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating terpolymers, random terpolymers, and block terpolymers. can

PEG will typically include multiple (OCH ₂ CH ₂ ) monomers [or (CH ₂ CH ₂ O) monomers, depending on how PEG is defined]. As used herein, the number of repeat units is identified by the subscript “n” in “(OCH ₂ CH ₂ ) _n ”. Thus, the value of (n) will generally fall within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, about 225 to about 1930, about 450 to about 1930, about 1200 to about 1930, about 568 to about 2727, about 660 to about 2730, about 795 to about 2730, about 795 to about 2730, about 909 to about 2730, and about 1,200 to about 1,900. For any given polymer of known molecular weight, the total weight average molecular weight of the polymer can be divided by the molecular weight of the repeating monomers to determine the number of repeat units (i.e., "n").

In some cases, PEG is an end-capped polymer, ie a polymer having at least one end capped with a relatively inactive group such as a lower C _1-6 alkoxy group or a hydroxyl group. When the polymer is PEG, for example, methoxy-PEG (commonly referred to as mPEG) can be used, which is a linear form of PEG wherein one end of the polymer is a methoxy (-OCH ₃ ) group and the other The terminal is a hydroxyl group or other functional group that can be chemically modified as desired.

In some embodiments, the PEG groups included in the IL-2 conjugates disclosed herein are linear or branched PEG groups. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. In some embodiments, the PEG group is a linear or branched PEG group having an average molecular weight between about 100 Da and about 150,000 Da. Exemplary ranges include, for example, a range of greater than 5,000 Da to about 100,000 Da, a range of about 6,000 Da to about 90,000 Da, a range of about 10,000 Da to about 85,000 Da, a range of greater than 10,000 Da to about 85,000 Da, and a range of about 20,000 Da to about 85,000 Da, from about 53,000 Da to about 85,000 Da, from about 25,000 Da to about 120,000 Da, from about 29,000 Da to about 120,000 Da, from about 35,000 Da to about 120,000 Da, and from about 40,000 and a weight average molecular weight ranging from Da to about 120,000 Da. Exemplary weight average molecular weights for the PEG group are about 100 Da, about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 750 Da, about 800 Da, about 900 Da, about 1,000 Da, about 1,500 Da, about 2,000 Da, about 2,200 Da, about 2,500 Da, about 3,000 Da, about 4,000 Da, about 4,400 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about 6,000 Da, about 7,000 Da , about 7,500 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, about 11,000 Da, about 12,000 Da, about 13,000 Da, about 14,000 Da, about 15,000 Da, about 20,000 Da, about 22,500 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, about 40,000 Da, about 45,000 Da, about 50,000 Da, about 55,000 Da, about 60,000 Da, about 65,000 Da, about 70,000 Da, about 75,000 Da, about 80,000 Da, about 90,000 Da, about 95,000 , and about 100,000 Da. In some embodiments, the PEG group is a linear PEG group having an average molecular weight as described above. In some embodiments, the PEG group is a branched PEG group having an average molecular weight as described above. In some embodiments, the PEG groups included in the IL-2 conjugates disclosed herein are linear or branched PEG groups having a defined molecular weight ± 10%, or 15% or 20% or 25%. For example, IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da ± 3000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 6,000 Da are included within the scope of the present invention.

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

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

In some embodiments, the PEG groups included in the IL-2 conjugates disclosed herein are branched methoxy PEG groups with an average molecular weight of about 5,000 Da to about 60,000 Da. In some embodiments, the PEG group is about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, about 8,000 daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons , about 14,000 daltons, about 15,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 0 daltons, about 60 daltons branched methoxy PEG groups having average molecular weights of 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group having an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy PEG group with an average molecular weight of about 5,000 Da. In some embodiments, the PEG group is a branched methoxy PEG group with an average molecular weight of about 10,000 Da. In some embodiments, the PEG group is a branched methoxy PEG group with an average molecular weight of about 20,000 Da. In some embodiments, the PEG group is a branched methoxy PEG group with an average molecular weight of about 30,000 Da. In some embodiments, the PEG group is a branched methoxy PEG group with an average molecular weight of about 50,000 Da. In some embodiments, the PEG group is a branched methoxy PEG group with an average molecular weight of about 60,000 Da. In some embodiments, the PEG groups included in the IL-2 conjugates disclosed herein are branched methoxy PEG groups having a defined molecular weight ± 10%, or 15% or 20% or 25%. For example, IL-2 conjugates comprising a branched methoxy PEG group having a molecular weight of 30,000 Da ± 3000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 6,000 Da are included within the scope of the present invention.

In some embodiments, exemplary PEG groups include linear or branched diacid PEG (dPEG) from Quanta Biodesign, Ltd; linear, branched, or forked PEG from Nektar Therapeutics; and Y-type PEG derivatives from JenKem Technology.

In any embodiment or modification of Formula I described herein and pharmaceutical compositions comprising it, the average molecular weight includes both the weight average molecular weight and the number average molecular weight; That is, for example, both a number average molecular weight of 30 kDa and a weight average molecular weight of 30 kDa are considered to be a molecular weight of 30 kDa. In some embodiments, the average molecular weight is a weight average molecular weight. In another embodiment, the average molecular weight is a number average molecular weight. In the methods provided herein, administering an IL-2 conjugate as described herein to a subject comprises administering more than a single molecule of the IL-2 conjugate; As such, use of the term "average" to describe the molecular weight of a PEG group refers to the average molecular weight of a PEG group of an IL-2 conjugate molecule at a dose administered to a subject.

conjugation chemistry

Linkers, conjugation moieties, and unnatural amino acids (incorporated into the IL-2 polypeptides described herein) are conjugated using a variety of conjugation reactions. Such conjugation reactions are usually suitable for aqueous conditions such as "bioorthogonal" reactions. In some embodiments, the conjugation reaction is a chemical reagent such as a catalyst; shed; or by reactive chemical groups found in linkers, conjugation moieties, or non-natural amino acids. In some embodiments, the conjugation reaction is enzyme mediated. In some embodiments, conjugation reactions as used herein are described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123, the disclosure of which is incorporated herein by reference. In some embodiments, conjugation reactions as used herein are described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417, the disclosure of which is incorporated herein by reference.

In some variations, the IL-2 conjugates described herein can be prepared by conjugation reactions including 1,3-dipolar cycloaddition reactions. In some embodiments, the 1,3-dipolar cycloaddition reaction involves a reaction of an azide with a phosphine (a “click” reaction). In some embodiments, the conjugation reaction is catalyzed by copper. In some embodiments, a conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached through a triazole. In some embodiments, the conjugation reactions described herein include the reaction of an azide with a modified olefin. In some embodiments, the conjugation reactions described herein include the reaction of an azide with a modified alkyne. In some embodiments, the conjugation reactions described herein include the reaction of an azide with a cycloalkyne, such as DBCO.

In some embodiments described herein, the conjugation reactions described herein include the reactions outlined in Scheme 1, wherein X is SEQ ID NO: 5, 6, 7, 8, 9, 30, 31, 32, 33 , and 34, is a position in an IL-2 conjugate comprising an unnatural amino acid.

Scheme 1.

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

In some embodiments, the conjugation reactions described herein include the reactions outlined in Scheme 2, wherein X is any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34 As in either case, it is a position in an IL-2 conjugate that includes an unnatural amino acid.

Scheme 2.

In some embodiments, the conjugation reactions described herein include the reactions outlined in Scheme 3, wherein X is any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34 As in either case, it is a position in an IL-2 conjugate that includes an unnatural amino acid.

Scheme 3.

In some embodiments described herein, the conjugation reactions described herein include the reaction outlined in Scheme 4, wherein X is SEQ ID NO: 5, 6, 7, 8, 9, 30, 31, 32, 33 , and 34, is a position in an IL-2 conjugate comprising an unnatural amino acid.

Scheme 4.

In some embodiments, the conjugation reactions described herein are performed on proteins containing amino acid residues derived from azide moieties, such as N6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK). and those derived from DBCO, a chemical moiety containing a modified cycloalkyne, such as a dibenzocyclooctyne group. PEG groups comprising DBCO moieties are commercially available or can be prepared by methods known to those skilled in the art. Exemplary reactions are shown in Schemes 5 and 6.

Scheme 5.

Scheme 6.

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

IL-2 polypeptide production

In some cases, IL-2 conjugates described herein comprising natural amino acid mutations or non-natural amino acid mutations are recombinantly produced or chemically synthesized. In some cases, an IL-2 conjugate described herein is produced recombinantly, eg, by a host cell system or in a cell-free system. In any of the embodiments or variations described herein, the amino acid may be an L-amino acid or a D-amino acid. In some embodiments, the amino acids are L-amino acids. In another embodiment the amino acid is a D-amino acid.

In some cases, IL-2 conjugates are recombinantly produced via host cell systems. In some cases, the host cell is a eukaryotic cell (eg, a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (eg, a gram-positive bacterium or a gram-negative bacterium). In some cases, eukaryotic host cells are mammalian host cells. In some cases, a mammalian host cell is a stable cell line or a cell line that has the ability to integrate the genetic material of interest into its genome and express the product of the genetic material after several generations of cell division. In other cases, the mammalian host cell is a transient cell line, or a cell line that does not integrate the genetic material of interest into its genome and is incapable of expressing the product of the genetic material after several generations of cell division.

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

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

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

In some embodiments, eukaryotic host cells are plant host cells. In some cases, plant cells include cells of algae. Exemplary plant cell lines include Chlamydomonas reinhardtii 137c, or strains of Synechococcus elongatus PPC 7942.

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

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

Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVLA1393 M11, pVLAG3L such as vectors pPolh-FLAG1 or pPolh-MAT 2, or a MAT vector 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, pBAD-DEST49 Gateway ^® target vector, pAO815 Pichia vector, pFLD1 Pichia pastoris (K. phaffii) vector, pGAPZA, B, & C Pichia pastoris (K. phaffii) vector, pPIC3.5K Pichia vector, pPIC6 A, B , &C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.

Avian vectors include, for example, pChlamy-4 vectors or MCS vectors.

Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors are p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3 or pBICEP-CMV 4. Exemplary mammalian stable expression vectors are 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, cell-free systems are used for the production of IL-2 polypeptides described herein. In some cases, cell-free systems include a mixture of cytoplasmic and/or nuclear components from cells and are suitable for in vitro nucleic acid synthesis. In some cases, cell-free systems use prokaryotic cell components. In other cases, cell-free systems use eukaryotic cell components. Nucleic acid synthesis is obtained in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, archaea, or HeLa cells. Exemplary cell-free systems include the E. coli S30 Extract system, E. coli T7 S30 system or PURExpress®, XpressCF and XpressCF+.

Cell-free translation systems include components such as plasmids, mRNA, DNA, tRNAs, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and/or unnatural amino acids, and/or proteins used for expression. Contains various other ingredients. These components are modified as necessary to improve yield, increase synthesis rate, increase protein product reliability, or incorporate non-natural amino acids. In some embodiments, cytokines described herein are described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or using the cell-free translation system described in US 8,778,631. In some embodiments, the cell-free translation system includes a modified release factor, or even removal of one or more release factors from the system. In some embodiments, the cell-free translation system includes a reduced protease concentration. In some embodiments, the cell-free translation system comprises a modified tRNA with a reassigned codon used to encode an unnatural amino acid. In some embodiments, the synthetases described herein for incorporation of non-natural amino acids are used in cell-free translation systems. In some embodiments, the tRNA is preloaded with an unnatural amino acid using enzymatic or chemical methods before being added to the cell-free translation system. In some embodiments, components for cell-free translation systems are obtained from modified organisms, such as modified bacteria, yeast, or other organisms.

In some embodiments, the IL-2 polypeptide is produced in circularly rearranged form via an expression host system or via a cell-free system.

Production of IL-2 Polypeptides Containing Non-Natural Amino Acids

Orthogonal or extended genetic codes can be used in the present invention, wherein one or more specific codons present in the nucleic acid sequence of an IL-2 polypeptide are assigned to encode an unnatural amino acid to generate IL using an orthogonal tRNA synthetase/tRNA pair. Can be genetically integrated into -2. An orthogonal tRNA synthetase/tRNA pair can charge the tRNA with an unnatural amino acid and incorporate the unnatural amino acid into a polypeptide chain in response to a codon.

In some cases, the codon is the codon amber, ocher, opal, or quadruplet codon. In some cases, codons correspond to orthogonal tRNAs to be used to carry unnatural amino acids. In some cases, the codon is amber. In other cases, the codon is an orthogonal codon.

In some cases, the codon is a quadruplet codon that can be translated by the orthogonal ribosome ribo-Q1. In some cases, quadruplet codons are those exemplified by Neumann, et al ., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature , 464 (7287): 441-444 (2010). As is, the disclosure content of which is incorporated herein by reference.

