TW202216203A - Immuno oncology combination therapy with il-2 conjugates and anti-egfr antibodies - Google Patents

Abstract

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

Description

Immuno-Oncology Combination Therapy with IL-2 Conjugates and Anti-EGFR Antibodies

The present invention discloses methods for treating cancer in an individual in need thereof, comprising administering an IL-2 conjugate and an anti-EGFR antibody.

Different T cell populations regulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses of the immune system by avoiding pathological autoreactivity, while cytotoxic T cells target and destroy infected and/or cancer cells. In some cases, modulating distinct T cell populations provides an option for treating a disease or indication. The regulation of different T cell populations can be enhanced by the presence of additional agents or methods in combination therapy.

Cytokines comprise a group of cell signaling proteins such as chemokines, interferons, interleukins, lymphoid interleukins, tumor necrosis factors, and other growth factors that play a role in innate and adaptive immune cell homeostasis. Cytokines are produced by immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, endothelial cells, fibroblasts and various stromal cells. In some instances, 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 monocytic lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4+ T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue residents.

In some instances, interleukin 2 (IL-2) signaling is used to modulate T cell responses, which are subsequently used to treat cancer. Accordingly, in one aspect, provided herein are methods of treating cancer in an individual comprising administering an IL-2 conjugate in combination with an anti-EGFR antibody.

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

Illustrative specific examples include the following.

式(I)； 其中： Z是CH ₂且Y是

； Y是CH ₂且Z是

； Z是CH ₂且Y是

；或 Y是CH ₂且Z是

； W是具有平均分子量為約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa，50 kDa或60 kDa的PEG基團；且 X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點； 其中式(I)結構在IL-2接合物的胺基酸序列中的位置是選自K34、F41、F43、K42、E61、P64、R37、T40、E67、Y44、V68、和L71。 Specific Example 1. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody, wherein the IL-2 conjugate Comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is substituted with a structure of formula (I):

Formula (I); wherein: Z is _CH and Y is

; Y is _CH and Z is

; Z is _CH and Y is

; or Y is _CH and Z 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; and X is a PEG group having the following Structure of L-amino acids:

X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue; wherein the structure of formula (I) is at the amine of the IL-2 conjugate The positions in the amino acid sequence are selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71.

。 Example 2. The method of Example 1, wherein in the IL-2 conjugate, Z is _CH and Y is

.

。 Example 3. The method of Example 1, wherein in the IL-2 conjugate, Y is _CH and Z is

.

。 Example 4. The method of Example 1, wherein in the IL-2 conjugate, Z is _CH and Y is

.

。 Example 5. The method of Example 1, wherein in the IL-2 conjugate, Y is _CH and Z is

.

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

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

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

式(IV)；

式(V)； 其中： W是具有平均分子量為約25 kDa、30 kDa或30 kDa的PEG基團； q是1、2或3； X是具有以下結構的L-胺基酸：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 Specific example 9. The method of specific example 1, wherein the structure of formula (I) has the structure of formula (IV) or formula (V), or is a mixture of structures of formula (IV) and formula (V):

formula (IV);

Formula (V); wherein: 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 is an L-amino acid having the structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next 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 1-10, wherein the anti-EGFR antibody is cetuximab.

式(XII)；

式(XIII)； 其中： n是一個使得-(OCH ₂CH ₂) _n-OCH ₃具有約30 kDa的分子量的整數； q是1、2或3；以及 波浪線表示與SEQ ID NO：50內未被取代的胺基酸殘基的共價鍵。 Specific example 12. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) cetuximab, wherein the IL-2 The conjugate comprises the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG30kD] has a structure of formula (XII) or formula (XIII), or a mixture of structures of formula (XII) and formula (XIII):

Formula (XII);

Formula (XIII); wherein: 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; and the wavy line indicates within SEQ ID NO: 50 Covalent bonding of unsubstituted amino acid residues.

Example 13. The method of any one of Examples 1-12, wherein q is 1.

Example 14. The method of any one of Examples 1-12, wherein q is 2.

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

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

Example 17. The method of any one of Examples 1-15, wherein the average molecular weight is a weight average molecular weight.

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

Example 19. The method of any one of Examples 1-18, wherein the IL-2 conjugate is administered to the individual about once every two weeks, about once every three weeks, or about once every four weeks.

Example 20. The method of any one of Examples 1-19, wherein the anti-EGFR antibody is administered to the individual about once a week, about once every two weeks, about once every three weeks, or about once every four weeks.

Example 21. The method of any one of Examples 1-20, wherein the IL-2 conjugate is administered to the individual by intravenous administration.

Embodiment 22. The method of any one of Embodiments 1-21, 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.

Specific example 24. The method of any one of specific examples 1-23, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), typical Hodge Gold lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable carcinoma, microsatellite stable carcinoma, gastric cancer, colon cancer, colorectal cancer (CRC), sub- Cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophagus, 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) deficiency, bladder cancer, ovarian cancer, moderate to low mutation tumor burden, cutaneous squamous cell carcinoma (CSCC), squamous cell skin carcinoma (SCSC), tumors with low to no expression of PD-L1, tumors with systemic diffuse to the liver and CNS beyond their primary anatomical origin, and diffuse large B-cell lymphoma.

Example 25. The method of any one of Examples 1-24, comprising administering to the individual about 16 μg/kg of the IL-2 conjugate.

Example 26. The method of any one of Examples 1-24, comprising administering to the individual about 24 μg/kg of the IL-2 conjugate.

Specific example 27. The method of any one of specific examples 1-10 or 12-26, wherein the anti-EGFR antibody is selected from panitumumab (Vectibix), necitumumab (necitumumab) ( Portrazza), JNJ-61186372 (Amivantamab), IMC-C225, ABX-EGF, ICR62, and EMD 55900.

Example 28. An IL-2 conjugate for use in the method of any one of Examples 1-27.

Example 29. Use of an IL-2 conjugate for the manufacture of a medicament for use in the method of any one of Examples 1-27.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the foregoing general description and the following embodiments are exemplary and explanatory only and do not limit any claimed subject matter. To the extent that any material incorporated herein by reference is inconsistent with the statement of the present invention, the statement shall control. In this application, unless expressly stated otherwise, the singular is used to include the plural. It must be noted that, as used in the specification and the appended claims, the singular forms "a (a, an)" and "the (the)" include the plural forms unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" and "included" is not intended to be limiting.

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

As used herein, ranges and amounts can be expressed as "about" a particular value or range. Approx. also includes the exact amount. Therefore, "about 5 µL" means "about 5 µL" and "5 µL". Generally, the term "about" includes amounts expected to be within experimental error, such as, for example, within 15%, 10%, or 5%.

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

As used herein, the terms "individual", "subject" and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of these terms require or are limited to situations characterized by supervision (eg, continuous or intermittent) by a health care worker (eg, physician, registered nurse, nurse practitioner, clinician assistant, hospital staff, or hospice worker).

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

In some instances, the terms "significant" or "significantly" in reference to activation of one or more cell populations via cytokine signaling complexes refer to changes sufficient to activate the cell populations. In some cases, changes in activated cell populations are measured as receptor signaling potency. In this case, an EC50 value can be provided. In other cases, an ED50 value may be provided. In additional instances, concentrations or doses of cytokines can be provided.

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

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

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

The term "monoclonal antibody (etc.)" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for variant antibodies (e.g. , containing natural mutations or arising during the production of monoclonal antibody preparations), which are usually present in small 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" denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be made by a variety of techniques including, but not limited to, fusion tumor methods, recombinant DNA methods, phage display methods, and the use of transgenes containing all or part of human immunoglobulin loci Animal methods, such methods and other exemplary methods for 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, but are not limited to, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), Uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP) and guanosine Monophosphate (GMP), Deoxyadenosine Triphosphate (dATP), Deoxythymidine Triphosphate (dTTP), Deoxycytidine Triphosphate (dCTP), Deoxyguanosine Triphosphate (dGTP), Deoxyadenosine Diphosphate (dADP), Thymidine Diphosphate (dTDP), Deoxycytidine Diphosphate (dCDP), Deoxyguanosine Diphosphate (dGDP), Deoxyadenosine Monophosphate (dAMP), Deoxythymidine Monophosphate (dTMP), deoxycytidine monophosphate (dCMP) and deoxyguanosine monophosphate (dGMP). 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 containing ribose as the sugar moiety include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.

As used herein, "base" or "nucleobase" refers to at least the nucleobase portion of a nucleoside or nucleotide (nucleosides and nucleotides include ribose or deoxyribose variants), which in some cases The following may contain further modifications to the sugar moiety of the nucleoside or nucleotide. In some instances, "base" is also used to refer to an entire nucleoside or nucleotide (eg, a "base" can be incorporated into DNA by a DNA polymerase, or into RNA by an RNA polymerase). However, unless the context requires it, the term "base" should not be construed as necessarily representing an entire nucleoside or nucleotide. In the chemical structures of bases or nucleobases provided herein, only the nucleoside or nucleotide base is shown, with sugar moieties and optional phosphate residues omitted for clarity. As used in the chemical structures of bases or nucleobases provided herein, wavy lines represent linkages to nucleosides or nucleotides, wherein the sugar moiety of the nucleosides or nucleotides may be further modified. In some embodiments, a wavy line represents the attachment of a base or nucleobase to a sugar moiety (such as a pentose) of a nucleoside or nucleotide. In some embodiments, the pentose sugar is ribose or deoxyribose.

In some embodiments, the nucleobase is typically the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring, may be modified, may have no similarity to natural bases, and/or may have been synthesized (eg, through organic synthesis). In certain embodiments, a nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide that is capable of interacting with a base of another nucleic acid, with or without the use of hydrogen bonding. In certain embodiments, the unnatural nucleobase is not derived from a natural nucleobase. It should be noted that unnatural nucleobases do not necessarily have basic properties, but for simplicity, they are referred to as nucleobases. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase can be attached to deoxyribose or ribose, while "d" without parentheses indicates that the nucleobase is attached to deoxyribose .

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

As used herein, an "analog" of a chemical structure refers to a chemical structure that retains substantial similarity to the parent structure, although it may not be readily synthetically derived from the parent structure. In some embodiments, the nucleotide analogs are non-natural nucleotides. In some embodiments, the nucleoside analogs are non-natural nucleosides. Related chemical structures that are readily derived synthetically from the parent chemical structure are called "derivatives".

As used herein, "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 that is not clearly or indisputably related to an exogenous factor relationship and comply with the criteria specified for DLT in Example 5.

As used herein, "Cetuximab" refers to a chimeric (mouse/human) anti-EGFR antibody sold by Eli Lilly and Co. under the tradename "Erbitux".

Although various features of the invention may be described in the context of a single specific example, these features can also be provided separately or in any suitable combination. Conversely, although the invention may be described in the context of individual embodiments for clarity, the invention may also be practiced in a single embodiment. IL-2 conjugate

Interleukin 2 (IL-2) is a pleiotropic type 1 cytokine whose structure contains a 15.5 kDa four α-helix bundle. The precursor form of IL-2 is 153 amino acid residues in length, the first 20 amino acids form the signal peptide, and residues 21-153 form the mature form. IL-2 is primarily produced by CD4+ T cells upon antigen stimulation, and to a lesser extent by CD8+ cells, natural killer (NK) cells and natural killer T (NKT) cells, activated dendritic cells (DC), and mast cells Production. IL-2 signaling is through interaction with the IL-2 receptor (IL-2R) subunits, namely IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132) ) interacts with a specific combination of . The interaction of IL-2 with IL-2Rα forms a "low affinity" IL-2 receptor complex with a _Kd of about ^10-8 M. The interaction of IL-2 with IL-2R[beta] and IL-2R[gamma] forms an "intermediate affinity" IL-2 receptor complex with a Kd of about 10&_lt;" 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 Kd of about > ^10-11 M.

In some instances, IL-2 regulates the activation and proliferation of regulatory T cells via signaling via the "high-affinity" IL-2Rαβγ complex. Regulatory T cells or CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T (Treg) cells mediate immune homeostasis by suppressing effector cells such as CD4 ⁺ T cells, CD8 ⁺ T cells, B cells, NK cells, and NKT cells maintenance. In some instances, Treg cells are generated from the thymus (tTreg cells) or induced by peripheral naive T cells (pTreg cells). In some cases, Treg cells are considered mediators of peripheral tolerance. Indeed, in one study, the transfer of CD25-depleted peripheral CD4 ⁺ T cells produced multiple autoimmune diseases in nude mice, while cotransfer of CD4 ⁺ CD25 ⁺ T cells suppressed autologous disease Generation of immunity (Sakaguchi, et al., “Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25),” J. Immunol . 155(3): 1151-1164 (1995), The respective disclosures of which are hereby incorporated by reference in their entirety). Increased Treg cell population downregulates effector T cell proliferation and suppresses autoimmunity and T cell antitumor responses.

IL-2 regulates activation and proliferation of CD8 ⁺ effector T (Teff) cells, NK cells, and NKT cells via "moderate affinity" IL-2Rβγ complex signaling. 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 used to identify and kill damaged cells, cancer cells T lymphocytes of cells and pathogen-infected cells. NK and NKT cells are lymphocyte types similar to CD8 ⁺ Teff cells that target cancer cells and pathogen-infected cells.

In some instances, IL-2 signaling is used to modulate T cell responses, which are subsequently used to treat cancer. For example, IL-2 is administered in high doses to induce expansion of Teff cell populations for use in the treatment of cancer. However, high doses of IL-2 further concomitantly stimulate Treg cells, thereby suppressing antitumor immune responses. High doses of IL-2 also induce toxic adverse events in the vasculature by engaging cells expressing IL-2R alpha chains, including innate immune cells type 2 (ILC-2), Eosinophilic globules, and endothelial cells. This can lead to eosinophilia, capillary leakage, and Vascular Leak Syndrome (VLS).