In some cases, codons used in the present invention are recoded codons, eg, synonymous codons or rare codons replaced with replacement codons. In some cases, recoded codons are described in Napolitano, et al., "Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli ," PNAS , 113 (38): E5588-5597 (2016). As noted above, 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, non-natural nucleic acids are used to incorporate one or more non-natural amino acids into IL-2. Exemplary non-natural nucleic acids include uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, Xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5 -haluracil and cytosine, 5-propynyl uracil 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 adenine and guanine, 5-halo, specifically 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7 -methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Certain non-natural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5- Propynylcytosine, 5-methylcytosine, increasing stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-enlarged nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N- 2, N-6 and 0-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine). 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanine, other alkyl derivatives, 2-propyl of adenine and guanine, and others Alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-haluracil, 5-halocytosine, 5-propynyl (-C≡C-CH ₃ ) uracil, 5-propynyl cytosine, pyr Other alkynyl derivatives of midine 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 adenine and guanine, 5-halo, specifically 5-bromo, 5-trifluoromethyl, other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyl Adenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic Pyrimidine, phenoxazine cytidine ([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][l ,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l ,4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] indol-2-one), pyridoindole cytidine (H-pyrido [3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), purine or pyrimidine base replaced by another heterocycle, 7-deaza-adenine, 7-deaza Guanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoro Pyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil , and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine , 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2 -amino-2'-deoxyadenosine, and US Pat. No. 3,687,808; U.S. Patent No. 4,845,205; U.S. Patent No. 4,910,300; U.S. Patent No. 4,948,882; U.S. Patent No. 5,093,232; U.S. Patent No. 5,130,302; U.S. Patent No. 5,134,066; U.S. Patent No. 5,175,273; U.S. Patent No. 5,367,066; U.S. Patent No. 5,432,272; U.S. Patent No. 5,457,187; U.S. Patent No. 5,459,255; U.S. Patent No. 5,484,908; U.S. Patent No. 5,502,177; U.S. Patent No. 5,525,711; U.S. Patent No. 5,552,540; U.S. Patent No. 5,587,469; U.S. Patent No. 5,594,121; U.S. Patent No. 5,596,091; U.S. Patent No. 5,614,617; U.S. Patent No. 5,645,985; U.S. Patent No. 5,681,941; U.S. Patent No. 5,750,692; U.S. Patent No. 5,763,588; U.S. Patent Nos. 5,830,653 and 6,005,096; International Publication No. 99/62923; See 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 those described in Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Additional base modifications are disclosed in, for example, U.S. Patent Nos. 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 in some cases the nucleic acids contain one or more heterocyclic bases other than the major five base components of naturally occurring nucleic acids. contains bases. For example, in some cases the heterocyclic base is uracil-5-yl, cytosine-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-amino Pyrrolo [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 group, wherein the purine is through the 9-position, the pyrimidine through the 1-position, the pyrrolopyrimidine through the 7-position, and the pyrazolopyrimidine through the 1-position attached to the sugar moiety of the nucleic acid via

In some embodiments, the nucleotide analog is also modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those with modifications in the linkage between two nucleotides, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphorotriesters , aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-amino phosphonates It contains amidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates. These phosphate or modified phosphate linkages between two nucleotides are via 3'-5' linkages or 2'-5' linkages, where linkages are 3'-5' to 5'-3', or 2'-5' It is understood to include an inverted polarity such as 5'-2' in . Various salts, mixed salts and free acid forms are also included. A number of US patents teach methods of making and using nucleotides containing modified phosphates; 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, the disclosures of each of which are incorporated herein by reference.

In some embodiments, the non-natural nucleic acid is a 2',3'-dideoxy-2',3'-didehydro-nucleoside (PCT/US2002/006460), 5'-substituted DNA and RNA derivatives (PCT/US2002/006460). 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 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/JP2484; PCT/JP2009/056718; PCT/JP2010/067560), or 5'-substituted monomers prepared as monophosphates with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337), the disclosures of each of which are incorporated herein by reference.

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

In some embodiments, the non-natural nucleic acid also includes a modification of the sugar moiety. In some cases, a nucleic acid comprises one or more nucleosides with modified sugar groups. Such sugar modified nucleosides may confer improved nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include addition of substituents (including 5' and/or 2'substituents); bridging of two ring atoms to form a bicyclic nucleic acid (BNA); replacement of a ribosyl ring oxygen atom by S, N(R), or C(R ₁ )(R ₂ ) (R = H, C ₁ -C ₁₂ alkyl or a protecting group); and combinations thereof, without limitation. 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, modified nucleic acids include 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 the sugar "analogue" cyclopentyl group . Sugars can be in the pyranosyl or furanosyl form. The sugar moiety can be the furanoside of ribose, deoxyribose, arabinose or 2'-O-alkylribose, and the sugar is attached to each heterocyclic base in [alpha] or [beta] anomeric configurations. It can be. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA/DNA chimeras. For example, sugar modifications may include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of such sugars or sugar analogs and each "nucleoside" in which such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known. Sugar modifications can also be made and combined with other modifications.

Modifications to the sugar moiety include natural and non-natural modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, 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 (alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl). Modifications per 2' are also -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n ONH ₂ , and -O(CH ₂ ) _n ON[(CH ₂ )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: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly Alkylamino, substituted silyl, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties . Similar modifications can also be made at other positions on the sugar, specifically the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. Modified sugars also include those containing modifications at bridging ring oxygens such as CH ₂ and S. A nucleotide sugar analogue may also have a sugar mimetic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. A number of U.S. patents, such as U.S. Patent No. 4,981,957, which teach the preparation of such modified sugar structures and detail and describe a series of base modifications; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; and 5,700,920, the disclosures of each of which are incorporated herein by reference in their entirety.

Examples of nucleic acids with modified sugar moieties include 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ₃ , and 2'-O(CH ₂ ) ₂ OCH ₃ substituents. Substituents at the 2' position may also be allyl, amino, azido, thio, O-allyl, O-(C ₁ -C ₁₀ alkyl), OCF ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ -ON(R _m )(R _n ), and O-CH ₂ -C(=0)-N(R _m )(R _n ), wherein each R _m and R _n is independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

In certain embodiments, the nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, a nucleic acid provided herein comprises one or more bicyclic nucleic acids, wherein the crosslink comprises a 4′→2′ bicyclic nucleic acid. Examples of such 4′→2′ bicyclic nucleic acids are those of the formula 4′-(CH ₂ )-O-2′(LNA); 4′-(CH ₂ )-S-2′; 4′-(CH ₂ ) ₂ -O-2′(ENA); 4′-CH(CH ₃ )-O-2′ and 4′-CH(CH ₂ OCH ₃ )-O-2′, and analogs thereof (see US Pat. No. 7,399,845); 4′-C(CH ₃ )(CH ₃ )-O-2′ and analogs thereof (International Publication No. 2009/006478, International Publication No. 2008/150729, US Patent Application Publication No. 2004/0171570, US Patent 7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134, and International Publication No. 2008/154401). See also, for example: Singh et al., Chem. Commun., 1998, 4, 455-456]; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; See Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638]; See Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222]; See Singh et al., J. Org. Chem., 1998, 63, 10035-10039]; See Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; See Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; See Braasch et al., Chem. Biol, 2001, 8, 1-7]; See Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243]; U.S. Patent No. 4,849,513; U.S. Patent No. 5,015,733; U.S. Patent No. 5,118,800; U.S. Patent No. 5,118,802; U.S. Patent No. 7,053,207; U.S. Patent No. 6,268,490; U.S. Patent No. 6,770,748; U.S. Patent No. 6,794,499; U.S. Patent No. 7,034,133; U.S. Patent No. 6,525,191; U.S. Patent No. 6,670,461; and U.S. Patent Nos. 7,399,845; International Publication No. 2004/106356, International Publication No. 1994/14226, International Publication No. 2005/021570, International Publication No. 2007/090071, and International Publication No. 2007/134181; US Patent Application Publication No. 2004/0171570, US Patent Application Publication No. 2007/0287831, and US Patent Application Publication No. 2008/0039618; U.S. Provisional Application No. 60/989,574, U.S. Provisional Application No. 61/026,995, U.S. Provisional Application No. 61/026,998, U.S. Provisional Application No. 61/056,564, U.S. Provisional Application No. 61/086,231, U.S. Provisional Application No. 61/097,787, and U.S. Provisional Application No. 61/099,844; and international applications PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922, and PCT/DK98/00393. The disclosures of each reference listed in this section are hereby incorporated by reference.

In certain embodiments, nucleic acids include linked nucleic acids. Nucleic acids may be linked to each other using any internucleic acid linkage. The two main classes of internucleic acid linkers are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus linkers between nucleic acids include methylenemethylimino (-CH ₂ -N(CH ₃ )-O-CH ₂ -), thiodiester (-OC(O)-S-), thionocarbamate ( -OC(O)(NH)-S-); Siloxane (-O-Si(H) ₂ -O-); and N,N*-dimethylhydrazine (-CH ₂ -N(CH ₃ )-N(CH ₃ )). In certain embodiments, internucleic acid linkages with chiral atoms can be prepared as racemic mixtures, separate enantiomers, such as alkylphosphonates and phosphorothioates. A non-natural nucleic acid may contain a single modification. A non-natural nucleic acid may contain multiple modifications within one of its moieties or between different moieties.

Backbone phosphate modifications to nucleic acids include methyl phosphonates, phosphorothioates, phosphoramidates (crosslinked or uncrosslinked), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, but , but is not limited thereto, and may be used in any combination. Other non-phosphate linkages may also be used.

In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate, and phosphorodithioate internucleotidic linkages) confer immunomodulatory activity to the modified nucleic acid and/or improve its stability. can improve (in vivo).

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

In some cases, backbone modifications include replacement of phosphodiester linkages with alternative moieties, such as anionic, neutral or cationic groups. Examples of such modifications include: anionic internucleoside linkages; N3'→P5' phosphoramidate modification; boranophosphate DNA; pro-oligonucleotides; neutral internucleoside linkages such as methylphosphonate; amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligo; peptide nucleic acids (PNA); and positively charged deoxyribonucleic acid guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). A modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, eg, a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

Substituents of phosphate may be, for example, short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatomic or heterocyclic internucleoside linkages. include These have morpholino linkages (formed in part from the sugar moiety of the nucleoside); siloxane backbone; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbone; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbone; and others having mixed N, O, S, and CH ₂ component parts. A number of US patents disclose methods of making and using phosphate substitutes of this type, including US Pat. No. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. It is also understood that in a nucleotide substitution both the sugar and phosphate moieties of a nucleotide may be replaced, for example by an amide type linkage (aminoethylglycine) (PNA). U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262 teach methods of making and using PNA molecules, each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg Med. Chem. Let., 1994, 4, 1053-1060]), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992 , 660, 306-309; 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al. ., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or tri Ethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (literature [ Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237]), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937]), but are not limited thereto. A number of US patents teach the preparation of such conjugates, including US Pat. No. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, but are not limited thereto. The disclosures of each reference listed in this section are hereby incorporated by reference.

In some cases, non-natural nucleic acids further form non-natural base pairs. Exemplary non-natural nucleotides capable of forming unnatural DNA or RNA base pairs (UBPs) under in vivo conditions include TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and Combinations of, but not limited to. In some embodiments, non-natural nucleotides include:

.

.

Exemplary unnatural base pairs include: (d)TPT3-(d)NaM; (d)5SICS-(d)NaM; (d) CNMO-(d) TAT1; (d) NaM-(d) TAT1; (d) CNMO-(d) TPT3; and (d) 5FM-(d) TAT1.

Other examples of non-natural nucleotides capable of forming non-natural UBPs that can be used to prepare the IL-2 conjugates disclosed herein are described 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; See 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; See Lavergne et al., J Am Chem Soc. 2013, 135:5408-5419]; and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010, the disclosures of each of which are incorporated herein by reference. In some embodiments, non-natural nucleotides include:

.

.

In some embodiments, the non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be derived from compounds of the formula:

wherein R ₂ is selected from hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azido;

The wavy line represents the linkage to ribosyl or 2'-deoxyribosyl, the 5'-hydroxy group of the ribosyl or 2'-deoxyribosyl moiety is in free form, monophosphate, diphosphate, triphosphate , α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate groups, or included in RNA or DNA or RNA analogues or DNA analogues.

In some embodiments, the non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein can be derived from compounds of the formula:

here,

each X is independently carbon or nitrogen;

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

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

E is oxygen, sulfur or selenium;

The wavy line indicates the point of attachment to the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof is in free form. , linked to a mono-phosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate or γ-thiotriphosphate group, or included in RNA or DNA or RNA analogues or DNA analogues.

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 2 X's are carbon. In some embodiments, both X's are carbon. In some embodiments, at least one X is nitrogen. In some embodiments, one X is nitrogen. In some embodiments, at least 2 X's are nitrogen. In some embodiments, two X's 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 an alkyl such as methyl, ethyl or propyl. In some embodiments, R ₂ is alkenyl, such as -CH ₂ =CH ₂ . In some embodiments, R ₂ is alkynyl, such as ethynyl. In some embodiments, R ₂ is methoxy. In some embodiments, R ₂ is methanethiol. In some embodiments, R ₂ is methaneseleno. In some embodiments, R ₂ is 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, the non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein are

로부터 유도될 수 있다.

can be derived from

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

, 또는 이들의 염을 포함한다.

, or salts thereof.