Adoptive cell therapy enables clinicians to effectively use a patient's own immune cells to fight diseases such as proliferative diseases (eg, cancer) and infectious diseases. The utility of IL-2 signaling can 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 a variety of cancers. Activation of EGFR promotes cell proliferation and survival, as well as angiogenesis, leading to 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 resulting in slower cell growth. Anti-EGFR antibodies can also have the same effect by competitively inhibiting the binding of transforming growth factor alpha (TGF-alpha) to EGFR.

Provided herein are methods of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody. Certain exemplary IL-2 polypeptides and IL-2 conjugates are provided in Table 1. Table 1. name sequence SEQ ID NO: IL-2 (Homo sapiens) (mature form) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 1 IL-2 (Homo sapiens) (precursor) NCBI Accession Number: AAB46883.1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 2 aldesleukin PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 3 IL-2_C125S APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 4 IL-2_P65X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK X LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 5 IL-2_E62X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE X LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 6 IL-2_F42X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT X KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 7 IL-2_K43X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF X FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 8 IL-2_K35X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP X LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 9 IL-2_P65[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 10 IL-2_E62[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE[ AzK] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 11 IL-2_F42[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT[ AzK] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 12 IL-2_K43[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF[ AzK] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 13 IL-2_K35[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP[ AzK] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 14 IL-2_P65 [AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 15 IL-2_E62 [AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 16 IL-2_F42 [AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 17 IL-2_K43 [AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 18 IL-2_K35 [AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 19 IL-2_P65 [AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 20 IL-2_E62 [AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT twenty one IL-2_F42 [AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT twenty two IL-2_K43 [AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT twenty three IL-2_K35 [AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT twenty four IL-2_P65 [AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 25 IL-2_E62 [AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 26 IL-2_F42 [AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 27 IL-2_K43 [AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 28 IL-2_K35 [AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 29 IL-2_P65X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK X LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 30 IL-2_E62X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE X LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 31 IL-2_F42X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT X KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 32 IL-2_K43X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF X FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 33 IL-2_K35X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP X LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 34 IL-2_P65[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 35 IL-2_E62[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE[ AzK] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 36 IL-2_F42[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT[ AzK] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 37 IL-2_K43[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF[ AzK] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 38 IL-2_K35[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP[ AzK] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 39 IL-2_P65[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 40 IL-2_E62[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 41 IL-2_F42[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 42 IL-2_K43[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 43 IL-2_K35[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 44 IL-2_P65[AzK_L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 45 IL-2_E62[AzK_L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 46 IL-2_F42[AzK_L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 47 IL-2_K43[AzK_L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 48 IL-2_K35[AzK_L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 49 IL-2_P65[AzK_L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 50 IL-2_E62[AzK_L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 51 IL-2_F42[AzK_L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 52 IL-2_K43[AzK_L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 53 IL-2_K35[AzK_L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 54 IL-2_P65[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 55 IL-2_E62[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 56 IL-2_F42[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 57 IL-2_K43[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 58 IL-2_K35[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 59 IL-2_P65[AzK_L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 60 IL-2_E62[AzK_L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 61 IL-2_F42[AzK_L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 62 IL-2_K43[AzK_L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 63 IL-2_K35[AzK_L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 64 IL-2_P65[AzK_L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 65 IL-2_E62[AzK_L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 66 IL-2_F42[AzK_L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 67 IL-2_K43[AzK_L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 68 IL-2_K35[AzK_L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 69 IL-2_P65[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 70 IL-2_E62[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 71 IL-2_F42[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 72 IL-2_K43[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 73 IL-2_K35[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 74 IL-2_P65[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 75 IL-2_E62[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 76 IL-2_F42[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 77 IL-2_K43[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 78 IL-2_K35[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 79 IL-2_P65[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 80 IL-2_E62[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 81 IL-2_F42[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 82 IL-2_K43[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 83 IL-2_K35[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 84 IL-2_F44X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK X YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 85 IL-2_F44X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFK X YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 86 IL-2_R38X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT X MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 87 IL-2_R38X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT X MLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 88 IL-2_T41X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML X FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 89 IL-2_T41X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML X FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 90 IL-2_E68X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE X VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 91 IL-2_E68X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE X VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 92 IL-2_Y45X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF X MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 93 IL-2_Y45X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF X MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 94 IL-2_V69X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE X LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 95 IL-2_V69X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE X LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 96 IL-2_L72X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN X AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 97 IL-2_L72X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN X AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 98 X = sites containing unnatural amino acids. [AzK]=N6-((2-azidoethoxy)-carbonyl)-L-lysine, with CAS number 1167421-25-1. [AzK_PEG]=N6-((2-azidoethoxy)-carbonyl)-L-lysine acid, stably conjugated to PEG via DBCO-mediated click chemistry to form compounds comprising formula (II) or (III) structural compounds. For example, if specified, PEG5kD represents a linear polyethylene glycol chain having an average molecular weight of 5 kilodaltons, terminated with methoxy groups. The ratio of regioisomers produced by the click reaction is about 1:1 or greater. The term "DBCO" denotes a chemical moiety comprising a dibenzocyclooctyne group. [AzK_L1_PEG]=N6-((2-azidoethoxy)-carbonyl)-L-lysine acid, stably conjugated to PEG via DBCO-mediated click chemistry to form structures comprising formula (IV) or formula (V) compounds of the structure. For example, if specified, PEG5kD represents a linear polyethylene glycol chain having an average molecular weight of 5 kilodaltons, terminated with methoxy groups. The ratio of regioisomers produced by the click reaction is about 1:1 or greater. The term "DBCO" denotes a chemical moiety comprising a dibenzocyclooctyne group.

(I) 其中： Z是CH ₂且Y是

； Y是CH ₂且Z是

； Z是CH ₂且Y是

；或 Y是CH ₂且Z是

； W是具有平均分子量選自約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa，50 kDa和60 kDa的PEG基團； q為1、2或3； X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點； 其中式(I)結構在IL-2接合物的胺基酸序列中的位置是選自K34、F41、F43、K42、E61、P64、R37、T40、E67、Y44、V68、和L71。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is substituted with a structure of formula (I):

(I) where: Z is _CH and Y is

; Y is _CH and Z is

; Z is _CH and Y is

; or Y is _CH and Z is

; W is a PEG group having an average molecular weight selected from about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; q is 1, 2 or 3; X has the following structure:

X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue; wherein the structure of formula (I) is at the amine of the IL-2 conjugate The positions in the amino acid sequence are selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71.

In any of the embodiments or variations of formula (I) described herein, the IL2 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 any of the embodiments or variations of formula (I) described herein, X is an L-amino acid.

。 在式(I)的一些具體例中，Z是CH ₂且Y是

。 在式(I)的一些具體例中，Y是CH ₂且Z是

。 In some specific examples of formula (I), Z is _CH2 and Y is. In some specific examples of formula (I), Y is _CH and Z is

. In some specific examples of formula (I), Z is _CH and Y is

. In some specific examples of formula (I), Y is _CH and Z is

.

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

式(Ia)； 其中： Z是CH ₂且Y是

； Y是CH ₂且Z是

； Z是CH ₂且Y是

；或 Y是CH ₂且Z是

； W是具有平均分子量選自約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa，50 kDa及60 kDa的PEG基團；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點； 其中式(I)結構在IL-2接合物的胺基酸序列中的位置是選自K34、F41、F43、K42、E61、P64、R37、T40、E67、Y44、V68、和L71。 In some embodiments of formula (I), q is 1 and the structure of formula (I) is the structure of formula (Ia):

Formula (Ia); wherein: Z is _CH and Y is

; Y is _CH and Z is

; Z is _CH and Y is

; or Y is _CH and Z is

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

X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue; wherein the structure of formula (I) is at the amine of the IL-2 conjugate The positions in the amino acid sequence are selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71.

。 在式(Ia)的一些具體例中，Y是CH ₂且Z是

。 在式(Ia)的一些具體例中，Z是CH ₂且Y是

。 在式(Ia)的一些具體例中，Z是CH ₂且Y是

。 在式(Ia)的一些具體例中，Y是CH ₂且Z是

。 In some specific examples of formula (Ia), Z is _CH and Y is

. In some specific examples of formula (Ia), Y is _CH and Z is

. In some specific examples of formula (Ia), Z is _CH and Y is

. In some specific examples of formula (Ia), Z is _CH and Y is

. In some specific examples of formula (Ia), Y is _CH and Z is

.

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

In some embodiments, the structure of formula (I) is selected from the group consisting of K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71 in the amino acid sequence of the IL-2 conjugate , wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers 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 structure of formula (I) is at the amino group of the IL-2 conjugate. The positions in the acid sequence refer to the positions in SEQ ID NO:3. In some embodiments, the structure of formula (I) is K34 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. In some embodiments, the structure of formula (I) is F41 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. 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 referenced Position in SEQ ID NO:3. In some embodiments, the structure of formula (I) is K42 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. In some embodiments, the structure of formula (I) is E61 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. In some embodiments, the structure of formula (I) is P64 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. In some embodiments, the structure of formula (I) is R37 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. In some embodiments, the structure of formula (I) is T40 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. 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 referenced Position in SEQ ID NO:3. In some embodiments, the structure of formula (I) is Y44 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. In some embodiments, the structure of formula (I) is V68 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3. In some embodiments, the structure of formula (I) is L71 in the amino acid sequence of the IL-2 conjugate, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to SEQ ID NO: Position in 3.

式(II)；

式(III)； W是具有平均分子量選自約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa、50 kDa和60 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 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 formula ( II) and mixtures of formula (III):

formula (II);

Formula (III); W is a PEG group having an average molecular weight selected from the group consisting of 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; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

Here and throughout, structures of formula (II) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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.

(IIa)；

式(IIIa)； 其中： W是具有平均分子量選自約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa、50 kDa和60 kDa的PEG基團；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 In some embodiments, q is 1 and the structures of Formula (II) and Formula (III) are structures of Formula (IIa) and Formula (IIIa):

(IIa);

Formula (IIIa); wherein: W is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa clique; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

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

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

式(II)；

式(III)； 其中： W是具有平均分子量約5 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_PEG5kD]具有式(IIa)或式(IIIa)的結構，或式(IIa)與式(IIIa)的混合物。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kD] has the structure of formula (II) or formula (III), or formula ( Mixtures of II) and formula (III).

formula (II);

Formula (III); wherein: W is a PEG group having an average molecular weight of about 5 kDa; q is 1, 2, or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_PEG5kD] has the 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.

式(II)。 在一些具體例中，q為1且[AzK_PEG5kD]具有式(IIa)的結構。 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).

式(III)。 在一些具體例中，q為1且[AzK_PEG5kD]具有式(IIIa)的結構。 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).

式(II)；

式(III)； 其中： W是具有平均分子量為約30 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q是1且[AzK_PEG30kD]具有式(IIa)或式(IIIa)的結構，或為式(IIa)和式(IIIa)結構的混合物。 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 is of formula A mixture of structures (II) and formula (III).

formula (II);

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

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_PEG30kD] has a structure of formula (IIa) or formula (IIIa), or a mixture of structures of formula (IIa) and formula (IIIa).

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

式(II)。 在一些具體例中，q為1且[AzK_PEG30kD]具有式(IIa)的結構。 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).

式(III)。 在一些具體例中，q為1且[AzK_PEG30kD]具有式(IIIa)的結構。 In some embodiments, the methods use an IL-2 conjugate, wherein [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).

式(II)；

式(III)； 其中： W是具有平均分子量選自5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa，50 kDa、和60 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_PEG]為式(IIa)和式(IIIa)結構的混合物。 In some embodiments, [AzK_PEG] is a mixture of structures of formula (II) and formula (III):

formula (II);

Formula (III); wherein: W is a PEG group having an average molecular weight selected from the group consisting of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa group; q is 1, 2, or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_PEG] is a mixture of structures of formula (IIa) and (IIIa).

In some embodiments, the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) that includes the total amount of [AzK_PEG] in the IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) that includes the total amount of [AzK_PEG] in the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) that includes 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 methoxyPEG group. In some embodiments, the methoxyPEG group is linear or branched. In some embodiments, the methoxyPEG group is linear. In some embodiments, the methoxyPEG groups are branched.

式(II)；

式(III)； 其中： W是具有平均分子量為5 kDa的PEG基團； q是1、2或3； X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_PEG5kD]為式(IIa)和式(IIIa)結構的混合物。 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); wherein: W is a PEG group having an average molecular weight of 5 kDa; q is 1, 2 or 3; X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_PEG5kD] is a mixture of structures of formula (IIa) and (IIIa).

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

式(II)；

式(III)； 其中： W是具有平均分子量為30 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_PEG30kD]為式(IIa)和式(IIIa)結構的混合物。 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); wherein: W is a PEG group having an average molecular weight of 30 kDa; q is 1, 2 or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_PEG30kD] is a mixture of structures of formula (IIa) and (IIIa).

In some embodiments, the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) that includes the total amount of [AzK_PEG30kD] in the IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) that includes the total amount of [AzK_PEG30kD] in the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) that includes 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), wherein W is a linear or branched PEG group group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, W in the structure of formula (II) or formula (III) is a linear PEG group. In some embodiments, W in the structure of formula (II) or formula (III) is a branched PEG group. In some embodiments, W in the structure of formula (II) or formula (III) is a methoxyPEG group. In some embodiments, W in the structure of formula (II) or formula (III) is a linear or branched methoxyPEG group. In some embodiments, the methoxyPEG group in the structure of formula (II) or formula (III) is linear. In some embodiments, the methoxyPEG group in the structure of formula (II) or formula (III) is branched.