로부터 유도될 수 있다.In some embodiments, the non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein are

can be derived from

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

을 포함한다.

includes

In some embodiments, the unnatural base pair is a non-natural amino acid as described in Dumas et al., "Designing logical codon reassignment - Expanding the chemistry in biology," Chemical Science , 6 : 50-69 (2015). generate

In some embodiments, the non-natural amino acid is incorporated into the IL-2 polypeptide by a synthetic codon comprising the non-natural nucleic acid. In some cases, unnatural amino acids are incorporated into IL-2 by orthogonal transforming synthetase/tRNA pairs. Such an orthogonal pair is a ratio that allows unnatural amino acids to charge non-natural tRNAs while minimizing a) charging of non-natural tRNAs with other endogenous amino acids and b) charging of other endogenous tRNAs with non-natural amino acids. Includes natural synthetases. Such orthogonal pairs include: a) tRNAs that can be charged by the non-natural synthetase while avoiding being charged by the endogenous synthetase to other endogenous amino acids. In some embodiments, such pairs are identified from a variety of organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, the orthogonal synthetase/tRNA pair includes components from two different organisms. In some embodiments, orthogonal synthetase/tRNA pairs include components that promote translation of two different amino acids prior to modification. 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. 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 alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamic acid tRNA. In some embodiments, the orthogonal tRNA is a modified alanine glycine. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

In some embodiments, the non-natural amino acid is incorporated into the IL-2 polypeptide by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs are Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. E. coli TyrRS (Ec-Tyr)/b. Stearothermophilus ( B. stearothermophilus ) tRNACUA pair, E. coli LeuRS (Ec-Leu) / b. Stearophilus tRNACUA pairs and pyrrolysyl-tRNA pairs, but are not limited thereto. In some cases, unnatural amino acids are incorporated into IL-2 polypeptides by Mj-Tyr RS/tRNA pairs. Exemplary UAAs that can be incorporated by the Mj-Tyr RS/tRNA pair include 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 -voronophenylalanine; and o -nitrobenzyltyrosine.

In some cases, the unnatural amino acid is incorporated into an IL-2 polypeptide by an Ec-Tyr /tRNA _CUA or Ec-Leu /tRNA _CUA pair. Exemplary UAAs that can be incorporated by an Ec-Tyr /tRNA _CUA or Ec-Leu /tRNA _CUA pair include phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O -propargyltyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3-(naphthalen-2-ylamino)-2-amino-propanoic acid.

In some cases, unnatural amino acids are incorporated into IL-2 polypeptides by pyrrolysyl-tRNA pairs. In some cases, PylRS is obtained from archaea, such as methanogenic archaea. In some cases, PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei , or Methanosarcina acetivorans . Exemplary UAAs that can be incorporated by pyrrolysyl-tRNA pairs are amide and carbamate substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid , N-ε-D-prolyl-L-lysine, and N-ε-cyclopentyloxycarbonyl-L-lysine; N -ε-acryloyl-L-lysine; N -ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine; and N-ε-(1-methylcyclopro-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein are M. M. selectively charged with unnatural amino acids such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) by Barkeri pyrrolysyl-tRNA synthetase ( Mb PylRS) . It can be prepared using M. mazei tRNA. Other methods are known to those skilled 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, non-natural amino acids are incorporated into IL-2 polypeptides by the synthetases disclosed in US 9,988,619 and US 9,938,516, the disclosures of each of which are incorporated herein by reference.

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

An orthogonal tRNA synthetase/tRNA pair charges the tRNA with an unnatural amino acid and incorporates the unnatural amino acid into a polypeptide chain in response to a codon. Exemplary aaRS-tRNA pairs are Methanococcus janaschi ( Mj-Tyr ) aaRS/tRNA pairs, E. E. coli TyrRS ( Ec-Tyr )/b. Stearomophilus tRNA _CUA pair, E. E. coli LeuRS ( Ec-Leu )/b. Stearophilus tRNA _CUA pairs and pyrrolysyl-tRNA pairs, but are not limited thereto. Other aaRS-tRNA pairs that can be used according to the present invention are M. from Majei, Feldman et al., J Am Chem Soc., 2018 140:1447-1454; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322, the disclosures of each of which are incorporated herein by reference.

In some embodiments, a method of making an IL-2 conjugate disclosed herein in a cell system expressing NTT and tRNA synthetase is provided. In some embodiments described herein, NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is Methanococcus jannaski , E. E. coli TyrRS ( Ec-Tyr )/b. stearothermophilus, and M. Mazei (M. mazei ). In some embodiments, NTT is PtNTT1 and the tRNA synthetase is Methanococcus jannaskii, E. E. coli TyrRS ( Ec-Tyr )/b. Stearothermophilus, or M. It is derived from Majei. In some embodiments, NTT is PtNTT2 and the tRNA synthetase is Methanococcus jannaskii, E. E. coli TyrRS ( Ec-Tyr )/b. Stearothermophilus, or M. It is derived from Majei. In some embodiments, NTT is PtNTT3 and the tRNA synthetase is Methanococcus jannaskii, E. E. coli TyrRS ( Ec-Tyr )/b. Stearothermophilus, or M. It is derived from Majei. In some embodiments, NTT is PtNTT3 and the tRNA synthetase is Methanococcus jannaskii, E. E. coli TyrRS ( Ec-Tyr )/b. Stearothermophilus, or M. It is derived from Majei. In some embodiments, NTT is PtNTT4 and the tRNA synthetase is Methanococcus jannaskii, E. E. coli TyrRS ( Ec-Tyr )/b. Stearothermophilus, or M. It is derived from Majei. In some embodiments, NTT is PtNTT5 and the tRNA synthetase is Methanococcus jannaskii, E. E. coli TyrRS ( Ec-Tyr )/b. Stearothermophilus, or M. It is derived from Majei. In some embodiments, NTT is PtNTT6 and the tRNA synthetase is Methanococcus jannaskii, E. E. coli TyrRS ( Ec-Tyr )/b. Stearothermophilus, or M. It is derived from Majei.

In some embodiments, an IL-2 conjugate disclosed herein comprises (a) the nucleotide triphosphate transporter Pt NTT2 (including truncated variants in which the first 65 amino acid residues of the full-length protein are deleted), (b) having the desired amino acid sequence. A first non-natural amino acid to encode an IL-2 variant and provide a codon at the desired position into which an unnatural amino acid such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) will be incorporated. A plasmid comprising a double-stranded oligonucleotide containing an unnatural base pair comprising a nucleotide and a second unnatural nucleotide, (c) M. A plasmid encoding a tRNA derived from Majei and containing an unnatural nucleotide to provide a recognized anticodon (for the codon of the IL-2 variant) instead of the native sequence, and (d) the same plasmid or a different plasmid encoding the tRNA Methanosarcica Barkeri ( M. barkeri ), which may be, E., which includes a plasmid encoding pyrrolysyl-tRNA synthetase ( Mb PylRS) . It can be produced in cells such as E. coli . In some embodiments, the cells are further supplemented with deoxyribotriphosphate comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with ribotriphosphate comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with one or more non-natural amino acids, such as N 6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the double-stranded oligonucleotide encoding the amino acid sequence of the desired IL-2 variant is, for example, at positions 34, 37, 40, 41, 42, 43, 44, 61, 64, 68 or 71 or at positions 35, 38, 41, 42, 43, 45, 62, 65, 69 or 72 of the sequence encoding the protein having SEQ ID NO: 4, wherein X is an unnatural nucleotide. In some embodiments, the cell contains the AXC-matching anticodon GYT instead of the native sequence of M. Further comprising a plasmid, which may be a protein expression plasmid or another plasmid, encoding an orthogonal tRNA gene from Majei, wherein Y is a non-natural nucleotide that is complementary and may be the same as or different from the non-natural nucleotide of the codon. In some embodiments, the non-natural nucleotide of a codon is different and complementary to the non-natural nucleotide of an anticodon. In some embodiments, the unnatural nucleotides of a codon are identical to the unnatural nucleotides of an anticodon. In some embodiments, the first and second non-natural nucleotides comprising non-natural base pairs in the double-stranded oligonucleotide are

로부터 유도될 수 있다. 일부 실시 형태에서, 이중가닥 올리고뉴클레오티드에 비천연 염기쌍을 포함하는 제1 및 제2 비천연 뉴클레오티드는

로부터 유도될 수 있다. 일부 실시 형태에서, 제1 및 제2 비천연 뉴클레오티드의 트리포스페이트는

can be derived from In some embodiments, the first and second non-natural nucleotides comprising non-natural base pairs in the double-stranded oligonucleotide are

can be derived from In some embodiments, the triphosphates of the first and second non-natural nucleotides are

, 또는 이들의 염을 포함한다. 일부 실시 형태에서, 제1 및 제2 비천연 뉴클레오티드의 트리포스페이트는

, or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides are

, 또는 이들의 염을 포함한다. 일부 실시 형태에서, 제1 비천연 뉴클레오티드 및 제2 비천연 뉴클레오티드를 포함하는 mRNA 유래 이중 가닥 올리고뉴클레오티드는

, or salts thereof. In some embodiments, the mRNA-derived double-stranded oligonucleotide comprising the first non-natural nucleotide and the second non-natural nucleotide is

로부터 유도된 비천연 뉴클레오티드를 포함하는 코돈을 포함할 수 있다. 일부 실시 형태에서, 엠. 마제이 tRNA는 mRNA의 비천연 뉴클레오티드를 포함하는 코돈을 인식하는 비천연 뉴클레오티드를 포함하는 안티코돈을 포함할 수 있다. 엠. 마제이 tRNA의 안티코돈은

It may contain codons comprising non-natural nucleotides derived from In some embodiments, M. Majay A tRNA may include an anticodon comprising an unnatural nucleotide that recognizes a codon comprising an unnatural nucleotide of mRNA. M. Majay The anticodon of tRNA is

로부터 유래된 비천연 뉴클레오티드를 포함할 수 있다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함하고, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함하고, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함하고, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 일부 실시 형태에서, mRNA는

로부터 유도된 비천연 뉴클레오티드를 포함하고, tRNA는

로부터 유도된 비천연 뉴클레오티드를 포함한다. 숙주 세포는 적절한 영양소가 포함된 배지에서 배양되며, (a) 코돈을 포함하는 사이토카인 유전자를 코딩하는 플라스미드(들)의 복제에 필요한 하나 이상의 비천연 염기를 포함하는 데옥시리보 뉴클레오시드의 트리포스페이트, (b) (i) 하나 이상의 비천연 염기를 포함하는 코돈을 포함하고 사이토카인의 코딩 서열에 상응하는 mRNA, 및 (ii) 하나 이상의 비천연 염기를 포함하는 안티코돈을 포함하는 tRNA의 전사에 필요한 하나 이상의 비천연 염기를 포함하는 리보 뉴클레오시드의 트리포스페이트, 및 (c) 관심 사이토카인의 폴리펩티드 서열에 혼입될 비천연 아미노산(들)으로 보충된다. 이어서, 숙주 세포는 관심 단백질의 발현을 허용하는 조건 하에서 유지된다.

It may contain non-natural nucleotides derived from. In some embodiments, the mRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It includes non-natural nucleotides derived from In some embodiments, the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It contains a non-natural nucleotide derived from, and the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It contains a non-natural nucleotide derived from, and the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It contains a non-natural nucleotide derived from, and the tRNA is

It includes non-natural nucleotides derived from In some embodiments, the mRNA is

It contains a non-natural nucleotide derived from, and the tRNA is

It includes non-natural nucleotides derived from The host cells are cultured in a medium containing appropriate nutrients and (a) a tree of deoxyribonucleosides containing one or more unnatural bases necessary for replication of the plasmid(s) encoding the cytokine gene containing codons. phosphate, (b) for transcription of (i) an mRNA comprising a codon comprising one or more unnatural bases and corresponding to the coding sequence of a cytokine, and (ii) a tRNA comprising an anticodon comprising one or more unnatural bases. triphosphate of a ribonucleoside comprising the required one or more unnatural bases, and (c) the unnatural amino acid(s) to be incorporated into the polypeptide sequence of the cytokine of interest. The host cell is then maintained under conditions permissive for expression of the protein of interest.

The resulting expressed AzK-containing protein can be purified by methods known to those skilled in the art and then allowed to react with an alkyne, such as DBCO, comprising a PEG chain having a desired average molecular weight as disclosed herein under conditions known to those skilled in the art. The IL-2 conjugates disclosed herein can be provided. Zhang et al., Nature 2017, 551(7682): 644647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO 2019014262; WO 2019014267; WO 2019028419; and other methods, such as disclosed in WO2019/028425, are known to those skilled in the art, the disclosures of each of which are incorporated herein by reference.

The resulting protein comprising, for example, the one or more non-natural amino acids, AzK, expressed, can be purified by methods known to those skilled in the art, followed by PEG having the desired average molecular weight, as disclosed herein, under conditions known to those skilled in the art. It can be reacted with an alkyne such as DBCO containing chain to provide the IL-2 conjugates disclosed herein. Zhang et al., Nature 2017, 551(7682): 644647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO 2019014262; WO 2019014267; WO 2019028419; and other methods, such as disclosed in WO2019/028425, are known to those skilled in the art, the disclosures of each of which are incorporated herein by reference.

Alternatively, IL-2 polypeptides comprising non-natural amino acid(s) are disclosed herein comprising tRNAs and aminoacyl tRNA synthetases and comprising a nucleic acid sequence of interest together with one or more in-frame orthogonal (termination) codons. It is prepared by introducing the described nucleic acid construct into a host cell. The host cells are cultured in a medium containing appropriate nutrients and (a) deoxyribonucleotides containing one or more unnatural bases required for replication of the plasmid(s) encoding cytokine genes, including new codons and anticodons. triphosphate of seed, (b) triphosphate of ribonucleoside required for transcription of mRNA corresponding to (i) cytokine sequence containing codon, and (ii) orthogonal tRNA containing anticodon, and (c) ratio Supplemented with natural amino acid(s). The host cell is then maintained under conditions permissive for expression of the protein of interest. The unnatural amino acid(s) are incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more non-natural amino acids are incorporated into an IL-2 polypeptide. Alternatively, two or more non-natural amino acids may be incorporated into an IL-2 polypeptide at two or more sites in the protein.