式(IV)；

式(V)； 其中： W是具有平均分子量選自5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa，50 kDa、和60 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 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] has the structure of formula (IV) or formula (V), or formula ( Mixtures of IV) and formula (V):

formula (IV);

Formula (V); wherein: W is a PEG group having an average molecular weight selected from the group consisting of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa group; q is 1, 2, or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next 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.

Here and throughout, structures of formula (VI) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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.

式(IVa)；

式(Va)； 其中： W是具有平均分子量選自約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、 35 kDa、40 kDa、45 kDa，50 kDa和60 kDa的PEG基團；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 In some embodiments, q is 1 and the structures of formula (IV) and formula (V) are formula (IVa) and formula (Va):

formula (IVa);

Formula (Va); wherein: W is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa clique; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

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

式(IV)。 在一些具體例中，q為1且[AzK_L1_PEG]具有式(IVa)的結構。 In some specific examples, [AzK_L1_PEG] has the structure of formula (IV):

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

In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO:40. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (IV) 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 the structure of formula (IV) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (IV) 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 the structure of formula (IV) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (IV) 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 the structure of formula (IV) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (IV) 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:44. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (IV) is a PEG group having an average molecular weight of about 30 kDa.

式(V)。 在一些具體例中，q為1且[AzK_L1_PEG]具有式(V)的結構。 In some specific examples, [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 the structure of formula (V) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (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 the structure of formula (V) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (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 the structure of formula (V) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (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 the structure of formula (V) is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight selected from about 5 kDa and 30 kDa. In some embodiments, W in the structure of formula (V) is a PEG group having an average molecular weight of about 5 kDa. In some embodiments, W in the structure of formula (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] contains an amino acid sequence having an average molecular weight selected from about PEG groups of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an amino acid sequence having an average molecular weight of about 5 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an amino acid sequence having an average molecular weight of about 10 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an amino acid sequence having an average molecular weight of about 15 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an amino acid sequence having an average molecular weight of about 20 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an average molecular weight of about 25 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an average molecular weight of about 30 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an average molecular weight of about 35 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an average molecular weight of about 40 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an average molecular weight of about 45 kDa PEG group. 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, [AzK_L1_PEG] contains an average molecular weight of about 50 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an average molecular weight of about 60 kDa PEG group. In some embodiments, the IL-2 conjugate has an amino acid sequence selected from any one of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] contains an amino acid sequence having an average molecular weight selected from about PEG groups of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa, and the PEG group is a methoxy PEG group , straight chain methoxy PEG groups, or branched chain methoxy PEG groups.

式(IV)；

式(V)； 其中： W是具有平均分子量約5 kDa的PEG基團；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_L1_PEG5kD]具有式(IVa)或式(Va)的結構，或為式(IVa)與式(Va)的混合物。 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] has the structure of formula (IV) or formula (V), or is of formula Mixtures of (IV) and formula (V):

formula (IV);

Formula (V); wherein: W is a PEG group having an average molecular weight of about 5 kDa; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_L1_PEG5kD] has 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.

式(IV)。 在一些具體例中，q為1且[AzK_L1_PEG5kD]具有式(IVa)的結構。 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 of formula (IVa).

式(V)。 在一些具體例中，q為1且[AzK_L1_PEG5kD]具有式(Va)的結構。 In some specific examples, [AzK_L1_PEG5kD] has the structure of formula (V)

Formula (V). In some embodiments, q is 1 and [AzK_L1_PEG5kD] has a structure of formula (Va).

式(IV)；

式(V)； 其中： W是具有平均分子量約30 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_L1_PEG30kD]具有式(IVa)或式(Va)的結構，或為式(IVa)和式(Va)結構的混合物。 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 is of formula Mixtures of (IV) and formula (V) structures:

formula (IV);

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

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_L1_PEG30kD] has a structure of formula (IVa) or formula (Va), or a mixture of structures of formula (IVa) and 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.

式(IV)。 在一些具體例中，q為1且[AzK_L1_PEG30kD]具有式(IVa)的結構。 In some specific examples, [AzK_L1_PEG30kD] has the structure of formula (IV):

Formula (IV). In some embodiments, q is 1 and [AzK_L1_PEG30kD] has the structure of formula (IVa).

式(V)。 在一些具體例中，q為1且[AzK_L1_PEG30kD]具有式(Va)的結構。 In some specific examples, [AzK_L1_PEG30kD] has the structure of formula (V):

Formula (V). In some embodiments, q is 1 and [AzK_L1_PEG30kD] has a structure of formula (Va).

式(IV)；

式(V)； 其中： W是具有平均分子量選自約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa、50 kDa和60 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 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); wherein: W is a PEG group having an average molecular weight selected from the group consisting of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa group; q is 1, 2, or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

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

式(IV)；

式(V)； 其中： W是具有平均分子量為5 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_L1_PEG5kD]為式(IVa)和式(Va)結構的混合物。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of any one of SEQ ID NOs: 45 to 49, wherein [AzK_L1_PEG5kD] is a mixture of structures of formula (IV) and formula (V):

formula (IV);

Formula (V); wherein: W is a PEG group having an average molecular weight of 5 kDa; q is 1, 2, or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_L1_PEG5kD] is a mixture of structures of formula (IVa) and formula (Va).

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

式(IV)；

式(V)； 其中： W是具有平均分子量為30 kDa的PEG基團； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 在一些具體例中，q為1且[AzK_L1 PEG30kD]為式(IVa)和式(Va)結構的混合物。 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); wherein: W is a PEG group having an average molecular weight of 30 kDa; q is 1, 2, or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. In some embodiments, q is 1 and [AzK_L1 PEG30kD] is a mixture of structures of formula (IVa) and formula (Va).

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

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

In some embodiments, the IL-2 conjugates described herein comprise a structure of formula (IV) or formula (V), or a mixture of formula (IV) or formula (V), wherein W is a linear or branched PEG group. In some embodiments, W in the structure of formula (IV) or formula (V) is a linear PEG group. In some embodiments, W in the structure of formula (IV) or formula (V) is a branched PEG group. In some embodiments, W in the structure of formula (IV) or formula (V) is a methoxyPEG group. In some embodiments, W in the structure of formula (IV) or formula (V) is a linear or branched methoxyPEG group. In some embodiments, the methoxyPEG group in the structure of formula (IV) or formula (V) is linear. In some embodiments, the methoxyPEG group in the structure of formula (IV) or formula (V) is branched.

For the IL-2 conjugates used in the methods described herein, exemplary structures of methoxyPEG groups are listed in the mPEG-DBCO structure below.

式(VI)；

式(VII)； 其中： n是約2至約5000範圍內的整數； q是1、2或3；且 X具有以下結構：

X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a compound of formula (VI) or (VII) Structure, or a mixture of (VI) and formula (VII) substituted:

formula (VI);

Formula (VII); wherein: n is an integer in the range of about 2 to about 5000; q is 1, 2, or 3; and X has the following structure:

X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

In some embodiments of formula (VI) or formula (VII), or mixtures of formula (VI) or formula (VII), q is 1. In some embodiments of formula (VI) or formula (VII), or mixtures of formula (VI) or formula (VII), q is 2. In some embodiments of formula (VI) or formula (VII), or mixtures of formula (VI) or formula (VII), q is 3.

Here and throughout, structures of formula (VI) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout, structures of formula (VII) include pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

式(VIa)；

式(VIIa)； 其中： n是約2至約5000範圍內的整數； X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 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); wherein: n is an integer in the range of about 2 to about 5000; X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

In some embodiments, n in compounds of formula (VI) and (VII) ranges from about 5 to about 4600, or about 10 to about 4000, or about 20 to about 3000, or about 100 to about 3000, or 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 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, n in compounds of formula (VI) and (VII) is selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568 , 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478 ,1589,1590,1591,1703,1704,1705,1817,1818,1819,1930,1931,1932,2044,2045,2046,2158,2159,2160,2271,2272,2273,2839,2840,2841,2953 , 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546 integers.

In some embodiments, the position of the structure of formula (VI), formula (VII), or the mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is with reference to that in SEQ ID NO:3 Location. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is Selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location K34. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location F41. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location F43. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location K42. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location E61. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location P64. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location R37. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location T40. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location E67. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position Y44. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At position V68. In some embodiments, the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location L71.

In some embodiments, the ratio of the amount of the structure of formula (VI) including the total amount of IL-2 conjugate to the amount of the structure of formula (VII) is about 1:1. In some embodiments, the ratio of the amount of the structure of formula (VI) including the total amount of IL-2 conjugate to the amount of the structure of formula (VII) is greater than 1:1. In some embodiments, the ratio of the amount of the structure of formula (VI) including the total amount of IL-2 conjugate to the amount of the structure of formula (VII) is less than 1:1.

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

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VI) or (VII) , or a mixture of (VI) and (VII) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from E61 and P64, and 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 (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VI) or (VII) , or a mixture of (VI) and (VII) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is E61, and wherein n is the following integer: 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 (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VI) or (VII) , or a mixture of (VI) and (VII) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is P64, and wherein n is the following integer: 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 (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, n in the structures of formula (VI) and (VII) is an integer such that the molecular weight of the PEG moiety is in the range of about 1,000 Daltons to about 200,000 Daltons, or about 2,000 Daltons To about 150,000 Daltons, or about 3,000 Daltons to about 125,000 Daltons, or about 4,000 Daltons to about 100,000 Daltons, or about 5,000 Daltons to about 100,000 Daltons, or about 6,000 Daltons 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 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 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 Daltons, about 9,000 Daltons to about 40,000 Daltons, or about 9,000 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 30,000 Daltons, or about 9,500 Daltons to about 35,000 Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons To about 40,000 Daltons, or about 10,000 Daltons to about 35,000 Daltons, or about 10,000 Daltons to about 30,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 15,000 Daltons 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 about 20,000 Daltons to about 45,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons, or about 20,000 Daltons to about 35,000 Daltons, Or about 20,000 Daltons to about 30,000 Daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VI) or (VII) , or a mixture of (VI) and (VII), where n is an integer such that the PEG moiety has a molecular weight of about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons, approximately 35,000 Daltons, approximately 40,000 Daltons, approximately 45,000 Daltons, approximately 50,000 Daltons, approximately 60,000 Daltons, approximately 70,000 Daltons Daltons, approximately 80,000 Daltons, approximately 90,000 Daltons, approximately 100,000 Daltons, approximately 125,000 Daltons, approximately 150,000 Daltons, approximately 175,000 Daltons, or approximately 200,000 Daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VI) or (VII) , or a mixture of (VI) and (VII), where n is an integer such that the PEG moiety has a molecular weight of about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

式(VIII)；

式(IX)； 其中： n是約2至約5000範圍內的整數； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a compound of formula (VIII) or formula (IX). Structure, or a mixture of formula (VIII) and formula (IX) substituted:

formula (VIII);

Formula (IX); wherein: n is an integer in the range of about 2 to about 5000; q is 1, 2, or 3; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

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

Here and throughout, structures of formula (VIII) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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.

式(VIIIa)；

式(IXa)； 其中： n是約2至約5000範圍內的整數；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 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); wherein: n is an integer in the range of about 2 to about 5000; and X has the structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

In some embodiments, n in compounds of formula (VIII) and (IX) ranges from about 5 to about 4600, or about 10 to about 4000, or about 20 to about 3000, or about 100 to about 3000, or 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 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, n in compounds of formula (VIII) and (IX) is selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568 , 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478 ,1589,1590,1591,1703,1704,1705,1817,1818,1819,1930,1931,1932,2044,2045,2046,2158,2159,2160,2271,2272,2273,2839,2840,2841,2953 , 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546 integers.

In some embodiments, the position of the structure of formula (VIII), formula (IX), or the mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the structures of formula (VIII), formula (IX), or mixtures thereof are in the amino acid sequence of the IL-2 conjugate The positions are referenced to the positions in SEQ ID NO:3. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is Selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location K34. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location F41. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location F43. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location K42. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location E61. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location P64. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location R37. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location T40. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location E67. In some embodiments, the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate 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 (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 (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is At location L71.