Once IL-2 polypeptides comprising unnatural amino acid(s) are produced in host cells, they can be extracted from the host cells by a variety of techniques known in the art, including enzymatic, chemical, and/or osmotic lysis and physical disruption. can IL-2 polypeptides can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobicity chromatography, affinity chromatography, or any other suitable technique known to those skilled in the art.

Suitable host cells may include bacterial cells (eg E. coli, BL21(DE3)), but most suitably the host cells are eukaryotic cells, such as insect cells (eg Drosophila melanocytes). gaster ( Drosophila melanogaster ), yeast cells, nematodes (eg C. elegans ), mice (eg Mus musculus ), or mammalian cells (eg, Chinese hamster ovary cells (CHO) 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 are E. coli. contains coli.

Other suitable host cells that may generally be used in embodiments of the present invention are those mentioned in the Examples section. Vector DNA can be introduced into host cells through conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to a foreign nucleic acid molecule (e.g., eg DNA) into a host cell. Suitable methods for transforming or transfecting host cells are well known in the art.

When generating a cell line, it is generally desirable that a stable cell line be prepared. For example, for stable transfection of mammalian cells, it is known that only a small fraction of cells are able to integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select for these integrants, genes encoding selectable markers (eg, for resistance to antibiotics) are generally introduced into the host cell along with the gene of interest. Preferred selectable markers include those conferring resistance to drugs such as G418, hygromycin or methotrexate. Nucleic acid molecules encoding the selectable markers can be introduced into the host cell on the same vector or can be introduced on separate vectors. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (eg, cells into which the selectable marker gene has been integrated 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 uniformity between individual cells or clones is achieved. Another advantage is that the optimal producer can be selected. Therefore, it is desirable to generate stable cell lines. In another embodiment, a construct described herein transfects a host cell. An advantage of transfecting host cells with the construct is that protein yield can be maximized. In one aspect, a cell comprising a nucleic acid construct or vector described herein is described.

Anti-EGFR antibody

The methods of treating cancer described herein include administering an anti-EGFR antibody in combination with an IL-2 conjugate described herein.

In some embodiments, the anti-EGFR antibody is an inhibitory antibody. In some embodiments, the anti-EGFR inhibitory antibody is cetuximab (Erbitux), panitumumab (Vectibix), nesitumumab (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 nesitumumab (Portrazza). In some embodiments, the anti-EGFR inhibitory antibody is JNJ-61186372 (Amivantamab). In some embodiments, the anti-EGFR inhibitory antibody is IMC-C225. In some embodiments, the anti-EGFR inhibitory antibody is ABX-EGF. In some embodiments, the anti-EGFR inhibitory antibody is ICR62. In some embodiments, the anti-EGFR inhibitory antibody is EMD 55900.x

treatment method

In one aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising an effective amount of (a) an IL-2 conjugate described herein, and (b) an anti-EGFR. Administering to a subject.

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

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

cancer type

In some embodiments, the cancer is renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma , microsatellite unstable cancer, microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer, 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 DNA damage response (DDR) defects, bladder cancer, ovarian cancer , moderate to low mutation burden tumors, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), low to non-expressing PD-L1 tumors, tumors disseminated systemically beyond the anatomical primary site of origin to the liver and CNS , 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 an advanced or metastatic solid tumor. In some embodiments, the cancer is in the form of a liquid tumor.

administration

In some embodiments, the response is complete response, partial response, or stable 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. do. 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 up to 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 extended up to 24 months.

In some embodiments, the IL-2 conjugate is administered to the subject prior to administering the anti-EGFR antibody to the subject. In some embodiments, the anti-EGFR antibody is administered to the subject prior to administering the IL-2 conjugate to the subject. In some embodiments, the IL-2 conjugate and 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 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 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 2 weeks, once every 3 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, 23 It is administered once a week, 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 the IL-2 conjugate is administered to a subject in need thereof once every 3 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 the 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 the 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 the 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 the IL-2 conjugate is administered to a subject in need thereof once every 23 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 24 weeks. In some embodiments, the effective amount of the IL-2 conjugate is administered once about every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. In some embodiments, the effective amount of the IL-2 conjugate is administered once about every 14, 15, 16, 17, 18, 19, 20, or 21 days.

In some embodiments, the amount of a given agent corresponding to such amount varies depending on factors such as the particular compound, the severity of the disease, the identity (eg, body weight) of the subject or host in need of treatment, but nonetheless, for example It is usually determined in a manner known in the art depending on the particular agent being administered, the route of administration, and the particular circumstances surrounding the case, including the subject or host being treated. In some cases, the desired dose is conveniently presented as a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, eg as two, three, four or more sub-doses daily.

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 as described herein every 3 weeks.

In some embodiments, an anti-EGFR antibody can be administered alone or in combination with an IL-2 conjugate at a dose determined to be safe and efficacious for the antibody and using a dosing regimen. 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 a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks. once every 6 weeks, once every 7 weeks, 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 in 23 weeks, once in 24 weeks, once in 25 weeks, once in 26 weeks, once in 27 weeks, or once in 28 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered once a week to a subject in need thereof. 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 3 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 every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. administered once.

In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, cetuximab is administered at a loading dose of about 100 mg/m ² to about 500 mg/m ² by intravenous infusion. In any of the embodiments described herein, the loading dose of cetuximab is mg per m ² body surface area of the subject. In some embodiments, cetuximab is administered at a loading dose of about 100 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 150 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 200 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 250 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 300 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 350 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 400 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 450 mg/m ² by intravenous infusion. In some embodiments, cetuximab is administered at a loading dose of about 500 mg/m ² by intravenous infusion. In some embodiments, an initial dose of cetuximab is administered at a loading dose of about 400 mg/m ² by intravenous infusion and all subsequent doses of cetuximab are administered by intravenous infusion at a loading dose of about 250 mg/m ² Administered as a loading dose. In any of these embodiments, cetuximab is infused over about 30-240 minutes. In some embodiments, cetuximab is infused over about 30 minutes. In some embodiments, cetuximab is infused over about 60 minutes. In some embodiments, cetuximab is infused over about 90 minutes. In some embodiments, cetuximab is infused over about 120 minutes. In some embodiments, cetuximab is infused over about 150 minutes. In some embodiments, cetuximab is infused over about 180 minutes. In some embodiments, cetuximab is infused over about 210 minutes. In some embodiments, cetuximab is infused over about 240 minutes. In any of these embodiments, cetuximab is administered at 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. administered at an infusion rate of 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 the doses of 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 a dose described herein every 3 weeks. In some embodiments, cetuximab is administered at a dose described herein every two weeks. In some embodiments, cetuximab is administered at a dose described herein weekly.

Additional agents/premedication

In some embodiments, any method 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 include administering an analgesic 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 dolacetron. 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 about 8 mg to 0.15 mg/kg.

In some embodiments, any of the methods described herein may include an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine), an analgesic (such as acetaminophen), and/or a serotonin 5-HT ₃ receptor antagonist ( eg 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 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 an analgesic (such as acetaminophen) and a serotonin 5-HT ₃ receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). do. In some embodiments, any of the methods described herein may include an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine), an analgesic (such as acetaminophen), and a serotonin 5-HT ₃ receptor antagonist (such as ranisetron, dolasetron, tropisetron, palonosetron or ondansetron).

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

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

In some embodiments, any of the methods described herein further comprises administering a premedication prior to administering the IL-2 conjugate. In some embodiments, the IL-2 conjugate premedication 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 IL-2 conjugate predose is a serotonin 5-HT ₃ receptor antagonist (eg 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 IL-2 conjugate premedication is an analgesic (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 premedication prior to administering the anti-EGFR antibody (eg, cetuximab). In some embodiments, the anti-EGFR antibody premedication 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 cetuximab pre-dose is a serotonin 5-HT ₃ receptor antagonist (eg, 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 anti-EGFR antibody premedication is an analgesic (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 administers a first dose of a premedication prior to administering the IL-2 conjugate and administers a second dose prior to administering the anti-EGFR antibody (eg, cetuximab). Further comprising administering a premedication. In some embodiments, the premedication for the IL-2 conjugate is the same as the premedication for the anti-EGFR antibody (eg cetuximab). In some embodiments, the premedication for the IL-2 conjugate is different than the premedication for the anti-EGFR antibody (eg cetuximab). In some embodiments, the premedication 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 is a serotonin 5-HT ₃ receptor antagonist (eg, 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 is an analgesic (such as acetaminophen). In some embodiments, acetaminophen is administered orally at a dose of about 650 to 1000 mg. In some embodiments, the premedication includes an antihistamine and a serotonin 5-HT ₃ receptor antagonist. In some embodiments, the premedication includes an antihistamine and an analgesic. In some embodiments, the premedication comprises a serotonin 5-HT ₃ receptor antagonist and an analgesic agent. In some embodiments, the premedication includes an antihistamine, a serotonin 5-HT ₃ receptor antagonist, and an analgesic. In some cases, the premedication for the IL-2 conjugate and the anti-EGFR antibody (such as cetuximab) is the same (such as diphenhydramine), only a single dose of the premedication is administered. In other cases where the premedication for the IL-2 conjugate and the anti-EGFR antibody (eg cetuximab) are the same, multiple doses of the premedication are administered.

In some embodiments of the methods described herein, the order of dosing is as follows: (i) a premedication to an anti-EGFR antibody (eg, cetuximab); (ii) anti-EGFR antibodies (such as cetuximab); (iii) a premedication agent for the IL-2 conjugate; and (iv) an IL-2 conjugate. In some variations where the premedication for the anti-EGFR antibody (eg cetuximab) is the same as the premedication for the IL-2 conjugate (eg diphenhydramine), administration of the premedication for the IL-2 conjugate may be omitted. In some embodiments, the dosing sequence is as follows: (i) a pre-dosing agent for the IL-2 conjugate; (ii) an IL-2 conjugate; (iii) a premedication for an anti-EGFR antibody (such as cetuximab); and (iv) an anti-EGFR antibody (such as cetuximab). In some variations where the premedication for the anti-EGFR antibody (eg cetuximab) is the same as the premedication for the IL-2 conjugate (eg diphenhydramine), administration of the premedication for the anti-EGFR antibody is omitted. It can be.

object

In some embodiments, the administration of the effective amount of the IL-2 conjugate and anti-EGFR antibody is for an adult. In some embodiments, the adult is male. In another embodiment, 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 old. In some embodiments, the administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody is administered 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 measurable disease (ie, cancer) as determined by RECIST v1.1. In some embodiments, the subject has been determined to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. In some embodiments, the subject has adequate cardiovascular, hematological, hepatic and renal function, as determined by a physician. In some embodiments, the subject has been determined (eg, by a physician) to have a life expectancy of at least 12 weeks.

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

In some embodiments, the subject is diagnosed with a diagnosis of an advanced and/or metastatic solid tumor having at least one tumor lesion with a location that allows safe biopsy access as per clinical judgment (ie, as determined by a physician). Histologically or cytologically confirmed. In some embodiments, the subject received prior anti-cancer therapy prior to administration of the first therapeutic dose. In some embodiments, treatment-related toxicity of prior anti-cancer therapy has been addressed to an appropriate level.

In some embodiments, the subject is a female of childbearing potential and is using a medically acceptable method of birth control during treatment and for at least 3 months after the last therapeutic dose is administered. In some embodiments, the subject is a premenopausal female who has tested negative for pregnancy (by serum pregnancy test) within 7 days prior to administration of the first therapeutic dose. In some embodiments, the subject is a postmenopausal female younger than 12 months who has tested negative for pregnancy (by serum pregnancy test) within 7 days prior to administration of the first therapeutic dose.

In some embodiments, the subject is a male who is not surgically sterile and has been using medically acceptable methods of birth control during treatment and for at least 3 months after the last dose is administered. In some embodiments, the male does not donate sperm or store in a sperm bank during the treatment period and for at least 3 months after administration of the last therapeutic dose.

In some embodiments, the subject has not received radiation therapy within 14 days of administering the first therapeutic dose. In some embodiments, the subject has not received conventional radiation or stereotactic radiosurgery within 7 days of administering the first therapeutic dose.

In some embodiments, the subject has not been treated with systemic anti-cancer therapy or study anti-cancer agent within 2 weeks of administering the first therapeutic dose. In some embodiments, the subject has not been treated with immunotherapy or tyrosine kinase inhibitor therapy within 4 weeks of administering the first therapeutic dose.

In some embodiments, the subject has not had major surgery within 30 days of administering the first therapeutic dose. In some embodiments, the subject underwent major surgery more than 30 days prior to administration of the first therapeutic dose and recovered to at least Grade 1 from any adverse events associated with the procedure. In some embodiments, the subject does not anticipate the need for major surgery during the course of treatment.

In some embodiments, the subject has not suffered from an active autoimmune disease requiring systemic treatment within 3 months prior to administration of the first therapeutic dose. In some embodiments, the subject has had no documented history of clinically severe autoimmune disease requiring systemic steroids or immunosuppressants prior to administration of the first therapeutic dose.

In some embodiments, the subject does not have primary central nervous system (CNS) disease or leptomeningeal disease. In some embodiments, the subject has known CNS metastasis but has received appropriate treatment, is asymptomatic without evidence of radiographic progression for at least 8 weeks prior to administration of the first therapeutic dose, and has received an anticonvulsant within 14 days prior to administration of the first therapeutic dose. There was no requirement for inducing steroids or enzymes.

In some embodiments, the subject has pneumonia, active pneumonia, interstitial lung disease requiring steroid use, idiopathic pulmonary fibrosis, confirmed pleural effusion, and severe dyspnea or supplementation at rest within 6 months of administering the first therapeutic dose. He did not have abnormal lung function including severe dyspnea requiring oxygen therapy.

In some embodiments, the subject has not taken a parenteral antibiotic within 14 days of administering the first therapeutic dose.

In some embodiments, the subject has no history of allogeneic or solid organ transplantation. In some embodiments, the subject does not have a human immunodeficiency virus (HIV) infection or an active infection of hepatitis C. In some embodiments, the subject does not have an uncontrolled hepatitis B virus (HBV) infection.

In some embodiments, the subject has had no clinically significant bleeding (eg, gastrointestinal bleeding, intracranial bleeding) within 2 weeks prior to administration of the first therapeutic dose. In some embodiments, the subject has not had a prior diagnosis of deep vein thrombosis or pulmonary embolism within 3 months of administering the first therapeutic dose.

In some embodiments, the subject has suffered from a severe or unstable cardiac condition (such as congestive heart failure (New York Heart Association Class III or IV), heart bypass surgery or coronary stent placement, angioplasty, Cardiac ejection fraction below the lower limit of normal, unstable angina, medically uncontrolled hypertension (eg, ≥160 mmHg systolic or ≥100 mmHg diastolic), uncontrolled cardiac arrhythmias requiring medication (2 per NCI CTCAE v5.0) grade abnormal) or myocardial infarction).

In some embodiments, the subject has a history of a non-pharmacologically induced prolonged corrective QT interval (QTcF), determined using Fridericia's formula, of > 450 milliseconds (msec) in males or > 470 msec in females. does not exist.

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

In some embodiments, the subject does not have an active secondary malignancy. In some embodiments, the subject has no history of prior malignancies. In some embodiments, the subject had curatively surgically resected non-melanoma skin cancer or cervical cancer prior to administration of the first therapeutic dose.

In some embodiments, the subject has any serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concomitant medical condition that precludes treatment or renders treatment inappropriate. have no disease

In some embodiments, the subject is not pregnant or breastfeeding. In some embodiments, the subject is not expected to conceive or father a child during the course of treatment and up to 3 months after administration of the last therapeutic dose.

In some embodiments, the subject is not receiving concomitant therapy with any investigational agent, vaccine or device during the course of treatment. In some embodiments, the subject is receiving concomitant therapy with an investigational agent, vaccine, or device during a course of treatment after approval by a physician.

dosing effect

In some embodiments, after administration of the IL-2 conjugate and the anti-EGFR antibody, the subject experiences a response as measured by the Criteria for the Evaluation of Immune-Related Responses in Solid Tumors (iRECIST). In some embodiments, after administration of the IL-2 conjugate and anti-EGFR antibody, the subject experiences an objective response rate (ORR) according to RECIST version 1.1. In some embodiments, after administration of the IL-2 conjugate and anti-EGFR antibody, the subject experiences a duration of response (DOR) according to RECIST version 1.1. In some embodiments, after administration of the IL-2 conjugate and anti-EGFR antibody, the subject experiences progression-free survival (PFS) according to RECIST version 1.1. In some embodiments, after administration of the IL-2 conjugate and anti-EGFR antibody, the subject experiences overall survival according to RECIST version 1.1. In some embodiments, after administration of the IL-2 conjugate and anti-EGFR antibody, the subject experiences a time to response (TTR) according to RECIST version 1.1. In some embodiments, after administration of the IL-2 conjugate and anti-EGFR antibody, the subject experiences a disease control rate (DCR) according to RECIST version 1.1. In any of these embodiments, the subject's experience is based on the physician's review of the subject's taken radiographic images.

In some embodiments, treatment is discontinued based on the physician's review of the subject's taken radiographic image.

In some embodiments, treatment is discontinued based on the physician's review of immunophenotyping of peripheral blood at various time points. In some embodiments, treatment is discontinued based on the physician's review of immunophenotyping of the tumor sample at various time points. In some embodiments, treatment is discontinued based on a physician's review of the presence of antibodies to any of the IL-2 conjugates disclosed herein at various time points. In some embodiments, treatment is discontinued based on a physician's review of plasma concentrations of any of the IL-2 conjugates disclosed herein at various time points. In any of these embodiments, the physician's review is based on an appropriate analysis of relevant parameters.

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

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

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

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not result in hypotension and reduced organ perfusion in the subject.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not result in impairment of neutrophil function in the subject. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not result in a decrease in chemotaxis in the subject.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject 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 infection prior to administration of the IL-2 conjugate and anti-EGFR antibody. 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 conjugate and anti-EGFR antibody.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not exacerbate existing or early symptoms of an autoimmune disease or inflammatory disorder in the subject. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not exacerbate existing or early symptoms of an autoimmune disease in the subject. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not exacerbate existing or early symptoms of an inflammatory disorder in the subject. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, ocular-bulbar myasthenia gravis, meniscal IgA glomerulonephritis, cholecystitis, cerebrovascular 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 ocular-bulb 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 cerebrovascular 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, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject results in a change in mental status, speech impairment, cortical blindness, limb or gait ataxia, hallucinations, agitation, stupor, or coma in the subject. does not cause In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not cause seizures in the subject. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject is not contraindicated in a subject with a known seizure disorder.

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

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not result in a decrease in mean arterial blood pressure in the subject after administration of the IL-2 conjugate to the subject. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject results in hypotension in the subject. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not cause the subject to experience a systolic blood pressure of less than 90 mm Hg or a 20 mm Hg drop from baseline systolic blood pressure.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not result in edema or impairment of kidney or liver function in the subject.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not result in eosinophilia in the subject. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject results in an eosinophil count in the subject's peripheral blood not exceeding 500/μL. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject results in an eosinophil count in the subject's peripheral blood that does not exceed 500 μL to 1500/μL. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject results in an eosinophil count in the subject's peripheral blood not exceeding 1500/μL to 5000/μL. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject results in an eosinophil count in the subject's peripheral blood not exceeding 5000/μL. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject is not contraindicated in subjects receiving existing psychotropic drug therapy.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject is not contraindicated in a subject receiving an existing therapy of a nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drug. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject is contraindicated in subjects receiving existing therapy of aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase. no. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject is not contraindicated in a subject receiving combination therapy containing an anti-neoplastic agent. In some embodiments, the antineoplastic agent is selected from dacarbazine, cis-platinum, tamoxifen, and interferon-alpha.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not result in one or more Grade 4 adverse events in the subject following administration of the IL-2 conjugate to the subject. In some embodiments, Grade 4 adverse events include hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; faint; bleeding; atrial arrhythmias; phlebitis; 2nd degree AV block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disease; stomatitis; nausea and vomiting; abnormal liver function tests; gastrointestinal bleeding; hematemesis; bloody diarrhea; gastrointestinal disorders; 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; convulsion; grand mal seizures; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; Shandong; pupillary disorders; renal dysfunction; renal failure; and acute tubular necrosis. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a group of subjects results in one or more Grade 4 adverse events in no more than 1% of subjects after administration of the IL-2 conjugate to the subject. . In some embodiments, Grade 4 adverse events include hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; faint; bleeding; atrial arrhythmia; phlebitis; 2nd degree AV block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disease; stomatitis; nausea and vomiting; abnormal liver function tests; gastrointestinal bleeding; hematemesis; bloody diarrhea; gastrointestinal disorders; bowel 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; convulsion; grand mal seizures; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; Shandong; pupillary disorders; renal dysfunction; renal failure; and acute tubular necrosis.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a group of subjects causes one or more adverse events in no more than 1% of subjects after administration of the IL-2 conjugate to the subject, wherein , wherein the one or more adverse events include duodenal ulcer; intestinal necrosis; myocarditis; supraventricular tachycardia; permanent or temporary 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 conjugate and an anti-EGFR antibody to a group of subjects results in one or more adverse events in no more than 1% of subjects after administration, wherein the one or more adverse events are malignant. hyperthermia; cardiac arrest; myocardial infarction; pulmonary embolism; stroke; bowel perforation; liver or kidney failure; severe depression leading to suicide; pulmonary edema; respiratory arrest; from respiratory failure.

In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral CD4+ regulatory T cells in the subject. In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject. In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody to the subject does not increase the number of CD8+ T and NK cells in the tumor in the subject without increasing the number of CD4+ regulatory T cells in the tumor in the subject. while increasing the number of peripheral CD8+ T and NK cells in a subject.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject does not require the availability of an intensive care facility or the availability of a skilled practitioner in cardiopulmonary or intensive care medicine. In some embodiments, administration of an effective amount of an IL-2 conjugate to a subject does not require the availability of an intensive care facility or the availability of a skilled practitioner in cardiopulmonary or intensive care medicine. Administration of an effective amount of an IL-2 conjugate to a subject does not require the availability of an intensive care facility. In some embodiments, administration of an effective amount of an IL-2 conjugate to a subject does not require the availability of an expert in cardiopulmonary or intensive care medicine.

In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody combination therapy improves the ADCC response to cancer, for example by improving the ADCC function of the anti-EGFR antibody. In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody combination therapy expands innate and adaptive immune cells. In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody combination therapy promotes immune activation within the tumor microenvironment. In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody results in a synergistic improvement in the anti-cancer activity of a combination of the two agents compared to the anti-cancer activity of either agent alone. In some embodiments, administration of the IL-2 conjugate increases the amount and number of activation of NK cells, which enhances ADCC triggered by anti-EGFR antibodies.

additional formulation

In some embodiments, the method further comprises 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 include one or more platinum-based chemotherapeutic agents. In some embodiments, the one or more chemotherapeutic agents include carboplatin and pemetrexed. In some embodiments, the one or more chemotherapeutic agents include carboplatin and nab-paclitaxel. In some embodiments, the one or more chemotherapeutic agents include carboplatin and docetaxel. In some embodiments, the subject's cancer is non-small cell lung cancer (NSCLC).

Kits/Articles of Manufacture

In certain embodiments, disclosed herein are kits and articles of manufacture for use with one or more methods and compositions described herein. Such kits include carriers, packages, or containers compartmentalized to receive one or more containers, such as vials, tubes, and the like, each container including one of the individual elements to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from various materials such as glass or plastic. This kit includes an IL-2 conjugate and an anti-EGFR antibody.

Kits typically include a label listing contents and/or instructions for use, and a package insert with instructions for use. Typically a set of instructions will also be included.

In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container where letters, numbers, or other characters comprising the label are attached, molded, or etched into the container itself, and the label is a receptacle that also receives the container, for example as a package insert. Or, if present in a carrier, the label is associated with the container. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic purpose. Labels also indicate directions for use of the contents, such as in the methods described herein.

In certain embodiments, pharmaceutical compositions are provided in the form of a pack or dispenser device containing one or more unit dosage forms containing a compound provided herein. The pack contains, for example, metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is provided with instructions for administration. In one embodiment, the pack or dispenser is also provided with a notice relating to the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notices are provided by the agency for the form of the drug for human or veterinary administration. reflects the approval of Such notices are, for example, the labeling approved by the US Food and Drug Administration for the drug, or the approved product insert. In one embodiment, a composition containing a compound provided herein formulated in a suitable pharmaceutical carrier is also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Exemplary Embodiments

The present invention is further illustrated by the following embodiments. Features of each embodiment may be combined with other embodiments where appropriate and practical.

Embodiment P1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody; The IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues of the IL-2 conjugate are replaced with the structure of Formula I (or a pharmaceutically acceptable salt, solvate, or hydrate thereof): method:

[Formula I]

here,

이거나;Z is CH ₂ and Y is

is;

이거나;Y is CH ₂ and Z is

is;

이거나; 또는Z is CH ₂ and Y is

is; or

이며;Y is CH ₂ and Z is

is;

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

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue;

The position of structure 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.

인, 방법.Embodiment P2. According to embodiment P1, in the IL-2 conjugate Z is CH ₂ and Y is

in, how.

인, 방법.Embodiment P3. According to embodiment P1, in the IL-2 conjugate Y is CH ₂ and Z is

in, how.

Embodiment P4. The method of any one of Embodiments P1 to P3, wherein the PEG groups in the IL-2 conjugate have an average molecular weight of 25 kDa, 30 kDa, or 35 kDa.

Embodiment P5. The method of Embodiment 4 wherein the PEG groups in the IL-2 conjugate have an average molecular weight of 30 kDa.

Embodiment P6. The method according to any one of Embodiments P1 to P5, wherein the position of structure I in the amino acid sequence of the IL-2 conjugate is P64.