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

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VIII) or (IX) , or a mixture of formula (VIII) and formula (IX), wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from the group consisting of F41, F43, K42, E61, and P64, and wherein n is the following integer : 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 (VIII) and (IX) is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022 , 1023, 1135, 1136, 1137, and 1249 integers.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VIII) or (IX) , or a mixture of formula (VIII) and formula (IX), wherein the substituted amino acid residue in SEQ ID NO: 3 is selected from E61 and P64, and 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 (VIII) and (IX) is an integer selected from the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VIII) or (IX) , or a mixture of formula (VIII) and formula (IX) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is E61, and wherein n is the following integer: 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 (VIII) and (IX) is an integer selected from the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (VIII) or (IX) , or a mixture of formula (VIII) and formula (IX) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is P64, and wherein n is the following integer: 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 (VIII) and (IX) is an integer selected from the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a compound of formula (VIII) or formula (IX). structure, or a mixture of (VIII) and formula (IX) substituted, wherein n is an integer such that the molecular weight of the PEG moiety is in the range of about 1,000 Daltons to about 200,000 Daltons, or about 2,000 Daltons to About 150,000 Daltons, or about 3,000 Daltons to about 125,000 Daltons, or about 4,000 Daltons to about 100,000 Daltons, or about 5,000 Daltons to about 100,000 Daltons, or about 6,000 Daltons 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 Daltons to about 65,000 Daltons, or about 5,000 Daltons to about 60,000 Daltons, or about 5,000 Daltons to about 50,000 Daltons, or about 6,000 Daltons to about 50,000 Daltons, or About 7,000 Daltons to about 50,000 Daltons, or about 7,000 Daltons to about 45,000 Daltons, or about 7,000 Daltons to about 40,000 Daltons, or about 8,000 Daltons to about 40,000 Daltons , or about 8,500 Daltons to about 40,000 Daltons, or about 8,500 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 50,000 Daltons, or about 9,000 Daltons to about 45,000 Daltons Daltons, about 9,000 Daltons to about 40,000 Daltons, or about 9,000 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 30,000 Daltons, or about 9,500 Daltons to about 35,000 Daltons Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons to About 40,000 Daltons, or about 10,000 Daltons to about 35,000 Daltons, or about 10,000 Daltons to about 30,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 15,000 Daltons 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 Daltons to about 50,000 Daltons, or about 20,000 Daltons to about 45,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons, or about 20, 000 Daltons to about 35,000 Daltons, or about 20,000 Daltons to about 30,000 Daltons. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a compound of formula (VIII) or formula (IX). structure, or a mixture of formula (VIII) and formula (IX) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Daltons, approximately 20,000 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons, approximately 35,000 Daltons, approximately 40,000 Daltons, approximately 45,000 Daltons, approximately 50,000 Daltons, approximately 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 at least one amino acid residue in the IL-2 conjugate is represented by a compound of formula (VIII) or formula (IX). structure, or a mixture of formula (VIII) and formula (IX) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Dalton, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

式(X)；

式(XI)； 其中： n是約2至約5000範圍內的整數； q是1、2或3；且 波浪線表示與SEQ ID NO：3內未被取代的胺基酸殘基的共價鍵。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (X) or formula (XI) structure, or a mixture of formula (X) and formula (XI) substituted:

formula (X);

Formula (XI); wherein: n is an integer in the range of about 2 to about 5000; q is 1, 2, or 3; and the wavy line represents covalentity with the unsubstituted amino acid residue within SEQ ID NO:3 key.

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

Here and throughout, structures of formula (X) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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.

式(Xa)；

式(XIa)； 其中： n是約2至約5000範圍內的整數；且 波浪線表示與SEQ ID NO：3內未被取代的胺基酸殘基的共價鍵。 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); wherein: n is an integer in the range of about 2 to about 5000; and the wavy line represents a covalent bond to the unsubstituted amino acid residue within SEQ ID NO:3.

In some embodiments, the stereochemistry of the chiral centers within formula (X) and formula (XI) is racemic, (R) rich, (S) rich, substantially (R), substantially Is (S), is (R) or (S). In some embodiments, the stereochemistry of the chiral centers within Formula (X) and Formula (XI) is racemic. In some embodiments, the stereochemistry of the chiral centers within formula (X) and formula (XI) is (R) rich. In some embodiments, the stereochemistry of the chiral centers within Formula (X) and Formula (XI) is (S) rich. In some embodiments, the stereochemistry of the chiral centers within formula (X) and formula (XI) is substantially (R). In some embodiments, the stereochemistry of the chiral centers within formula (X) and formula (XI) is substantially (S). In some embodiments, the stereochemistry of the chiral center within Formula (X) and Formula (XI) is (R). In some embodiments, the stereochemistry of the chiral center within Formula (X) and Formula (XI) is (S).

In some embodiments, n in compounds of formula (X) and (XI) ranges from about 5 to about 4600, or about 10 to about 4000, or about 20 to about 3000, or about 100 to about 3000, or 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 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, n in compounds of formula (X) and (XI) is selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568 , 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478 ,1589,1590,1591,1703,1704,1705,1817,1818,1819,1930,1931,1932,2044,2045,2046,2158,2159,2160,2271,2272,2273,2839,2840,2841,2953 , 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546 integers.

In some embodiments, the position of the structure of formula (X), formula (XI), or the mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41 , F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the structure of formula (X), formula (XI), or a mixture thereof is in the amino acid sequence of the IL-2 conjugate The positions of are referenced to the positions in SEQ ID NO:3. 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 selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location 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 location 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 location 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 location 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 location E61. In some embodiments, the position of the structure of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position P64. In some embodiments, the position of the structure of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location 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 location E67. In some embodiments, the position of the structure of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position Y44. In some embodiments, the position of the structure of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is 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 location L71.

In some embodiments, the ratio of the amount of the structure of formula (X) including the total amount of IL-2 conjugate to the amount of the structure of formula (XI) is about 1:1. In some embodiments, the ratio of the amount of the structure of formula (X) including the total amount of IL-2 conjugate to the amount of the structure of formula (XI) is greater than 1:1. In some embodiments, the ratio of the amount of the structure of formula (X) including the total amount of IL-2 conjugate to the amount of the structure of formula (XI) is less than 1:1.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (X), formula (XI) Structure, or a mixture of Formula (X) and Formula (XI) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from the group consisting of F41, F43, K42, E61, and P64, and wherein n is the following Integer: 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 selected from Integers of 1022, 1023, 1135, 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (X), formula (XI) Structure, or a mixture of formula (X) and formula (XI) substitution, wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from E61 and P64, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 .

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (X), formula (XI) Structure, or a mixture of formula (X) and formula (XI) substitution, wherein the substituted amino acid residue in SEQ ID NO: 3 is E61, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (X), formula (XI) Structure, or a mixture of formula (X) and formula (XI) substitution, wherein the amino acid residue substituted in SEQ ID NO: 3 is P64, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, n in the structures of Formula (X) and Formula (XI) is an integer such that the molecular weight of the PEG moiety is in the range of about 1,000 Daltons to about 200,000 Daltons, or about 2,000 Daltons Daltons to about 150,000 Daltons, or about 3,000 Daltons to about 125,000 Daltons, or about 4,000 Daltons to about 100,000 Daltons, or about 5,000 Daltons to about 100,000 Daltons, or about 6,000 Daltons 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 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 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, about 9,000 Daltons to about 40,000 Daltons, or about 9,000 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 30,000 Daltons, or about 9,500 Daltons to About 35,000 Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons 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 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 about 20,000 Daltons to about 45,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons, or about 20,000 Daltons to about 35,000 Daltons , or from about 20,000 Daltons to about 30,000 Daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (X), formula (XI) Structure, or a mixture of Formula (X) and Formula (XI) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Daltons, approximately 20,000 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons, approximately 35,000 Daltons, approximately 40,000 Daltons, approximately 45,000 Daltons, approximately 50,000 Daltons, approximately 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 at least one amino acid residue in the IL-2 conjugate is defined by formula (X), formula (XI) Structure, or a mixture of Formula (X) and Formula (XI) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Dalton, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

式(XII)；

式(XIII)； 其中： n是約2至約5000範圍內的整數； q是1、2或3；且 波浪線表示與SEQ ID NO：3內未被取代的胺基酸殘基的共價鍵。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a compound of formula (XII) or formula (XIII) structure, or a mixture of formula (XII) and formula (XIII) substituted:

Formula (XII);

Formula (XIII); wherein: n is an integer in the range of about 2 to about 5000; q is 1, 2, or 3; and the wavy line represents covalentity with the unsubstituted amino acid residue within SEQ ID NO:3 key.

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

Here and throughout, structures of formula (XII) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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.

式(XIIa)；

式(XIIIa)； 其中： n是約2至約5000範圍內的整數；且 波浪線表示與SEQ ID NO：3內未被取代的胺基酸殘基的共價鍵。 In some embodiments, q is 1 and the structures of Formula (XII) and Formula (XIII) are structures of Formula (XIIa) and Formula (XIIIa):

Formula (XIIa);

Formula (XIIIa); wherein: n is an integer in the range of about 2 to about 5000; and the wavy line represents a covalent bond to the unsubstituted amino acid residue within SEQ ID NO:3.

In some embodiments, the stereochemistry of the chiral centers within formula (XII) and formula (XIII) is racemic, (R) rich, (S) rich, substantially (R), substantially Is (S), is (R), or (S). In some embodiments, the stereochemistry of the chiral centers within Formula (XII) and Formula (XIII) is racemic. In some embodiments, the stereochemistry of the chiral centers within Formula (XII) and Formula (XIII) is (R) rich. In some embodiments, the stereochemistry of the chiral centers within Formula (XII) and Formula (XIII) is (S) rich. In some embodiments, the stereochemistry of the chiral centers within Formula (XII) and Formula (XIII) is substantially (R). In some embodiments, the stereochemistry of the chiral centers within Formula (XII) and Formula (XIII) is substantially (S). In some embodiments, the stereochemistry of the chiral center within Formula (XII) and Formula (XIII) is (R). In some embodiments, the stereochemistry of the chiral center within Formula (XII) and Formula (XIII) is (S).

In some embodiments, n in compounds of formula (XII) and formula (XIII) ranges from about 5 to about 4600, or about 10 to about 4000, or about 20 to about 3000, or about 100 to about 3000, or 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 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, n in compounds of formula (XII) and formula (XIII) is selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, Integers of 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

In some embodiments, the position of the structure of formula (XII), formula (XIII), or mixtures thereof 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 structure of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) is in the amino acid sequence of the IL-2 conjugate The positions of are referenced to the positions in SEQ ID NO:3. In some embodiments, the position of the structure of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location 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 location 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 location F43. In some embodiments, the position of the structure of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location 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 location E61. In some embodiments, the position of the structure of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is 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 location R37. In some embodiments, the position of the structure of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is 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 location E67. In some embodiments, the position of the structure of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is 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 location L71.

In some embodiments, the ratio of the amount of structure of formula (XII) to the amount of structure of formula (XIII) that make up the total amount of IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of structure of formula (XII) to the amount of structure of formula (XIII) that make up the total amount of IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the amount of structure of formula (XII) to the amount of structure of formula (XIII) that make up 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 at least one amino acid residue in the IL-2 conjugate is defined by formula (XII), formula (XIII) Structure, or a mixture of formula (XII) and formula (XIII) substitution, wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from the group consisting of K34, F41, F43, K42, E61, P64, R37, T40 , E67, Y44, V68, and L71, wherein n is 100 to about 1150, or about 100 to about 1100, or about 100 to about 1000, or about 100 to about 900, or about 100 to about 750, or about 100 to about 700, or about 100 to about 600, or about 100 to about 575, or about 100 to about 500, or about 100 to about 450, or about 100 to about 350, or about 100 to about 275, or about 100 to about 230, or about 150 to about 475, or about 150 to about 340, or about 113 to about 340, or about 450 to about 800, or about 454 to about 796, or about 454 to about 682, or about 340 to about 795 , or about 341 to about 682, or about 568 to about 909, or about 227 to about 1500, or about 225 to about 2280, or about 460 to about 2160, or about 460 to about 2050, or about 341 to about 1820, or about 341 to about 1710, or about 341 to about 1250, or about 225 to about 1250, or about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 An integer from about 690, or from about 114 to about 575. In some embodiments, n in compounds of formula (XII) and formula (XIII) is selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, Integers of 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XII), formula (XIII) Structure, or a mixture substitution of formula (XII) and formula (XIII), wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from the group consisting of F41, F43, K42, E61, and P64, and wherein n is the following Integer: 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 (XII) and formula (XIII) is selected from Integers of 1022, 1023, 1135, 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (XII), (XIII) , or a mixture of formula (XII) and formula (XIII), wherein the substituted amino acid residues in SEQ ID NO: 3 are selected from E61 and P64, and 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 (XII) and formula (XIII) is an integer selected from the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 .

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XII), formula (XIII) Structure, or a mixture of Formula (XII) and Formula (XIII) substituted, wherein the substituted amino acid residue in SEQ ID NO: 3 is E61, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XII), formula (XIII) Structure, or a mixture of Formula (XII) and Formula (XIII) substituted, wherein the substituted amino acid residue in SEQ ID NO: 3 is P64, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, n in the structure of Formula (XII) or Formula (XIII) is an integer such that the molecular weight of the PEG moiety is in the range of about 1,000 Daltons to about 200,000 Daltons, or about 2,000 Daltons Daltons to about 150,000 Daltons, or about 3,000 Daltons to about 125,000 Daltons, or about 4,000 Daltons to about 100,000 Daltons, or about 5,000 Daltons to about 100,000 Daltons, or about 6,000 Daltons 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 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 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, about 9,000 Daltons to about 40,000 Daltons, or about 9,000 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 30,000 Daltons, or about 9,500 Daltons to About 35,000 Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons 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 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 about 20,000 Daltons to about 45,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons, or about 20,000 Daltons to about 35,000 Daltons , or from about 20,000 Daltons to about 30,000 Daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XII), formula (XIII) Structure, or a mixture of Formula (XII) and Formula (XIII) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Daltons, approximately 20,000 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons, approximately 35,000 Daltons, approximately 40,000 Daltons, approximately 45,000 Daltons, approximately 50,000 Daltons, approximately 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 at least one amino acid residue in the IL-2 conjugate is defined by formula (XII), formula (XIII) Structure, or a mixture of Formula (XII) and Formula (XIII) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Dalton, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

式(VIII)；

式(IX)； 其中： n是一個整數，使得PEG基團的分子量為約15,000道耳頓至約60,000道耳頓； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is represented by formula (VIII) or formula ( IX), or a mixture of formula (VIII) and formula (IX) substituted:

formula (VIII);

Formula (IX); wherein: 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; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

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

Here and throughout, structures of formula (VIII) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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.