Embodiment P7. In embodiment P1, the structure of Formula I has the structure of Formula (XII) or Formula (XIII) (or a pharmaceutically acceptable salt, solvate, or hydrate thereof), or a structure of Formula (XII) and Formula (XIII) A mixture of the structures (or a pharmaceutically acceptable salt, solvate, or hydrate thereof):

[Formula XII]

[Formula XIII]

here,

n is an integer ranging from about 2 to about 5000;

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

Embodiment P8. The method of Embodiment P7 wherein n in the IL-2 conjugate is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 25 kDa, 30 kDa, or 35 kDa.

Embodiment P9. The method of embodiment P8 wherein n in the IL-2 conjugate is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 30 kDa.

Embodiment P10. The method of any one of Embodiments P7 to P9, wherein the position of the structure of Formula XII or Formula XIII in the amino acid sequence of the IL-2 conjugate is P64.

Embodiment P11. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody; The IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 50, [AzK_L1_PEG30kD] has the structure of Formula IV or Formula V (or a pharmaceutically acceptable salt, solvate, or hydrate thereof), or A mixture of a structure of Formula IV and a structure of Formula V (or a pharmaceutically acceptable salt, solvate, or hydrate thereof):

[Formula IV]

[Formula V]

here,

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

X has the following structure:

를 가지며;

has;

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

X+1 represents the point of attachment to the trailing amino acid residue.

Embodiment P12. The method of 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 of any one of embodiments P1 to P13, wherein the anti-EGFR antibody is cetuximab.

Embodiment P15. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) cetuximab, comprising: The -2 conjugate comprises the amino acid sequence of SEQ ID NO: 50, [AzK_L1_PEG30kD] has the structure of Formula XII or Formula XIII (or a pharmaceutically acceptable salt, solvate, or hydrate thereof), or A mixture of structure XII and structure XIII (or a pharmaceutically acceptable salt, solvate, or hydrate thereof):

[Formula XII]

[Formula XIII]

here,

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

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

Embodiment P16. The method according to any one of embodiments P1 to P15, wherein the cancer is 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 unstable cancer, microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer, 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 with DNA damage response (DDR) defects cancer, bladder cancer, ovarian cancer, moderate to low mutational burden tumors, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), low- to non-expressing PD-L1 tumors, liver and A tumor disseminated systemically to the CNS, and diffuse large B-cell lymphoma.

Embodiment P17. The method of any one of embodiments P1 to P16, wherein the IL-2 conjugate is administered to the subject once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, 6 administered once a week, once every 7 weeks or once every 8 weeks.

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

Example

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

Example 1. Preparation of IL-2 conjugate IL-2_P65_[AzK_L1_PEG30kD]-1.

IL-2 used for bioconjugation was E. coli using the methods disclosed herein, using the following. E. coli as inclusion bodies: (a) (i) A protein having the desired amino acid sequence, the gene containing the unnatural amino acid N 6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK) A protein containing the first unnatural base pair providing a codon at the desired position incorporated therein, and (ii) M. an expression plasmid encoding a tRNA derived from Majei Pyl, the gene comprising a second unnatural nucleotide providing a matching anticodon in place of its native sequence; (b) a plasmid encoding pyrrolysyl-tRNA synthetase ( Mb PylRS) from M. barkeri , (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 are deleted. The double-stranded oligonucleotide encoding the amino acid sequence of the IL-2 variant contained the codon AXC as codon 64 of the sequence encoding the protein having SEQ ID NO: 3 in which P64 was replaced with an unnatural amino acid described herein. M. The plasmid encoding the orthologous tRNA gene from Majei contains the AXC-matching anticodon GYT instead of its native sequence, where Y is a non-natural nucleotide disclosed herein. X and Y were selected from the non-natural nucleotides dTPT3 and dNaM as disclosed herein. Expressed proteins were extracted from inclusion bodies and refolded (using standard procedures), followed by site-specific pegylation of the AzK-containing IL-2 product using DBCO-mediated copper-free click chemistry to obtain a stable covalent mPEG. A moiety (methoxy, linear PEG group with an average molecular weight of 30 kDa) was attached to AzK (as outlined in Scheme 6 above).

The IL-2 conjugate "IL-2_P65_[AzK_L1_PEG30kD]-1" comprises SEQ ID NO: 50 in which the proline at position 64 is replaced by AzK_L1_PEG30kD, wherein AzK_L1_PEG30kD is a structure of Formula IV or Formula V, or a structure of Formula IV and Formula V and a 30 kDa, linear mPEG chain. The IL-2 conjugate "IL-2_P65_[AzK_L1_PEG30kD]-1" also comprises SEQ ID NO: 3 wherein the proline residue (P64) at position 64 is replaced with a structure of Formula VIII or Formula IX or a mixture of Formula VIII and Formula IX. where n is an integer such that the molecular weight of the PEG group is about 30 kDa. The IL-2 conjugate "IL-2_P65[AzK_L1_PEG30kD]-1" also comprises SEQ ID NO: 3 wherein the proline residue (P64) at position 64 is replaced by a structure of formula XII or formula XIII or a mixture of formula XII and formula XIII. where n is an integer such that the molecular weight of the PEG group is about 30 kDa.

Example 2. Antibody Dependent Cytotoxicity (ADCC) Assay with IL-2_P65_[AzK_L1_PEG30kD]-1 and Cetuximab.

The effect of IL-2_P65_[AzK_L1_PEG30kD]-1 on the ADCC function of cetuximab is examined using a Calcein-acetioxymethyl (Calcein-AM; Invitrogen) release assay.

ingredient.

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

The following reagents are used: Calcein-acetioxymethyl (Calcein-AM; Invitrogen), Probenecid (Invitrogen), ultra-low IgG fetal bovine serum (Thermofisher), and human isotype IgG1 antibody (Biolegend).

procedure.

Human primary NK cells are negatively selected from PBMCs using the RoboSep™ device according to the manufacturer recommended protocol. Purified NK cells were cultured at various concentrations (0.1 μg/mL, 0.01 μg/mL, 0.001 μg/mL, and 0 μg/mL) of IL-2_P65_ in a humidified incubator with 5% CO ₂ at 37°C for 18 hours. With [AzK_L1_PEG30kD]-1, culture in RPMI 1640 medium supplemented with 1% low IgG FBS. These cultured cells are used as effector cells. A human EGFR-positive cancer cell line (A431) was labeled with Calcein-AM for 30 minutes (50 μg was diluted in 25 μL DMSO to prepare a stock solution, after which 10 μL of the calcein stock solution was mixed with 4 mL of RPMI 1640 + 1% low IgG FBS + 1% probenecid (for staining of 4×10 ⁶ cells), then washed, 96 well round bottom plate at a density of 5×10 ³ cells/well plate on Add cetuximab and isotype human IgG1 antibody at various concentrations (10 μg/mL to 1 pg/mL) for 30 min to allow opsonization before adding NK cells. NK cells activated with IL-2_P65_[AzK_L1_PEG30kD]-1 are collected and added as effector cells at an E:T ratio of 3:1 (6×10 ⁴ NK cells to 2×10 ⁴ target cells). Plates were then incubated for 1 hour at 37 °C in a humidified incubator with 5% CO ₂ , and 90 μL of supernatant was harvested and transferred to an opaque 96-well microplate and read using an Envision 2104 plate reader (excitation: 492 nm, emission). : 515 nm) using a fluorescence measurement method.

Cells are lysed with 2% Triton X-100 for maximal release. Subtract the fluorescence value of the culture medium background from the fluorescence values of experimental emission (A), target cell spontaneous emission (B), and target cell maximal emission (C).

Calculate the percent cytotoxicity and ADCC for each plate (in duplicate) using the following formula:

Cytotoxicity (%) = (A - B) / (C - B) × 100

ADCC (%) = Cytotoxicity (%, with antibody) - Cytotoxicity (%, without antibody)

Measurements are performed in triplicate using three duplicate wells for each experiment. Each experiment is repeated at least three times. GraphPad Prism 5 (GraphPad Software, Inc., San Diego, Calif., USA) is used to fit the data points to a 4-parameter equation to calculate half maximum effective concentration (EC50) values.

result.

Without activation by IL-2_P65_[AzK_L1_PEG30kD]-1, cetuximab-treated human NK cells show cytotoxicity against EGFR expressing cancer cell lines (A431 and A549) but EGFR null expressing cells (NCI-H69) not so about After activation by IL-2_P65_[AzK_L1_PEG30kD]-1, cetuximab-treated human NK cells show enhanced cytotoxicity against EGFR expressing cancer cell lines (A431 and A549).

Example 3. In vitro studies of IL-2 conjugates and cetuximab (PBMC ADCC assay).

The effect of antibody-dependent cellular toxicity (ADCC) by the IL-2 conjugate of Example 1 in combination with cetuximab using human PBMCs co-cultured with calcein-labeled cancer cell lines (CAL27 and A431) A study was conducted to investigate.

CAL27 cells.

reagent.

Bioassay Buffer: 1% ultra low IgG FBS added to phenol red free RPMI. Complete Assay Buffer: 450 μL probenecid added to 45 mL Bioassay Buffer, where the final probenecid concentration is 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 complete assay buffer (final Calcein-AM concentration: 5 μg/mL). Triton-X-100 Lysis Buffer: 20 μL Triton-X-100 added to 4 mL Complete Assay Buffer (final concentration: 0.5%).

procedure.

On day 1, a 6-point, 1/5 dilution series (in PBS) of the IL-2 conjugate was prepared. IL-2 conjugate concentrations were 2, 0.4, 0.08, 0.016, 0.0032 and 0 μg/mL. PBMC were collected by centrifugation at 200 x g for 5 minutes and resuspended in phenol red free RPMI + 10% ultra low IgG at 20 million cells/mL. Appropriate volumes of these PBMCs were transferred to 6 sections of a multi-well reservoir to which various IL-2 conjugate dilutions were added. Pipette the PBMCs up and down to mix well with the IL-2 conjugate, and use a multichannel pipette to transfer 50 mL to a round-bottom 96-well plate (final PBMC count per well was 1 million). Six empty wells were set aside for control additions the next day. Plates were incubated overnight in a humidified 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 express dissociation buffer and collected by centrifugation at 200 x g for 5 minutes. Cells were counted, and 5 million cells were resuspended in 4 mL calcein-AM staining buffer and incubated at 37° C. for 30 minutes in the presence of 5% carbon dioxide. Cells were then collected and washed twice in complete assay buffer by centrifugation at 200 x g for 5 minutes. Cells were counted and resuspended at 400,000 cells/mL for a final target cell number 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) for final assay concentrations of 1, 0.1, 0.001, 0.0001 μg/mL. Isotype controls (hIgG1, Biolegend) were diluted to 3 μg/mL for a final concentration of 1 μg/mL in complete assay buffer. An equal volume of 400,000 cells/mL of stained CAL27 cells was mixed with antibody dilution or isotype control and incubated at 4° C. for 30 minutes to allow antibody to bind. After incubation, 100 μL of the antibody-CAL27 cell mixture was added to a 96-well plate containing 50 μL of IL-2 conjugate treated PBMCs from day 1.

50 μL calcein-AM stained CAL27 cells without PBMCs but treated with complete assay buffer (background signal) or stained CAL27 cells treated with 50 μL Triton-X-100 treatment (for maximal signal after cell lysis) (both complete assays) Control wells were prepared in triplicate (made up to a final volume of 150 μL with buffer). The plate was centrifuged at 200 x g for 1 minute, then incubated at 37°C for 60 minutes in the presence of 5% carbon dioxide. After incubation, the plate was briefly centrifuged again before 90 μL of the supernatant was transferred to a new black, clear-bottom plate and the fluorescence signal was read on an Envision 2104 plate reader (Excitation: 492 nm; Emission: 515 nm).

Cytotoxicity was calculated using the following formula.

Cytotoxicity (%) = (A - B) / (C - B) × 100

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

Data represent % cytotoxicity of IL-2 conjugate treated human PBMCs against target cancer cells in the presence of cetuximab. The average percentage from technical overlap was converted to a ratio. Analysis was performed using a two-way generalized linear mixed model (GLMM) using factors for IL-2 conjugate, cetuximab and their interactions, using random donor effect, treatment rate as quasibinomial variables. This was followed by a post hoc assay (using Dunnett-Hsu adjustment) to compare IL-2 conjugate treated groups to controls. Statistical analysis was performed using SAS (1) version 9.4 software. A probability of less than 5% (p<0.05) was considered significant.

result.

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

Tests of fixed effects from the GLMM model indicate that the factors IL-2 conjugate, cetuximab, and their interactions have a significant effect on cytotoxicity (i.e., differences between the IL-2 conjugate groups are different significantly different for cetuximab concentrations). Pairwise comparisons were made between the IL-2 conjugate 2 mg/mL group versus control group at a cetuximab concentration of 0.001 mg/mL (p=0.0001) and between the IL-2 conjugate 0.4 mg/mL group versus control group (p=0.0001). 0.0001) showed a significant difference. Pairwise comparisons were also made between the IL-2 conjugate 2 mg/mL group versus control group at a cetuximab concentration of 0.01 mg/mL (p<0.0001), and between the IL-2 conjugate 0.4 mg/mL group versus control group (p<0.0001). p<0.0001), and between the IL-2 conjugate 0.08 mg/mL group versus the control group (p=0.0003). In addition, pairwise comparisons were made between the IL-2 conjugate 2 mg/mL group versus the control group at a cetuximab concentration of 0.1 mg/mL (p<0.0001), and between the IL-2 conjugate 0.4 mg/mL group versus the control group (p<0.0001). p<0.0001), and between the IL-2 conjugate 0.08 mg/mL group versus the control group (p<0.0001). Finally, pairwise comparisons were made between the IL-2 conjugate 2 mg/mL group versus the control group at a cetuximab concentration of 1 mg/mL (p<0.0001) and between the IL-2 conjugate 0.4 mg/mL group versus the control group. (p<0.0001), and between the IL-2 conjugate 0.08 mg/mL group versus the control group (p<0.0001).