式(VIIIa)；

式(IXa)； 其中： n為一個整數，使得PEG基團的分子量為約15,000道耳頓至約60,000道耳頓；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 In some embodiments, q is 1 and the structures of Formula (VIII) and Formula (IX) are those of Formula (VIIIa) and Formula (IXa):

formula (VIIIa);

Formula (IXa); wherein: n is an integer such that the molecular weight of the PEG group is from about 15,000 Daltons to about 60,000 Daltons; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

In some embodiments, the amino acid residue at E61 in the IL-2 conjugate is substituted with a structure of formula (VIII) or formula (IX), or a mixture of formula (VIII) and formula (IX), and where n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 30,000 Daltons. In some embodiments, the amino acid residue at P64 in the IL-2 conjugate is substituted with a structure of formula (VIII) or formula (IX), or a mixture of formula (VIII) and formula (IX), and where n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 30,000 Daltons.

式(VIII)；

(IX)； 其中： n為一個整數，使得PEG基團的分子量為約15,000道耳頓至約60,000道耳頓； q是1、2或3；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at E61 or P64 in the IL-2 conjugate is represented by formula (VIII) or (IX) ), or a mixture of formula (VIII) and formula (IX) substituted:

formula (VIII);

(IX); wherein: 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; and X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue.

In some embodiments, the amino acid residue at E61 in the IL-2 conjugate is substituted with a structure of formula (VIII) or formula (IX), or a mixture of formula (VIII) and formula (IX), and where n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 30,000 Daltons. In some embodiments, the amino acid residue at P64 in the IL-2 conjugate is substituted with a structure of formula (VIII) or formula (IX), or a mixture of formula (VIII) and formula (IX), and where n is an integer such that the molecular weight of the PEG group is from about 20,000 Daltons to about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is 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, wherein at least one amino acid in the IL-2 conjugate The residue was replaced with a cysteine covalently bonded to the PEG group. In some embodiments, the PEG group has a molecular weight selected from the group consisting of 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 about 20 kDa. In some embodiments, the PEG group has a molecular weight of 25 kDa. In some embodiments, the PEG group has a molecular weight of 30 kDa. In some embodiments, the PEG group has a molecular weight of 35 kDa. In some embodiments, the PEG group has a molecular weight of 40 kDa. In some embodiments, the PEG group has a molecular weight of 45 kDa. In some embodiments, the PEG group has a molecular weight of 50 kDa. In some embodiments, the PEG group has a molecular weight of 60 kDa. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue substituted with cysteine in the IL-2 conjugate is selected from K34 , T36, R37, T40, F41, K42, F43, Y44, E60, E61, E67, K63, P64, V68, L71, and Y106. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, and at least one amino acid residue substituted with cysteine in the IL-2 conjugate is selected from K34 , T40, F41, K42, Y44, E60, E61, E67, K63, P64, V68, and L71. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 4, and at least one amino acid residue substituted with cysteine in the IL-2 conjugate is selected from K35 , T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one non-lysine residue is substituted 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 bonded via a non-releasable linkage. In some embodiments, the IL-2 conjugate comprises a non-releasable, covalently bonded PEG group.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the non-lysine amino acid in the IL-2 conjugate is substituted with a lysine residue, and wherein the lysine amino acid is substituted with a lysine residue. Amino acid residues comprise one or more water-soluble polymers and covalent linkers. In some embodiments, the lysine residues are located in the K34-Y106 region of SEQ ID NO:3. In some embodiments, the lysine residue is located at K34. In some embodiments, the lysine residue is located at F41. In some embodiments, the lysine residue is located at F43. In some embodiments, the lysine residue is located at K42. In some embodiments, the lysine residue is located at E61. In some embodiments, the lysine residue is located at P64. In some embodiments, the lysine residue is located at R37. In some embodiments, the lysine residue is located at T40. In some embodiments, the lysine residue is located at E67. In some embodiments, the lysine residue is located at Y44. In some embodiments, the lysine residue is located at V68. In some embodiments, the lysine residue is located at L71.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the non-lysine amino acid in the amino acid sequence of the IL-2 conjugate is comprised of the following amino group Acid substitution: (a) lysine acid; (b) covalent linker; and (3) and one or more water-soluble polymers. In some embodiments, the one or more water-soluble polymers comprise PEG groups.

式(XIV)；

式(XV)； 其中： m是0到20的整數； p是0到20的整數； n是約2至約5000範圍內的整數；且 波浪線表示與SEQ ID NO：3內未被取代的胺基酸殘基的共價鍵。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) structure, or a mixture of formula (XIV) and formula (XV) substituted:

Formula (XIV);

formula (XV); wherein: m is an integer from 0 to 20; p is an integer from 0 to 20; n is an integer in the range of about 2 to about 5000; Covalent bonds of amino acid residues.

Here and throughout, structures of formula (XIV) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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 the chiral centers within formula (XIV) and formula (XV) is racemic, (R) rich, (S) rich, substantially (R), substantially Is (S), is (R), or (S). In some embodiments, the stereochemistry of the chiral centers within Formula (XIV) and Formula (XV) is racemic. In some embodiments, the stereochemistry of the chiral centers within Formula (XIV) and Formula (XV) is (R) rich. In some embodiments, the stereochemistry of the chiral centers within Formula (XIV) and Formula (XV) is (S) rich. In some embodiments, the stereochemistry of the chiral centers within Formula (XIV) and Formula (XV) is substantially (R). In some embodiments, the stereochemistry of the chiral centers within formula (XIV) and formula (XV) is substantially (S). In some embodiments, the stereochemistry of the chiral center within Formula (XIV) and Formula (XV) is (R). In some embodiments, the stereochemistry of the chiral center within 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 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 compounds of formula (XIV) and formula (XV) is 2. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 3. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 4. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 5. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 6. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 7. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 8. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 9. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 10. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 11. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 12. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 13. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 14. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 15. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 16. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 17. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 18. In some embodiments, m in compounds of formula (XIV) and formula (XV) is 19. In some embodiments, m in 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 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 in compounds of formula (XIV) and formula (XV) is 1. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 2. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 3. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 4. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 5. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 6. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 7. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 8. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 9. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 10. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 11. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 12. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 13. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 14. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 15. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 16. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 17. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 18. In some embodiments, p in compounds of formula (XIV) and formula (XV) is 19. In some embodiments, p in 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 compounds of formula (XIV) and formula (XV) is in the range of about 5 to about 4600, or about 10 to About 4000, or about 20 to about 3000, or about 100 to about 3000, or 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 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 compounds of formula (XIV) and formula (XV) is an integer from 1 to 6, and p is 1 to 6 an integer, and n is selected from Integers of 1023, 1135, 1136, and 1137. In some specific examples of 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 selected from 113, 114, 227, 228, 340, 341, Integers of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of 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 selected from 113, 114, 227, 228, 340, 341, Integers of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 1, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 3, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 4, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 5, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 6, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 7, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 8, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 9, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 10, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 11, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 11, p is 2, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is selected from 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, Integers of 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 compounds of formula (XIV) and formula (XV) is selected from 2, 5, 10, 11, 22 , 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 ,1137,1249,1250,1251,1362,1363,1364,1476,1477,1478,1589,1590,1591,1703,1704,1705,1817,1818,1819,1930,1931,1932,2044,2045,2046 , 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546 integers. In some embodiments, the position of the structure of formula (XIV), formula (XV), or the mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41 , F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the structure of formula (XIV), formula (XV), or the mixture of formula (XIV) and formula (XV) in IL- 2 The positions in the amino acid sequence of the conjugate are referenced to the positions in SEQ ID NO:3. In some embodiments, the position of the structure of formula (XIV), formula (XV), or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of formula (XIV), formula (XV), or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location 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 location 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 location 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 location 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 location 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 location 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 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 location E67. In some embodiments, the position of the structure of formula (XIV), formula (XV), or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is 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 location L71. In some embodiments, the ratio of the amount of the structure of formula (XIV) including the total amount of IL-2 conjugate to the amount of the structure of formula (XV) is about 1:1. In some embodiments, the ratio of the amount of the structure of formula (XIV) including the total amount of IL-2 conjugate to the amount of the structure of formula (XV) is greater than 1:1. In some embodiments, the ratio of the amount of the structure of formula (XIV) including the total amount of IL-2 conjugate to the amount of the structure of formula (XV) is less than 1:1.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is represented by a structure of formula (XIV) or (XV) , or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from the group consisting of F41, F43, K42, E61, and P64, and wherein n is the following integer : 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 selected from the group consisting of Integers of 1022, 1023, 1135, 1136, 1137, and 1249.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of Formula (XIV) and Formula (XV) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from E61 and P64, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 .

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of Formula (XIV) and Formula (XV) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is E61, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of Formula (XIV) and Formula (XV) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is P64, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of Formula (XIV) and Formula (XV) substituted where n is an integer such that the molecular weight of the PEG moiety is in the range of about 1,000 Daltons to about 200,000 Daltons, or about 2,000 Daltons To about 150,000 Daltons, or about 3,000 Daltons to about 125,000 Daltons, or about 4,000 Daltons to about 100,000 Daltons, or about 5,000 Daltons to about 100,000 Daltons, or about 6,000 Daltons 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 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 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 Daltons, about 9,000 Daltons to about 40,000 Daltons, or about 9,000 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 30,000 Daltons, or about 9,500 Daltons to about 35,000 Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons To about 40,000 Daltons, or about 10,000 Daltons to about 35,000 Daltons, or about 10,000 Daltons to about 30,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 15,000 Daltons 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 about 20,000 Daltons to about 45,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons, or about 20,0 00 Daltons to about 35,000 Daltons, or about 20,000 Daltons to about 30,000 Daltons. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of formula (XIV) and formula (XV) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Daltons, approximately 20,000 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons, approximately 35,000 Daltons, approximately 40,000 Daltons, approximately 45,000 Daltons, approximately 50,000 Daltons, approximately 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 at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of formula (XIV) and formula (XV) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons Dalton, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of Formula (XIV) and Formula (XV) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from F41, F43, K42, E61, and P64, and m is 1 to 6 An integer of , p is an integer from 1 to 6, and 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, some embodiments of compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794 , 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249 integers.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of formula (XIV) and formula (XV) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is selected from E61 and P64, and wherein m is an integer from 1 to 6 and p 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 specific examples of compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, Integers of 908, 909, and 910.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of formula (XIV) and formula (XV) substituted, wherein the substituted amino acid residue in SEQ ID NO: 3 is E61, and wherein m is an integer from 1 to 6 and p is 1 to 6 integer, and n is the following integer: 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 specific examples of compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, Integers of 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XIV) or formula (XV) Structure, or a mixture of Formula (XIV) and Formula (XV) substituted, wherein the amino acid residue substituted in SEQ ID NO: 3 is P64, and wherein m is an integer from 1 to 6 and p is 1 to 6 integer, and n is the following integer: 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 specific examples of compounds of formula (XIV) and formula (XV), m is 2, p is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, Integers of 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

式(XVI)；

式(XVII)； 其中： m是0到20的整數； n是約2至約5000範圍內的整數；且 波浪線表示與SEQ ID NO：3內未被取代的胺基酸殘基的共價鍵。 In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XVI) or formula (XVII) structure, or a mixture of formula (XVI) and formula (XVII) substituted:

Formula (XVI);

Formula (XVII); wherein: m is an integer from 0 to 20; n is an integer in the range of about 2 to about 5000; and the wavy line represents covalentity with the unsubstituted amino acid residue within SEQ ID NO:3 key.

Here and throughout, structures of formula (XVI) include pharmaceutically acceptable salts, solvates, or hydrates thereof. Here 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 the chiral centers within Formula (XVI) and Formula (XVII) is racemic, (R) rich, (S) rich, substantially (R), substantially Is (S), is (R) or (S). In some embodiments, the stereochemistry of the chiral centers within Formula (XVI) and Formula (XVII) is racemic. In some embodiments, the stereochemistry of the chiral centers within Formula (XVI) and Formula (XVII) is (R) rich. In some embodiments, the stereochemistry of the chiral centers within Formula (XVI) and Formula (XVII) is (S) rich. In some embodiments, the stereochemistry of the chiral centers within Formula (XVI) and Formula (XVII) is substantially (R). In some embodiments, the stereochemistry of the chiral centers within Formula (XVI) and Formula (XVII) is substantially (S). In some embodiments, the stereochemistry of the chiral center within Formula (XVI) and Formula (XVII) is (R). In some embodiments, the stereochemistry of the chiral center within 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 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 in compounds of formula (XVI) and formula (XVII) is 1. 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 in compounds of formula (XVI) and formula (XVII) is 4. In some embodiments, m in compounds of formula (XVI) and formula (XVII) is 5. In some embodiments, m in compounds of formula (XVI) and formula (XVII) is 6. 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 compounds of formula (XVI) and formula (XVII) is in the range of about 5 to about 4600, or about 10 to About 4000, or about 20 to about 3000, or about 100 to about 3000, or 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 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 compounds of formula (XVI) and formula (XVII) is an integer from 1 to 6, and n is selected from the group consisting of 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 the integer. In some specific examples of compounds of formula (XVI) and formula (XVII), m is an integer from 2 to 6, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of compounds of formula (XVI) and formula (XVII), m is an integer from 2 to 4, and n is selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, Integers of 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 1, and n is selected from Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 2, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 3, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 4, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 5, and n is selected from Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 6, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 7, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 8, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 9, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 10, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 11, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 12, and n is selected from the group consisting of Integers of 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some specific examples of compounds of formula (XVI) and formula (XVII), m is 2, and n is selected from 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, Integers of 1135, 1136, and 1137.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein n in compounds of formula (XVI) and formula (XVII) is selected from 2, 5, 10, 11, 22 , 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 ,1137,1249,1250,1251,1362,1363,1364,1476,1477,1478,1589,1590,1591,1703,1704,1705,1817,1818,1819,1930,1931,1932,2044,2045,2046 , 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546 integers. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or the mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41 , F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the structure of formula (XVI), formula (XVII), or the mixture of formula (XVI) and formula (XVII) in IL- 2 The positions in the amino acid sequence of the conjugate are referenced to the positions in SEQ ID NO:3. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location K34. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location 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 location F43. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location 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 location E61. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is 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 location R37. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position T40. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location E67. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position Y44. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position V68. In some embodiments, the position of the structure of formula (XVI), formula (XVII), or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at location L71. In some embodiments, the ratio of the amount of the structure of formula (XVI) including the total amount of IL-2 conjugate to the amount of the structure of formula (XVII) is about 1:1. In some embodiments, the ratio of the amount of the structure of formula (XVI) including the total amount of IL-2 conjugate to the amount of the structure of formula (XVII) is greater than 1:1. In some embodiments, the ratio of the amount of the structure of formula (XVI) including the total amount of IL-2 conjugate to the amount of the structure of formula (XVII) is less than 1:1.

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

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the IL-2 conjugate is represented by a structure of formula (XVI), formula (XVII), or formula (XVI) and The at least one amino acid residue substituted by the mixture of formula (XVII) is selected from F41, F43, K42, E61, and P64, and wherein n is the following integer: 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 (XVII) is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, Integers of 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 the IL-2 conjugate is represented by a structure of formula (XVI), formula (XVII), or formula (XVI) and The at least one amino acid residue substituted by the mixture of formula (XVII) is selected from E61 and P64, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 .

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XVI), formula (XVII) Structure, or a mixture of formula (XVI) and formula (XVII) substituted, which is substituted E61, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XVI), formula (XVII) structure, or a mixture of formula (XVI) and formula (XVII) substituted, which is substituted with P64, and wherein n is the following integer: 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 the group consisting of 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910 . In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XVI), formula (XVII) structure, or a mixture of formula (XVI) and formula (XVII) substituted where n is an integer such that the molecular weight of the PEG moiety is in the range of 1,000 Daltons to about 200,000 Daltons, or about 2,000 Daltons to About 150,000 Daltons, or about 3,000 Daltons to about 125,000 Daltons, or about 4,000 Daltons to about 100,000 Daltons, or about 5,000 Daltons to about 100,000 Daltons, or about 6,000 Daltons 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 Daltons to about 65,000 Daltons, or about 5,000 Daltons to about 60,000 Daltons, or about 5,000 Daltons to about 50,000 Daltons, or about 6,000 Daltons to about 50,000 Daltons, or About 7,000 Daltons to about 50,000 Daltons, or about 7,000 Daltons to about 45,000 Daltons, or about 7,000 Daltons to about 40,000 Daltons, or about 8,000 Daltons to about 40,000 Daltons , or about 8,500 Daltons to about 40,000 Daltons, or about 8,500 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 50,000 Daltons, or about 9,000 Daltons to about 45,000 Daltons Daltons, about 9,000 Daltons to about 40,000 Daltons, or about 9,000 Daltons to about 35,000 Daltons, or about 9,000 Daltons to about 30,000 Daltons, or about 9,500 Daltons to about 35,000 Daltons Daltons, or about 9,500 Daltons to about 30,000 Daltons, or about 10,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 45,000 Daltons, or about 10,000 Daltons to About 40,000 Daltons, or about 10,000 Daltons to about 35,000 Daltons, or about 10,000 Daltons to about 30,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 15,000 Daltons 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 Daltons to about 50,000 Daltons, or about 20,000 Daltons to about 45,000 Daltons, or about 20,000 Daltons to about 40,000 Daltons, or about 2 0,000 Daltons to about 35,000 Daltons, or about 20,000 Daltons to about 30,000 Daltons. In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XVI), formula (XVII) structure, or a mixture of formula (XVI) and formula (XVII) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons Daltons, approximately 20,000 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons, approximately 35,000 Daltons, approximately 40,000 Daltons, approximately 45,000 Daltons, approximately 50,000 Daltons, approximately 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 at least one amino acid residue in the IL-2 conjugate is defined by formula (XVI), formula (XVII) structure, or a mixture of formula (XVI) and formula (XVII) substituted where n is an integer such that the molecular weight of the PEG moiety is about 5,000 daltons, about 7,500 daltons, about 10,000 daltons, about 15,000 daltons Dalton, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is defined by formula (XVI), formula (XVII) structure, or a mixture of formula (XVI) and formula (XVII) substituted, selected from F41, F43, K42, E61, and P64, m is an integer from 1 to 6, and n is an integer from about 450 to about 800, Or about 454 to about 796, or about 454 to about 682, or about 568 to about 909. In some embodiments of compounds of formula (XVI) and formula (XVII), m is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, Integers of 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 the IL-2 conjugate is represented by a structure of formula (XVI), formula (XVII), or formula (XVI) and The at least one amino acid residue substituted by the mixture of formula (XVII) is selected from E61 and P64, and wherein m is an integer from 1 to 6, and n is the following integer: 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 compounds of formula (XVI) and formula (XVII), m is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and an integer of 910.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the IL-2 conjugate is represented by a structure of formula (XVI), formula (XVII), or (XVI) and ( A mixture of XVII) substituted at least one amino acid residue, which is E61, and wherein m is an integer from 1 to 6, and n is the following integer: 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 compounds of formula (XVI) and formula (XVII), m is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and an integer of 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein the IL-2 conjugate is represented by a structure of formula (XVI), formula (XVII), or formula (XVI) and At least one amino acid residue substituted by the mixture of formula (XVII) is P64, and wherein m is an integer from 1 to 6, and n is the following integer: 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 compounds of formula (XVI) and formula (XVII), m is 2, and n is selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and an integer of 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some specific examples, n is about 681.

In some embodiments, the IL-2 conjugate includes SEQ ID NOs: 1-98. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 15-29. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 40-54. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 55-69. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 70-84. In some embodiments, the IL-2 conjugate comprises a structure of formula (I). In some embodiments, the IL-2 conjugate comprises a structure of formula (II). In some embodiments, the IL-2 conjugate comprises a structure of formula (III). In some embodiments, the IL-2 conjugate comprises a structure of formula (IV). In some embodiments, the IL-2 conjugate comprises a structure of formula (V). In some embodiments, the IL-2 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. 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: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 includes 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 the amino acid sequence of any one of SEQ ID NOs: 86, 88, 90, 92, 94, 96, and 98. In any of these embodiments, a structure of formula (I) or any variant thereof (such as formula (II) to formula (XV) or any variant thereof) is incorporated into a site comprising an unnatural amino acid .

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

In some embodiments, the IL-2 conjugate further comprises additional mutations. In some instances, the additional mutation is at an amino acid position selected from the group consisting of K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In this case, the amino acid is conjugated to additional conjugation 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 prior to incorporation into an additional linking moiety.

In some cases, the PEG group is not limited to a particular structure. In some cases, the PEG is linear (eg, end-capped, eg, alkoxy PEG or bifunctional PEG), branched, or multi-armed (eg, forked PEG or PEG attached to a polyol core) , dendritic (or star) configurations, each with or without one or more degradable linkages. Furthermore, the internal structure of water-soluble polymers can be organized in any number of different repeating patterns and can be selected from homopolymers, alternating copolymers, random copolymers, block copolymers, alternating terpolymers, random terpolymers A group consisting of polymers, and block trimers.

PEGs typically contain multiple ( _OCH2CH2 ) monomers [or ( _{CH2CH2O )} monomers, depending _on how PEG is defined]. As used herein, the number of repeating units is identified by the subscript "n" in "( _{OCH2CH2 ) n} ". Accordingly, the value of (n) generally falls within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, about 225 to about 1930, about 450 to About 1930, about 1200 to about 1930, about 568 to about 2727, about 660 to about 2730, about 795 to about 2730, about 795 to about 2730, about 909 to about 2730, and about 1,200 to about 1,900. For any given polymer of known molecular weight, the number of repeating units (ie, "n") can be determined by dividing the total weight average molecular weight of the polymer by the molecular weight of the repeating monomers.

In some cases, the PEG is a terminally capped polymer, ie, a polymer having at least one terminal capped with a relatively inert group, such as a low carbon C _1-6 alkoxy or hydroxyl group. For example, when the polymer is PEG, methoxy-PEG (commonly referred to as mPEG) can be used, which is a linear form of PEG in which one end of the polymer is a methoxy ( _-OCH3 ) group, and The other terminus is a hydroxyl group or other functional group optionally chemically modified.

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

In some embodiments, the PEG groups that make up the IL-2 conjugates disclosed herein are straight or branched chain PEG groups having an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG group is a linear or branched PEG group having the following average molecular weights: about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons Daltons, approximately 8,000 Daltons, approximately 9,000 Daltons, approximately 10,000 Daltons, approximately 11,000 Daltons, approximately 12,000 Daltons, approximately 13,000 Daltons, approximately 14,000 Daltons, approximately 15,000 Daltons Dalton, approximately 20,000 Dalton, approximately 22,500 Dalton, approximately 25,000 Dalton, approximately 30,000 Dalton, approximately 35,000 Dalton, approximately 40,000 Dalton, approximately 45,000 Dalton, approximately 50,000 Dalton, approximately 55,000 Daltons, approximately 60,000 Daltons, approximately 65,000 Daltons, approximately 70,000 Daltons, approximately 75,000 Daltons, approximately 80,000 Daltons, approximately 90,000 Daltons, approximately 95,000 Daltons, and approximately 100,000 Daltons. In some embodiments, the PEG group has 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 of linear or branched PEG groups. In some embodiments, the PEG group is a straight or branched chain 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 has 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 of straight-chain PEG groups. In some embodiments, the PEG group has 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 of branched PEG groups.

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

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

In some embodiments, exemplary PEG groups include, but are not limited to, linear or branched discrete PEG (dPEG) from Quanta Biodesign, Ltd; linear, branched, or forked PEG from Nektar Therapeutics; and from JenKem Technology Y-shaped PEG derivatives.

In any of any of the embodiments or variations of Formula (I) and pharmaceutical compositions comprising the same described herein, the average molecular weight includes both weight average molecular weight and number average molecular weight; in other words, for example, a 30 kDa number average molecular weight and The 30 kDa weight average molecular weights are all consistent with the 30 kDa molecular weight. In some embodiments, the average molecular weight is a weight average molecular weight. In other specific examples, the average molecular weight is the number average molecular weight. It is to be understood that in the methods provided herein, administering an IL-2 conjugate as described herein to an individual comprises administering more than a single molecule of IL-2 conjugate; thus, the term "average" is used to describe the Molecular weight refers to the average molecular weight of the PEG groups of the IL-2 conjugate molecule in a dose administered to an individual. bonding chemistry

Various ligation reactions are used to ligate linkers, ligation moieties, and unnatural amino acids that are incorporated into the IL-2 polypeptides described herein. These conjugation reactions are generally compatible with aqueous conditions, such as "bioorthogonal" reactions. In some embodiments, the conjugation reaction is mediated by chemical reagents, such as catalysts, light, or reactive chemical groups found on linkers, conjugation moieties, or unnatural amino acids. In some embodiments, the ligation reaction is mediated by an enzyme. In some embodiments, the ligation reactions 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, the ligation reactions used herein are described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417, the disclosures of which are incorporated herein by reference.

In some variations, the IL-2 conjugates described herein can be prepared by a conjugation reaction involving a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises the reaction of an azide and a phosphine (a "click" reaction). In some embodiments, the joining reaction is catalyzed by copper. In some embodiments, the conjugation reactions described herein result in cytokine peptides comprising linkers or conjugation moieties attached via triazoles. In some embodiments, the joining reactions described herein comprise the reaction of an azide with a strained olefin. In some embodiments, the conjugation reactions described herein comprise the reaction of an azide with a strained alkyne. In some embodiments, the conjugation reactions described herein comprise the reaction of an azide with a cycloalkyne (eg, DBCO).

In some embodiments described herein, the conjugation reactions described herein comprise the reactions outlined in Scheme 1, wherein X is the position in the IL-2 conjugate that comprises the unnatural amino acid, such as in SEQ ID NO:5, Any of 6, 7, 8, 9, 30, 31, 32, 33, and 34. plan 1.

In some embodiments, the engaging portion comprises a water-soluble polymer. In some embodiments, reactive groups include alkynes or azides.

In some embodiments, the conjugation reactions described herein comprise the reactions outlined in Scheme 2, wherein X is the position in the IL-2 conjugate that contains the unnatural amino acid, such as in SEQ ID NOs: 5, 6, 7, Any of 8, 9, 30, 31, 32, 33, and 34. Scenario 2.

In some embodiments, the conjugation reactions described herein comprise the reactions outlined in Scheme 3, wherein X is the position in the IL-2 conjugate that contains the unnatural amino acid, such as in SEQ ID NOs: 5, 6, 7, Any of 8, 9, 30, 31, 32, 33, and 34. Scenario 3.

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

方案6.

In some embodiments, the conjugation reactions described herein comprise an azide moiety (such as an azide moiety included in a protein containing N6 -((2-azidoethoxy)-carbonyl)- A cycloaddition reaction between L-lysine (the amino acid residue of AzK) and a strained cycloalkyne such as one derived from DBCO, which is a chemical moiety containing a dibenzocyclooctyne group. PEG groups comprising DBCO moieties are commercially available or can be prepared by methods known to those of ordinary skill in the art. Exemplary reactions are shown in Schemes 5 and 6. Scenario 5.

Scenario 6.

Conjugation reactions, such as the click reactions described herein, can produce single regioisomers or mixtures 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, the IL-2 conjugates described herein containing natural amino acid mutations or non-natural amino acid mutations are recombinantly produced or chemically synthesized. In some cases, the IL-2 conjugates described herein are recombinantly produced, eg, by a host cell system or in a cell-free system. In any embodiment or variation described herein, the amino acid can be an L-amino acid or a D-amino acid. In some embodiments, the amino acid is an L-amino acid. In other specific examples, the amino acid is a D-amino acid.