The data demonstrates 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 an isotype control.

A431 cells.

Studies were performed using EGFR expressing A431 cells (epidermoid carcinoma) according to the procedure outlined above for CAL27 cells. Figure 2b shows the enhanced ADCC function of cetuximab on EGFR expressing A431 cells (PBMC:A431 ratio 50:1). The data demonstrates that the IL-2 conjugate enhanced the ADCC function of cetuximab against EGFR expressing A431 cancer cells.

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

The effect of IL-2_P65_[AzK_L1_PEG30kD]-1 on the ADCC function of cetuximab was examined using a Calcein-acetioxymethyl (Calcein-AM; Invitrogen) release assay.

ingredient.

NK-92.CD16 V (high affinity variant) (Conkwest Inc., San Diego, CA) was used as an 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 isotype IgG1 antibody (Biolegend); Calcein-acetioxymethyl (Calcein-AM; Invitrogen C3100MP), and probenecid (Invitrogen; P36400). The bioassay medium was phenol red free RPMI with 1% ultra low IgG fetal bovine serum supplemented with 1% probenecid for complete assay medium. MyeloCult H5100 (Stemcell catalog number 05150) supplemented with IL-2 (100 U/mL) and hydrocortisone (Sigma H6909; 50 μM, 10 mL) was used to culture NK-92.CD16 V cells.

procedure.

IL-2 supplements were withdrawn from the NK-92.CD16 V cell cultures and incubated overnight prior to the start of the assay. The next day, IL-2_P65_[AzK_L1_PEG30kD]-[AzK_L1_PEG30kD]- Cells were plated in 96-well round-bottom plates in the presence of 1 (60,000 cells were plated for a 3:1 ratio of effector:target cells) (18 h at 37°C in a humidified incubator with 5% CO ₂ ). during). These cells are used as effector cells. The next day, human EGFR-positive cancer cell lines (A431, DLD-1, FaDu or CAL27) were labeled with calcein-AM for 30 minutes (50 μg was diluted in 25 μL DMSO to prepare a stock solution, after which 10 μL of Sain stock solution was added to 4 mL RPMI 1640 containing 1% low IgG FBS and 1% probenecid for staining of 5 x 10 ⁶ cells), then washed. Cells were divided into several labeled tubes for incubation with various concentrations of cetuximab or isotype control. Cetuximab and isotype human IgG1 antibody were added at 3X concentration (for a final assay concentration of 10 μg/mL to 1 pg/mL), labeled target cells and antibody were mixed and allowed to stand for 30 minutes to opsonize allowed. After this incubation, target cells (20,000) and antibodies were added onto 100 μL of NK-92.CD16 V cells. The plate was briefly centrifuged at 1100 rpm for 1 minute and then incubated for 1 hour at 37°C and 5% CO ₂ .

After incubation, the plate was briefly centrifuged again as before, and 90 μL of the supernatant was transferred from each well to a clear bottom black plate without disturbing the cells. Fluorescent signals were read using an Envision 2104 (Excitation: 492 nm; Emission: 515 nm).

Cells were lysed with 2% Triton X-100 for maximal release. Fluorescence values of the culture medium background were subtracted from the fluorescence values of experimental emission (A), target cell spontaneous emission (B), and target cell maximal emission (C).

Percent cytotoxicity and ADCC for each plate (in duplicate) were calculated using the following formula:

Cytotoxicity (%) = (A - B) / (C - B) × 100

ADCC (%) = Cytotoxicity (%, with antibody) - Cytotoxicity (%, without antibody)

Measurements were performed in triplicate using three duplicate wells for each experiment. Each experiment is repeated at least three times. GraphPad Prism 5 (GraphPad Software, Inc., San Diego, Calif., USA) is used to fit the data points to a 4-parameter equation to calculate half maximum effective concentration (EC50) values.

result.

Cytotoxicity data using the NK92 cell line ADCC assay were EGFR expressing A431 (epidermoid carcinoma) (NK92:A431 ratio 3:1), DLD-1 (adenocarcinoma, colorectal cancer) (NK92:DLD-1 ratio 3:1), 3A-3D for FaDu (epithelial squamous cell carcinoma) (NK92:FaDu ratio 3:1) and CAL27 (epithelial squamous cell carcinoma) (NK92:CAL27 ratio 3:1) cells. The data demonstrates that the IL-2 conjugate enhanced the ADCC function of cetuximab against EGFR expressing cancer cells.

Example 5. Clinical study of combination therapy using IL-2 conjugates and cetuximab.

outline. Monotherapy using the IL-2 conjugate of Example 1 has been demonstrated to promote a peripheral increase in the number of NK cells, important effector cells that mediate antibody-dependent cellular toxicity (ADCC) to IgG1 antibodies such as cetuximab. .

A Phase 1/2, open-label, multicenter study was conducted evaluating the clinical benefit of combining the IL-2 conjugate described in Example 1 with cetuximab for the treatment of participants with advanced or metastatic solid tumors.

Participants received IL-2 conjugate (16 or 24 μg/kg dose) by IV infusion once every 3 weeks. Herein and throughout the discussion of this cohort, drug mass per kg of subject (eg, 16 μg/kg) refers to IL-2 mass excluding PEG and linker mass . Cetuximab is given at an initial loading dose of 400 mg/m ² over 120 minutes on Day 1 of Cycle 1 (maximum infusion rate of 10 mg/min), followed by all follow-up beginning with administration on Day 8 of Cycle 1. The dose was given as 250 mg/m ² infused over 60 minutes (maximum infusion rate 10 mg/min). Cetuximab was given on days 1, 8 and 15 of each 21 day cycle. The injection time of the IL-2 conjugate was about 30 minutes each. For each treatment cycle, prior to administration of the IL-2 conjugate, all participants received an IL-2 conjugate premedication to prevent or reduce the acute effects of infusion-related reactions (IAR) or flu-like symptoms. Received 30-60 min prior to injection of the conjugate. IL-2 conjugate premedication was as follows: antipyretics (oral) and antihistamines (H1 blockers). Anti-emetics were given at the discretion of the attending physician. All participants were pre-dosed with diphenhydramine (approximately 25-50 mg, intravenous) prior to administration of the first dose of cetuximab. Premedication for subsequent doses of cetuximab was an option based on the evaluation of the attending physician. If the IL-2 conjugate and cetuximab were given on the same day, participants who received diphenhydramine as the cetuximab premedication may have skipped the diphenhydramine as the IL-2 conjugate premedication. The dosing sequence was as follows: (i) pre-dose for cetuximab (30-60 min before start of cetuximab infusion); (ii) cetuximab; (iii) a pre-dose for IL-2 conjugate (administered 30-60 minutes prior to start of IL-2 conjugate infusion); and (iv) an IL-2 conjugate. Treatment was repeated for up to a total of 35 cycles or for a duration of up to 735 days.

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

Eosinophilia (elevated peripheral eosinophil counts): a cell surrogate marker for IL-2 induced proliferation of cells (eosinophils) associated with vascular leak syndrome (VLS);

Interleukin 5 (IL-5) : a cytokine surrogate marker for IL-2 induced activation of type 2 innate lymphoid cells and release of this chemoattractant resulting in eosinophilia and potentially VLS;

Interleukin 6 (IL-6) : cytokine surrogate marker for IL-2 induced cytokine release syndrome (CRS); and

Interferon γ (IFN-γ): A cytokine surrogate marker for IL-2 induced activation of CD8+ cytotoxic T lymphocytes.

The following biomarkers serve as surrogate predictors of antitumor immune activity:

Peripheral CD8+ effector cells: markers for IL-2 induced proliferation of these target cells in the periphery, which upon infiltration are surrogate markers that induce potential therapeutic responses;

Peripheral CD8+ Memory Cells: A marker for IL-2 induced proliferation of these target cells in the periphery that, upon infiltration, is a surrogate marker leading to a potentially durable therapeutic potential and maintenance of memory populations;

Peripheral NK cells: a marker for IL-2 induced proliferation of these target cells in the periphery, which upon infiltration may be a surrogate marker leading to a potentially rapid therapeutic response; and

Peripheral CD4+ Regulatory Cells: A marker for IL-2 induced proliferation of these target cells in the periphery to induce an immunosuppressive TME upon infiltration and to be a surrogate marker to counterbalance the effects of effector-based therapy.

First cohort using IL-2 conjugate at a dose of 16 μg/kg

result. The 5 subjects included 2 males and 3 females with a median age of 69 years (range 65-72 years). All subjects had an Eastern United States Collaborative Oncology Group (ECOG) performance status of 0 or 1 and received 1-4 previous lines of systemic therapy. The cancers were anal cancer (1 subject), colon adenocarcinoma (1 subject), adrenocortical cancer (1 subject), lung squamous cell carcinoma (1 subject), and small intestine cancer (1 subject). All 5 subjects had metastatic disease.

Subjects received IL-2 conjugate (16 μg/kg) and cetuximab combination treatment for 2-7 cycles (2-7 doses of IL-2 conjugate). Two subjects (one with anal cancer and one with metastatic adrenocortical carcinoma) developed progressive disease (PD) after 2 cycles of combination treatment and discontinued IL-2 conjugate and cetuximab combination therapy. One subject with colon cancer showed disease progression after 5 cycles. Two subjects are progressing as follows: 1 at cycle 3 for lung squamous cell carcinoma and 1 at cycle 7 for small bowel carcinoma.

Peripheral CD8+ _Teff cell counts were measured ( FIGS. 4A-4B ). Some subjects observed prolonged CD8+ expansion (eg, a 2-fold or greater change) compared to baseline at 3 weeks after the previous dose.

Peripheral NK cell counts are illustrated in Figures 5A-5B . An increase in NK cell counts was observed in each subject. Subjects generally showed increased NK cell counts compared to baseline at 8 days and 3 weeks after previous administration.

Peripheral CD4+ T _reg counts are illustrated in FIGS. 6A-6B

Eosinophil counts were measured ( FIGS. 7A-7B ). The measured values did not exceed a 4-fold increase and were consistently below the range of 2328 to 15958 eosinophils/μL in patients with IL-2 induced eosinophilia, which is described in Pisani et al., Blood 1991 Sep 15;78 (6):1538-44]. Lymphocyte counts were also measured (FIGS. 8A-8B).

Results summary and discussion. All subjects tested had peripheral expansion after administration of CD8+ T effector (Teff) cells, NK cells and CD4+ T _reg cells.

An adverse event (AE) was an unforeseen medical occurrence that occurred in a clinical investigation subject who received a medicinal product without regard to causation. A dose-limiting toxicity was defined as an AE occurring within ±1 day from Day 1 to Day 29 (including endpoint) of the treatment cycle, which was unambiguous or incontrovertible and not entirely related to an external cause and met one or more of the following criteria: Satisfied:

Grade 3 neutropenia lasting >7 days (absolute neutrophil count < 1000/mm ³ > 500/mm ³ ), or Grade 4 neutropenia of any duration

· Grade 3+ febrile neutropenia

Grade 4+ thrombocytopenia (platelet count < 25,000/mm ³ )

Grade 3+ thrombocytopenia (platelet count < 50,000-25,000/mm ³ ) lasting longer than 5 days or associated with clinically significant bleeding or requiring platelet transfusion

Failure to meet recovery criteria of an absolute neutrophil count of at least 1,000 cells/mm ³ and a platelet count of at least 75,000 cells/mm ³ within 10 days

· Any other grade 4+ hematological toxicity lasting ≥5 days

· Grade 3+ ALT or AST combined with bilirubin greater than twice the ULN without evidence of cholestasis or other causes, such as viral infection or other drugs (ie, Hy's law)

· Grade 3 infusion-related reactions occurring with premedication; Grade 4 infusion-related reaction

· Grade 3 vascular leak syndrome, defined as hypotension associated with pulmonary edema and fluid retention

· Grade 3+ Anaphylaxis

· Grade 3+ hypotension

· Grade 3+ AEs not resolving to Grade < 2 within 7 days of starting accepted standard of care healthcare management

· Grade 3+ Cytokine Release Syndrome

The following exceptions apply to non-hematologic AEs:

· Grade 3 fatigue, nausea, vomiting or diarrhea that resolves to Grade 2 or less with optimal medical management within ≤ 3 days

· Grade 3 fever (defined as greater than 40°C for 24 hours or less)

· Grade 3 infusion-related reactions occurring without premedication; Subsequent doses should use a premedication and become a DLT if the reaction recurs.

· Grade 3 arthralgia or rash that resolves to Grade 2 or less within 7 days of starting accepted standard of care medical management (eg, systemic corticosteroid therapy)

If a subject has a Grade 1 or Grade 2 ALT or AST elevation at baseline to be considered indirectly affected by liver metastasis, the Grade 3 elevation must also be ≥ 3 times baseline and persist for more than 7 days.

A serious AE was defined as any AE resulting in any of the following outcomes: death; life-threatening AE; Inpatient hospitalization or extension of existing hospitalization; Persistent or significant incapacity or substantial disruption of the ability to perform normal life functions; or congenital malformations/birth defects. Significant medical events that may not result in death, are not life threatening, or require hospitalization may, in good medical judgment, endanger the subject and require medical or surgical intervention to prevent one of the consequences listed above. If intervention may be necessary, it may be considered serious. Examples of such medical events include allergic bronchospasm requiring intensive care in an emergency room or home, blood disorders or convulsions that do not result in inpatient admission, or occurrence of drug dependence or drug abuse.

There was no significant elevation of IL-5. There was no cumulative toxicity. There was no end organ toxicity. There was no QTc prolongation or other cardiac toxicity. Overall, IL-2 conjugates were considered to be well tolerated.

Four out of five subjects had at least one treatment-induced AE (TEAE). Most TEAEs were grade 1-2, 1 subject had at least one grade 3, and 1 subject had at least one grade 4 TEAE. Four subjects had treatment-related AEs. These included: one grade 1 infusion reaction; one grade 1 miscarriage; one grade 1 fatigue; one grade 2 diarrhea; and one grade 4 lymphocyte count reduction. Two subjects had three unrelated SAEs: one dysphagia and spinal cord compression; and one pleural effusion. TEAEs did not result in drug discontinuation, dose reductions, DLTs, and anaphylaxis or CRS. Treatment-related AEs resolved to accepted standards of care. TEAEs are detailed in Table 2 and treatment-related adverse events are summarized in Table 3.

[Table 2]

[Table 3]

Second cohort using IL-2 conjugate at a dose of 24 μg/kg

result. Three subjects were male with a median age of 71 years (range 65-72 years). All subjects had an Eastern United States Oncology Collaborative Group (ECOG) performance status of 1. One subject received 1 previous line of therapy and the second subject received 4 previous lines of therapy. Cancers were gastric cancer (one subject), head and neck squamous cell carcinoma (HNSCC) (one subject), and colon cancer (one subject). All subjects had metastatic disease. Subjects received IL-2 conjugate (24 μg/kg) and cetuximab combination treatment for one cycle (one dose of IL-2 conjugate). One subject developed progressive disease (PD) after the first cycle of combination therapy, preventing administration of an additional therapeutic dose of the IL-2 conjugate and cetuximab combination therapy.

Two of the subjects experienced at least one TEAE. One of the subjects experienced at least one Grade 3 treatment-related AE, which was Grade 3 chills. None of the subjects had an associated SAE. No DLTs were observed and there were no drug discontinuations due to TEAEs. TEAEs are detailed in Table 4 and treatment-related adverse events are summarized in Table 5.

[Table 4]

[Table 5]

Although the foregoing invention has been described in some detail by way of examples and examples for purposes of clarity and understanding, the descriptions and examples should not be construed as limiting the scope of the invention. Numerous variations, alterations, and substitutions will now occur to those skilled in the art without departing from this invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be embraced therein. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entirety. To the extent any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content of this disclosure controls.

SEQUENCE LISTING <110> SYNTHORX, INC. ABBADESSA, Giovanni et al. <120> IMMUNO ONCOLOGY COMBINATION THERAPY WITH IL-2 CONJUGATES AND ANTI-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> PRT <213> Homo sapiens <220> <221> misc_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> PRT <213> Homo sapiens <220> <221> misc_feature <223> IL-2 (homo sapiens) (precursor) NCBI Accession No.: 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 <211> 132 <212> PRT <213> artificial sequence <220> <223> Synthetic: aldesleukin <400> 3 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 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> PRT <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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK] <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK] <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK] <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK] <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK] <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_PEG] <220> <221> misc_feature <222> (65)..(65) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_PEG] <220> <221> misc_feature <222> (62)..(62) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_PEG] <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_PEG] <220> <221> misc_feature <222> (43)..(43) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_PEG] <220> <221> misc_feature <222> (35)..(35) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65 [AzK_PEG5kD] <220> <221> misc_feature <222> (65)..(65) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_PEG5kD] <220> <221> misc_feature <222> (62)..(62) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42 [AzK_PEG5kD] <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_PEG5kD] <220> <221> misc_feature <222> (43)..(43) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_PEG5kD] <220> <221> misc_feature <222> (35)..(35) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65 [AzK_PEG30kD] <220> <221> misc_feature <222> (65)..(65) <223> Xaa is [AzK_PEG30kD]: 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62 [AzK_PEG30kD] <220> <221> misc_feature <222> (62)..(62) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42 [AzK_PEG30kD] <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43 [AzK_PEG30kD] <220> <221> misc_feature <222> (43)..(43) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35 [AzK_PEG30kD] <220> <221> misc_feature <222> (35)..(35) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK]-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK]-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK]-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK]-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK]-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_L1_PEG]-1 <220> <221> misc_feature <222> (64)..(64) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_L1_PEG]-1 <220> <221> misc_feature <222> (61)..(61) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_L1_PEG]-1 <220> <221> misc_feature <222> (41)..(41) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_L1_PEG]-1 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_L1_PEG]-1 <220> <221> misc_feature <222> (34)..(34) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_L1_PEG5kD]-1 <220> <221> misc_feature <222> (64)..(64) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_L1_PEG5kD]-1 <220> <221> misc_feature <222> (61)..(61) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_L1_PEG5kD]-1 <220> <221> misc_feature <222> (41)..(41) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_L1_PEG5kD]-1 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_L1_PEG5kD]-1 <220> <221> misc_feature <222> (34)..(34) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_L1_PEG30kD]-1 <220> <221> misc_feature <222> (64)..(64) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_L1_PEG30kD]-1 <220> <221> misc_feature <222> (61)..(61) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_L1_PEG30kD]-1 <220> <221> misc_feature <222> (41)..(41) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_L1_PEG30kD]-1 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_L1_PEG30kD]-1 <220> <221> misc_feature <222> (34)..(34) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_L1_PEG]-2 <220> <221> misc_feature <222> (65)..(65) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_L1_PEG]-2 <220> <221> misc_feature <222> (62)..(62) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_L1_PEG]-2 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_L1_PEG]-2 <220> <221> misc_feature <222> (43)..(43) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_L1_PEG]-2 <220> <221> misc_feature <222> (35)..(35) <223> Xaa is [AzK_L1_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_L1_PEG5kD]-2 <220> <221> misc_feature <222> (65)..(65) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_L1_PEG5kD]-2 <220> <221> misc_feature <222> (62)..(62) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_L1_PEG5kD]-2 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_L1_PEG5kD]-2 <220> <221> misc_feature <222> (43)..(43) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_L1_PEG5kD]-2 <220> <221> misc_feature <222> (35)..(35) <223> Xaa is [AzK_ L1_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_L1_PEG30kD]-2 <220> <221> misc_feature <222> (65)..(65) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_L1_PEG30kD]-2 <220> <221> misc_feature <222> (62)..(62) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_L1_PEG30kD]-2 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_L1_PEG30kD]-2 <220> <221> misc_feature <222> (43)..(43) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_L1_PEG30kD]-2 <220> <221> misc_feature <222> (35)..(35) <223> Xaa is [AzK_ L1_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_PEG]-1 <220> <221> misc_feature <222> (64)..(64) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_PEG]-1 <220> <221> misc_feature <222> (61)..(61) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_PEG]-1 <220> <221> misc_feature <222> (41)..(41) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_PEG]-1 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_PEG]-1 <220> <221> misc_feature <222> (34)..(34) <223> Xaa is [AzK_PEG]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_PEG5kD]-1 <220> <221> misc_feature <222> (64)..(64) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_PEG5kD]-1 <220> <221> misc_feature <222> (61)..(61) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_PEG5kD]-1 <220> <221> misc_feature <222> (41)..(41) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_PEG5kD]-1 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_PEG5kD]-1 <220> <221> misc_feature <222> (34)..(34) <223> Xaa is [AzK_PEG5kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_P65[AzK_PEG30kD]-1 <220> <221> misc_feature <222> (64)..(64) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E62[AzK_PEG30kD]-1 <220> <221> misc_feature <222> (61)..(61) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F42[AzK_PEG30kD]-1 <220> <221> misc_feature <222> (41)..(41) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K43[AzK_PEG30kD]-1 <220> <221> misc_feature <222> (42)..(42) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_K35[AzK_PEG30kD]-1 <220> <221> misc_feature <222> (34)..(34) <223> Xaa is [AzK_PEG30kD]: N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to 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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F44X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_F44X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_R38X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_R38X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_T41X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_T41X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E68X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_E68X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_Y45X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_Y45X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_V69X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_V69X-1 <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_L72X <220> <221> misc_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> PRT <213> artificial sequence <220> <223> Synthetic: IL-2_L72X-1 <220> <221> misc_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 (29)

A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody; wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 wherein one or more amino acid residues of the IL-2 conjugate are replaced with the structure of Formula I:
[Formula I]

(here,
Z is CH ₂ and Y is

is;
Y is CH ₂ and Z is

is;
Z is CH ₂ and Y is

is; or
Y is CH ₂ and Z is

ego;
W is a PEG group having an average molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa;
X has the following structure:

It is an L-amino acid having;
X-1 represents the point of attachment to the preceding amino acid residue;
X+1 represents the point of attachment to the trailing amino acid residue;
The position of structure 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). 2. The method of claim 1, wherein in the IL-2 conjugate Z is CH ₂ and Y is

in, how. 2. The method of claim 1, wherein Y in the IL-2 conjugate is CH ₂ and Z is

in, how. 2. The method of claim 1, wherein in the IL-2 conjugate Z is CH ₂ and Y is

in, how. 2. The method of claim 1, wherein Y in the IL-2 conjugate is CH ₂ and Z is

in, how. 6. The method of any one of claims 1-5, wherein the PEG groups in the IL-2 conjugate have an average molecular weight of about 25 kDa, 30 kDa, or 35 kDa. 7. The method of claim 6, wherein the PEG groups in the IL-2 conjugate have an average molecular weight of about 30 kDa. 8. The method of any one of claims 1 to 7, wherein the position of the structure of Formula I in the amino acid sequence of the IL-2 conjugate is P64. The method of claim 1 , wherein the structure of Formula I has the structure of Formula IV or Formula V, or is a mixture of the structure of Formula IV and the structure of Formula V:
[Formula IV]

[Formula V]

(here,
W is a PEG group having an average molecular weight of about 25 kDa, 30 kDa, or 30 kDa;
q is 1, 2 or 3;
X has the following structure:

It is an L-amino acid having;
X-1 represents the point of attachment to the preceding amino acid residue;
X+1 indicates the point of attachment to the following amino acid residue). 10. The method of claim 9, wherein the position of the structure of Formula IV or Formula V in the amino acid sequence of the IL-2 conjugate is P64. 11. The method of any one of claims 1-10, wherein the anti-EGFR antibody is cetuximab. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (a) an IL-2 conjugate, and (b) cetuximab, comprising: -2 conjugate comprises the amino acid sequence of SEQ ID NO: 50, and [AzK_L1_PEG30kD] has the structure of Formula (XII) or Formula (XIII), or is a mixture of the structures of Formula (XII) and Formula (XIII):
[Formula XII]

[Formula XIII]

(here,
n is an integer, where n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 30 kDa;
q is 1, 2 or 3;
Wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 50 that are not replaced). 13. The method of any one of claims 1 to 12, wherein q is 1. 13. The method of any one of claims 1 to 12, wherein q is 2. 13. The method of any one of claims 1 to 12, wherein q is 3. 16. The method of any one of claims 1 to 15, wherein the average molecular weight is a number average molecular weight. 16. The method of any one of claims 1 to 15, wherein the average molecular weight is a weight average molecular weight. 18. The method of any one of claims 1-17, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. 19. The method of any one of claims 1-18, wherein the IL-2 conjugate is administered to the subject about once every 2 weeks, about once about every 3 weeks, or about once every 4 weeks. 20. The method of any one of claims 1 to 19, wherein the anti-EGFR antibody is administered about once every 1 week, about once every 2 weeks, about once every 3 weeks, or about once every 4 weeks, method. 21. The method of any one of claims 1-20, wherein the IL-2 conjugate is administered to the subject by intravenous administration. 22. The method of any one of claims 1-21, wherein the IL-2 conjugate and the anti-EGFR antibody are administered separately. 24. The method of claim 23, wherein the IL-2 conjugate and the anti-EGFR antibody are administered sequentially. 24. The method of any one of claims 1-23, wherein the cancer is 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 unstable cancer, microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer, 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 DNA damage response (DDR) defective Castration-resistant prostate cancer, bladder cancer, ovarian cancer, moderate to low mutation burden tumors, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), low to non-expressing PD-L1 tumors, beyond the anatomical primary site of origin A tumor disseminated systemically to the liver and CNS, and diffuse large B-cell lymphoma. 25. The method of any one of claims 1-24, comprising administering to the subject about 16 μg/kg of the IL-2 conjugate. 25. The method of any one of claims 1-24, comprising administering to the subject about 24 μg/kg of the IL-2 conjugate. The anti-EGFR antibody of any one of claims 1 to 10 or 12 to 26, wherein the anti-EGFR antibody is Panitumumab (Vectibix), Necitumumab (Portrazza), JNJ-61186372 (Amivantamab), IMC- C225, ABX-EGF, ICR62, and EMD 55900. 28. An IL-2 conjugate for use in the method of any one of claims 1-27. 28. Use of an IL-2 conjugate for the manufacture of a medicament for the method of any one of claims 1-27.

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