In some cases, the IL-2 conjugate is recombinantly produced by a host cell system. In some cases, the host cells are eukaryotic cells (eg, mammalian cells, insect cells, yeast cells, or plant cells), or prokaryotic cells (eg, Gram-positive bacteria or Gram-negative bacteria). In some instances, the eukaryotic host cell is a mammalian host cell. In some cases, a mammalian host cell is a stable cell line, or a cell line that incorporates the genetic material of interest into its own genome and is capable of expressing the product of the genetic material after multiple generations of cell division. In other cases, the mammalian host cell is a transient cell line, or one that does not incorporate the genetic material of interest into its own gene body and is unable to express the product of the genetic material after multiple generations of cell division cell line.

Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cell, 293H 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 line, T-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 yeast strains, such as INVSc1.

In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, the plant cells comprise cells from algae. Exemplary plant cell lines include cell lines from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

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

In some cases, suitable polynucleic acid molecules or vectors for the production of IL-2 polypeptides described herein include any suitable vector derived from a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors from bacterial (eg, E. coli), insect, yeast (eg, Pichia, methanolic yeast), algal, or mammalian sources. 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, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393, FLAG vectors such as pVL1393 M11, - FLAG1 or pPolh-MAT2), 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® destination vector, ^pAO815bi Pichia vector, pFLD1 Pichia (Methanol yeast) vector, pGAPZA, B, & C Pichia (Methanol yeast) 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.

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

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

In some cases, cell-free systems are used to produce the IL-2 polypeptides described herein. In some cases, cell-free systems comprise 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 utilize prokaryotic components. In other cases, cell-free systems utilize eukaryotic cellular components. Nucleic acid synthesis is obtained in cell-free systems based on eg Drosophila cells, Xenopus eggs, Archaea, or HeLa cells. Exemplary cell-free systems include the E. coli S30 Extract system, the E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.

Cell-free translation systems comprise a wide range of components such as plastids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperones, translation initiation and elongation factors, natural and/or unnatural amino acids, and /or other components for protein expression. Such components are optionally modified to increase yield, increase synthesis rate, increase protein product accuracy, or incorporate unnatural amino acids. In some embodiments, the cytokines described herein are synthesized using the cell-free translation systems described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or US 8,778,631. In some embodiments, the cell-free translation system includes modified release factors, or even removes one or more release factors from the system. In some embodiments, the cell-free translation system comprises a reduced protease concentration. In some embodiments, the cell-free translation system comprises modified tRNAs with reassigned codons for encoding unnatural amino acids. In some embodiments, the synthetases described herein for incorporation of unnatural amino acids are used in cell-free translation systems. In some embodiments, the tRNA is preloaded with unnatural amino acids using enzymatic or chemical methods before being added to the cell-free translation system. In some embodiments, the components of the cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organisms.

In some embodiments, IL-2 polypeptides are produced in a circular arrangement via expression host systems or via cell-free systems. Production of IL-2 Polypeptides Containing Unnatural 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 a non-natural amino acid such that it can be encoded by using an It is genetically incorporated into IL-2 by crossing the tRNA synthetase/tRNA pair. Orthogonal tRNA synthetase/tRNA pairs are capable of loading tRNAs with unnatural amino acids and incorporating the unnatural amino acids into polypeptide chains corresponding to codons.

In some cases, the codons are amber, ochre, opal, or quadruplet codons. In some cases, the codons correspond to orthogonal tRNAs that will be used to carry the unnatural amino acid. In some cases, the codons are of the amber type. In other cases, the codons are orthogonal codons.

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

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

In some cases, one or more non-natural amino acids are incorporated into IL-2 using a non-natural nucleic acid. Exemplary non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me- C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanine and other alkyl derivatives, 2- of adenine and guanine Propyl and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-even Azauracil, Cytosine, and Thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- Hydroxyl, and other 8-substituted adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methyl Guanine 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, those that increase the stability of double-strand formation, general nucleic acids, hydrophobic nucleic acids, hybrid nucleic acids, size-amplified nucleic acids, fluorinated Nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propane Alkynylcytosine. 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanine, other alkyl groups derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-Propynyl (-C≡C-CH ₃ )uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6- Azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8 substituted Adenines and guanines, 5-halo (especially 5-bromo), 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine , 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3 -Deazaadenine, tricyclic pyrimidine, cytidine ([5,4-b][l,4]benzoxazine-2(3H)-one), cytidine (1H-pyrimidine) [5,4-b][l,4]benzothiazin-2(3H)-one), G-clamp, cytidine (e.g. 9-(2-aminoethoxy)-H -pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazolcytidine (2H-pyrimido[4,5-b]indol-2-one) ), pyridoindolecytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), in which the purine or pyrimidine base is substituted with other Heterocycle, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorine Cytosine, Cyclocytosine, Cytosine Arabinoside, 5-Fluorocytosine, Fluoropyrimidine, Fluorouracil, 5,6-Dihydrocytosine, 5-Iodocytosine, Hydroxyurea, Iodouracil, 5-Nitro Cytosine, 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 Pyrimidine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and those described in: US Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5,093,232; 5,130,302; 5,134,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711；5,552,540；5,587,469；5,594,121；5,596,091；5,614,617；5,645,985；5,681,941；5,750,692；5,763,588；5,830,653和6,005,096；WO 99/62923；Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813；The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Additional base modifications can be found, for example, in US Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993; the respective disclosures of which are incorporated herein by reference.

Non-natural nucleic acids comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and in some cases, nucleic acids include one or several heterocyclic bases other than those of naturally occurring nucleic acids. The main five base components. For example, in some instances, heterocyclic bases include uracil-5-yl, cytosine-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanine- 8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxypyrrolo[2,3-d]pyrimidin-5-yl, 2-amino -4-Pendant oxypyrrolo[2.3-d]pyrimidin-3-yl group in which purines are via the 9-position, pyrimidines are via the 1-position, pyrrolopyrimidines are via the 7-position, and pyrazolopyrimidines are via the 1-position A position is attached to a sugar moiety of a nucleic acid.

In some embodiments, the nucleotide analogs are also modified in the phosphate moiety. Modified phosphate moieties include, but are not limited to, those with modifications at the linkage between two nucleotides, and contain, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters , aminoalkylphosphonates, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, aminophosphonates (including 3' - aminoaminophosphates and aminoalkylaminophosphates), thiocarbonoaminophosphates, thiocarbonoalkylphosphonates, thiocarbonoalkylphosphonates and borane phosphates. It is understood that these phosphate or modified phosphate linkages between two nucleotides are through a 3'-5' linkage or a 2'-5' linkage, and the linkage contains an inverted polarity, Such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are the various salts, mixed salts and free acid forms.許多美國專利教示如何製造和使用含有經修飾磷酸酯的核苷酸，包括但不限於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;

In some embodiments, non-natural nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleosides (PCT/US2002/006460), 5' substituted DNA and RNA derivatives (PCT/US2011/033961 Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; 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; or 5'-substituted monomers in the form of monophosphates with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23(1 & 2), 317-337); their respective The disclosures are incorporated herein by reference.

In some embodiments, the non-natural nucleic acid includes modifications at the 5'-position and the 2'-position of the sugar ring (PCT/US94/02993), such as 5'- _CH substituted 2'-O-protected (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). In some cases, the non-natural nucleic acid includes an amide-linked nucleoside dimer, prepared for incorporation into an oligonucleotide, wherein the 3'-linked nucleoside (5' to 3') in the dimer 2'- _OCH3 and 5'-(S) _-CH3 are included (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids can include 2'-substituted 5'- _CH2 (or O) modified nucleosides (PCT/US92/01020). Non-natural nucleic acids can include 5'-methylenephosphonate DNA and RNA monomers, as well as dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; with Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids may include 5'-phosphonate monomers with 2'-substitution (US2006/0074035) and other modified 5'-phosphonate monomers (WO1997/35869). Non-natural nucleic acids may include 5' modified methylene phosphonate monomers (EP614907 and EP629633). Non-natural nucleic acids can include analogs of 5' or 6'-phosphonic ribonucleosides containing hydroxyl groups at the 5' and/or 6'-positions (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783- 1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033). Non-natural nucleic acids may include 5'-phosphodeoxyribonucleoside monomers and dimers with 5'-phosphate groups (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids may include nucleosides with 6'-phosphonate groups in which the 5' or/and 6'-positions are unsubstituted or thio-tertiary butyl (SC(CH ₃ ) ₃ ) (and its analogues) substitution; methyleneamine (CH ₂ NH ₂ ) (and its analogues) or cyano (CN) (and its analogues) (Fairhurst et al., Synlett, 2001, 4, 467-472 ; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26) , 7638-7640; and Hampton et al., J. Am. Chem. Soc, 1973, 95(13), 4404-4414). The disclosures of each reference listed in this chapter are incorporated herein by reference.

In some embodiments, the non-natural nucleic acid also includes modifications of the sugar moiety. In some cases, the nucleic acid contains one or more nucleosides in which the sugar groups have been modified. Such sugar-modified nucleosides may confer increased 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 nucleofuranose rings include, but are not limited to, the addition of substituent groups (including 5' and/or 2' substituent groups; bridging of two ring atoms to form bicyclic nucleic acids (BNA); (R) or C(R ₁ )(R ₂ ) (R=H, C ₁ -C ₁₂ alkyl or protecting group) replacing a ribosyl epoxy atom); and combinations thereof. Examples of chemically modified sugars can be found in WO2008/101157, US2005/0130923 and WO2007/134181, the disclosures of each of which are incorporated herein by reference.

In some cases, the modified nucleic acid comprises a modified sugar or sugar analog. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or sugar "analog" ring amyl. The sugar can be in the pyranosyl or furanosyl form. The sugar moiety can be ribose, deoxyribose, arabinose, or a furanoside of 2'-O-alkyl ribose, and the sugar can be attached to the individual heterocyclic base in the [alpha] or [beta] isomer configuration base. 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 Chimera. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl substituted deoxyribonucleosides and 2'-O-glycol-like ribonucleosides. The preparation of these sugars or sugar analogs and the corresponding "nucleosides" in which such sugars or analogs are attached to heterocyclic bases (nucleobases) are known. Sugar modifications can also be made and combined with other modifications.

Modifications to the sugar moiety include natural modifications of ribose and deoxyribose as well as non-natural modifications. Sugar modifications include, but are not limited to, modifications at the following 2' positions: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl ; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted _C1 to _C10 , alkyl or _C2 to _C10 alkenyl and alkynyl. 2' sugar modifications also include but are not limited to -O[( _CH2 ) _nO ] _mCH3 , -O( _{CH2 ) nOCH3} , -O( _CH2 ) _nNH2 , _-O ( _{CH2 ) 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: _C1 to _C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkane base, 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, heterocycloalkane groups, aminoalkylamine groups, polyalkylamine groups, substituted silicon groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or Groups that improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions of the sugar, particularly at the 3' position of the sugar at the 3' terminal nucleotide or at the 2'-5' linking oligonucleotide and the sugar at the 5' terminal nucleotide. 5' position. Modified sugars also include sugars containing modifications such as _CH2 and S at the bridging epoxy. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties in place of pentofuranoses. There are a number of U.S. patents that teach the preparation of such modified sugar structures and detail a range of base modifications, such as U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 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, but are not limited to, include 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'- _OCH3 , and 2 Nucleic acids with '-O( _{CH2 ) 2OCH3} substituents. The substituent at the 2' position can also be selected from allyl, amine, azide, thio, O-allyl, O-(C ₁ -C ₁₀ alkyl), OCF ₃ , O(CH ₂ )2SCH3, O( _CH2 ) ₂ -ON( _Rm )( _Rn ), and O- _CH2 -C(=O)-N( _Rm )( _Rn ), wherein each _Rm and _Rn are independent is 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, the nucleic acids provided herein include one or more bicyclic nucleic acids, wherein the bridge comprises a 4' to 2' bicyclic nucleic acid. Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited to, one of the following formulae: 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 its analogs ( See US Patent No. 7,399,845); 4'-C( _CH3 )( _CH3 )-O-2' and analogs thereof (see WO2009/006478, WO2008/150729, US2004/0171570, US Patent No. 7,427,672, Chattopadhyaya et al ., J. Org. Chem., 209, 74, 118-134, and WO2008/154401). See also, e.g.: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci . USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035 -10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al ., Chem. Biol, 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 4,849,513; 5,015,733; 5,118,800; 6,770,748；6,794,499；7,034,133；6,525,191；6,670,461；和7,399,845；國際專利號WO2004/106356、WO1994/14226、WO2005/021570、WO2007/090071、和WO2007/134181；美國公開號US2004/0171570、US2007/0287831、和US2008 /0039618; US Provisional Application Nos. 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and International Application Nos. PCT/US2008/064591, PCT US2008/066154 , PCT US2008/068922, and PCT/DK98/00393. The disclosures of each reference listed in this chapter are incorporated herein by reference.

In certain embodiments, nucleic acids include linked nucleic acids. Nucleic acids can be linked together using any internucleic acid linkage. Two broad classes of internucleic acid linking groups are defined by the presence or absence of phosphorus atoms. Representative phosphorous-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linking groups include, but are not limited to, methylenemethylimino ( _-CH2 -N( _CH3 ) _-O -CH2-), thiodiester (-OC(O )-S-), thiocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H) ₂ -O-); and N,N*- Dimethylhydrazine ( _-CH2 -N( _CH3 )-N( _CH3 )). In certain embodiments, internucleic acid linkages with chiral atoms can be prepared as racemic mixtures, individual enantiomers, such as alkylphosphonates and phosphorothioates. A non-natural nucleic acid can contain a single modification. A non-natural nucleic acid may contain multiple modifications within one portion or between different portions.

Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters, phosphorodithioates, phosphorodithioates phosphodithioate, and borane phosphate, and can be used in any combination. Other non-phosphate linkages can also be used.

In some embodiments, backbone modifications (eg, methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity and/or Improve its in vivo stability.

In some cases, the phosphorus derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate ester, phosphorodithioate, phosphoramidate or the like. Exemplary polynucleotides comprising modified phosphate linkages or non-phosphate linkages can be found in Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24 :2318-2323; Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, "Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Patent No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem. 8: 1157-1179 ; their respective disclosures 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' to P5' phosphoramidate modifications; borane phosphate DNA; proto-oligonucleotides; neutral internucleoside linkages such as methylphosphonic acid esters; amide-linked DNA; methylene (methylimino) linkages; methylal and thioformal linkages; backbones containing sulfonyl groups; N-morpholino oligos ); peptide nucleic acids (PNA); and positively charged deoxyribonucleic acid guanidine (DNG) oligomers (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). Modified nucleic acids may comprise chimeric or mixed backbones comprising one or more modifications, eg, a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

Phosphate substituents include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic nuclei interglycoside linkages. These include those with N-morpholine linkages (partly formed from the sugar moieties of the nucleosides); siloxane backbones; sulfide, arylene, and arsenic backbones; methylal and thioformal backbones; methylene groups Formal and thioformal skeletons; olefin-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazine skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others with mixed N, O, S and CH ₂ component parts.許多美國專利揭示如何製造和使用這些類型的磷酸酯置換物，包括但不限於美國專利號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; It is also understood that in a nucleotide, the sugar and phosphate moieties of the nucleotide can be replaced by, for example, an amide-type linkage (aminoethylglycine) (PNA). US Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which are incorporated herein by reference, teach how to make and use PNA molecules. See also Nielsen et al., Science, 1991, 254, 1497-1500. Other types of molecules (conjugates) can also be attached to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acids (Manoharan et al., Bioorg. Med Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-trityl mercaptan (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306- 309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), sulfur cholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chain, 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 1-di-O-hexadecyl-rac-glycerol-SH -Triethylammonium phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene Diol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al. al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).許多美國專利教示了此類接合物的製備，包括但不限於美國專利號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和5,688,941。 The disclosures of each of the references listed in this chapter are incorporated herein by reference.

。 例示性非天然鹼基對包括：(d)TPT3-(d)NaM；(d)5SICS-(d)NaM；(d)CNMO-(d)TAT1；(d)NaM-(d)TAT1；(d)CNMO-(d)TPT3；及(d)5FM-(d)TAT1。 In some cases, the unnatural nucleic acid further forms unnatural base pairs. Exemplary unnatural nucleotides capable of forming unnatural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM , CNMO, dCNMO, and combinations thereof. In some embodiments, 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.

。 Additional examples of non-natural nucleotides capable of forming non-natural UBPs that can be used to prepare the IL-2 conjugates disclosed herein can be found in Dien et al., J Am Chem Soc., 2018, 140:16115-16123; Feldman et al. ., J Am Chem Soc, 2017, 139:11427-11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res. 2014, 42:10235-10244 ; Malyshev et al., Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc. 2013, 135:5408 -5419; and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010; their respective disclosures are incorporated herein by reference. In some embodiments, non-natural nucleotides include:

.

其中R ₂選自氫、烷基、烯基、炔基、甲氧基、甲硫醇、甲硒基、鹵素、氰基、和疊氮基；且 波浪線表示與核糖基或2'-去氧核糖基的鍵，其中核糖基或2'-去氧核糖基部分的5'-羥基為游離形式，連接至單磷酸酯、二磷酸酯、三磷酸酯、α-硫代三磷酸酯、β-硫代三磷酸酯、或γ-硫代三磷酸酯基團，或包含在RNA或DNA或RNA類似物或DNA類似物中。 In some embodiments, non-natural nucleotides useful in preparing 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, methylthiol, methylselenyl, halogen, cyano, and azido; An oxyribosyl bond in which the 5'-hydroxyl of the ribosyl or 2'-deoxyribosyl moiety is in free form, attached to a monophosphate, diphosphate, triphosphate, alpha-thiotriphosphate, beta - A thiotriphosphate, or gamma-thiotriphosphate group, or contained in RNA or DNA or RNA analogs or DNA analogs.

其中： 各X獨立地為碳或氮； 當X為氮時，則R ₂不存在，而當X為碳時，R ₂存在，且獨立地為氫、烷基、烯基、炔基、甲氧基、甲硫醇、甲硒基、鹵素、氰基、或疊氮化物； Y是硫、氧、硒或二級胺； E是氧、硫或硒；且 波浪線表示與核糖基、去氧核糖基或雙去氧核糖基部分或其類似物的鍵結點，其中核糖基、去氧核糖基或雙去氧核糖基部分或其類似物為游離形式，連接至單磷酸酯、二磷酸酯、三磷酸酯、α-硫代三磷酸酯、β-硫代三磷酸酯或、γ-硫代三磷酸酯基團，或包含在RNA或DNA或RNA類似物或DNA類似物中。 In some embodiments, non-natural nucleotides useful in preparing the IL-2 conjugates disclosed herein can be derived from compounds of the formula:

wherein: each X is independently carbon or nitrogen; when X is nitrogen, then R is absent, and when X is carbon, R _is present, and is independently _hydrogen , alkyl, alkenyl, alkynyl, methyl Oxygen, methylthiol, methylselenyl, halogen, cyano, or azide; Y is sulfur, oxygen, selenium, or a secondary amine; E is oxygen, sulfur, or selenium; Bonding point of an oxyribosyl or dideoxyribosyl moiety or analog thereof, wherein the ribosyl, deoxyribosyl or dideoxyribosyl moiety or analog thereof is in free form, attached to a monophosphate, diphosphate ester, triphosphate, alpha-thiotriphosphate, beta-thiotriphosphate or, gamma-thiotriphosphate groups, or included in RNA or DNA or RNA analogs or DNA analogs.

In some embodiments, each X is carbon. In some embodiments, at least one X is carbon. In some embodiments, an X is carbon. In some embodiments, at least two X's are carbon. In some specific examples, both X's are carbon. In some embodiments, at least one X is nitrogen. In some embodiments, an X is nitrogen. In some embodiments, at least two X's are nitrogen. In some embodiments, both X's are nitrogen.

In some embodiments, Y is sulfur. In some embodiments, Y is oxygen. In some specific examples, 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 specific examples, E is selenium.

_In some embodiments, when X is carbon, then R2 is present. ^In some embodiments, when X is nitrogen, R is absent. In some embodiments, when present, each R2 is _hydrogen . In some embodiments, R ₂ is an alkyl group 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 methyl mercaptan. In some embodiments, R ₂ is methylselenyl. In some embodiments, R ₂ is halogen, such as chlorine, bromine or fluorine. 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.

、

、

、

、

、

、

、

、

、

、

及

。 In some embodiments, non-natural nucleotides useful in preparing the IL-2 conjugates disclosed herein can be derived from

,

,

,

,

,

,

,

,

,

,

and

.

、

、

、

、

、

、

、

、

、

、

和

、或其鹽。 In some embodiments, non-natural nucleotides that can be used to prepare the IL-2 conjugates disclosed herein include

,

,

,

,

,

,

,

,

,

,

and

, or its salt.

和

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

and

.

和

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

and

.

In some specific examples, unnatural base pairs generate unnatural amino acids, as described in Dumas et al., "Designing logical codon reassignment - Expanding the chemistry in biology," Chemical Science , 6 : 50-69 (2015) described.

In some embodiments, the unnatural amino acid is incorporated into the IL-2 polypeptide via synthetic codons comprising the unnatural nucleic acid. In some cases, unnatural amino acids are incorporated into IL-2 by orthogonal, modified synthetase/tRNA pairs. This orthogonal pair comprises an unnatural synthetase capable of loading unnatural amino acids onto unnatural tRNAs while simultaneously loading a) other endogenous amino acids onto unnatural tRNAs and b) unnatural amino acids Loading onto other endogenous tRNAs is minimized. Such orthogonal pairs contain a) other endogenous amino acids that can be loaded by non-natural synthetases while avoiding loading by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, orthogonal synthase/tRNA pairs comprise components from a single organism. In some embodiments, the orthogonal synthase/tRNA pair comprises components from two different organisms. In some embodiments, the orthogonal synthase/tRNA pair comprises components that promote translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthase is a modified alanine synthase. In some embodiments, the orthogonal synthase is a modified arginine synthase. In some embodiments, the orthogonal synthase is a modified aspartate synthase. In some embodiments, the orthogonal synthase is a modified aspartate synthase. In some embodiments, the orthogonal synthase is a modified cysteine synthase. In some embodiments, the orthogonal synthase is a modified glutamic acid synthase. In some embodiments, the orthogonal synthase is a modified glutamate synthase. In some embodiments, the orthogonal synthase is a modified alanine glycine. In some embodiments, the orthogonal synthase is a modified histidine synthase. In some embodiments, the orthogonal synthase is a modified leucine synthase. In some embodiments, the orthogonal synthase is a modified isoleucine synthase. In some embodiments, the orthogonal synthase is a modified lysine synthase. In some embodiments, the orthogonal synthase is a modified methionine synthase. In some embodiments, the orthogonal synthase is a modified phenylalanine synthase. In some embodiments, the orthogonal synthase is a modified proline synthase. In some embodiments, the orthogonal synthase is a modified serine synthase. In some embodiments, the orthogonal synthase is a modified threonine synthase. In some embodiments, the orthogonal synthase is a modified tryptophan synthase. In some embodiments, the orthogonal synthase is a modified tyrosine synthase. In some embodiments, the orthogonal synthase is a modified valine synthase. In some embodiments, the orthogonal synthase is a modified phosphoserine synthase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartate tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamic acid 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 unnatural amino acid is incorporated into the IL-2 polypeptide by an aminoacyl group (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii ( Mj-Tyr ) aaRS/tRNA pairs, E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus tRNA _CUA pair, Escherichia coli LeuRS ( Ec-Leu )/Bacillus stearothermophilus tRNA _CUA pair, and pyrrolidinyl-tRNA pair. In some cases, the unnatural amino acid is incorporated into the IL-2 polypeptide via the Mj-Tyr RS/tRNA pair. Exemplary UAAs that can be incorporated by the Mj-Tyr RS/tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives, such as p-aminoampheline and p-methoxyphenylalanine; m-substituted tyramine Acid derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronphenylalanine ; and o-nitrobenzyltyrosine.

In some cases, the unnatural amino acid is incorporated into the IL-2 polypeptide via the Ec-Tyr /tRNA _CUA or Ec-Leu /tRNA _CUA pair. Exemplary UAAs that can be incorporated via Ec-Tyr /tRNA _CUA or Ec-Leu /tRNA _CUA pairs include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents compounds; O- propargyl tyrosine; alpha-aminocaprylic acid, O-methyltyrosine, O-nitrobenzylcysteine; and 3-(naphthalen-2-ylamino)- 2-Amino-propionic acid.

In some cases, the unnatural amino acid is incorporated into the IL-2 polypeptide via a pyrrolidinosyl-tRNA pair. In some cases, PylRS are obtained from archaea, such as methanogenic archaea. In some cases, the PylRS is from Methanosarcina barkeri , Methanosarcina mazei , or Methanosarcina acetivorans . Exemplary UAAs that can be incorporated via pyrrole lysinyl-tRNA pairs include, but are not limited to, amide- and carbamate-substituted lysine acids, such as 2-amino-6-((R)- Tetrahydrofuran-2-carbamido)hexanoic acid, N- ε- _D -prolinyl- _L -lysine, and N -ε-cyclopentyloxycarbonyl- _L -lysine; N -ε- - Acryloyl- _L- lysine; N- ε-[(1-(6-nitrobenzo[d][1,3]dioxolan-5-yl)ethoxy)carbonyl] _{- L-} lysine; and N -ε-(1-methylcycloprop-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein can be prepared by using M. mazei tRNA produced by M. pasteurii pyrrolidinosyl-tRNA synthetase ( Mb PylRS) selectively loaded with unnatural amino acids such as N6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK). Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647, the disclosures of which are incorporated herein by reference.

In some cases, unnatural amino acids are incorporated into IL-2 polypeptides by synthetases as disclosed in US 9,988,619 and US 9,938,516, the disclosures of each of which are incorporated herein by reference.

Host cells incorporating the constructs or vectors disclosed herein are grown or maintained in a suitable medium to produce tRNA, tRNA synthetase, and protein of interest. The medium also contains unnatural amino acids (etc.) such that the protein of interest incorporates the unnatural amino acids (etc.). In some embodiments, nucleoside triphosphate transporters (NTTs) from bacteria, plants or algae are also present in the host cell. In some embodiments, the IL-2 conjugates disclosed herein are prepared by using host cells expressing NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter used in the host cell can be selected from the group consisting of TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana (T. pseudonana) )), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, TpNTT3 (P. tricornutum), GsNTT (Galdieria sulfuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana) , CtNTT1, CtNTT2 (Chlamydia trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some embodiments, the NTT is selected from the group consisting of PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5 and PtNTT6. In some specific examples, the NTT is PtNTT1. In some embodiments, the NTT is PtNTT2. In some embodiments, the NTT is PtNTT3. In some embodiments, the NTT is PtNTT4. In some embodiments, the NTT is PtNTT5. In some embodiments, the NTT is PtNTT6. Zhang et al., Nature 2017, 551(7682): 644-647; Malyshev et al. Nature 2014 (509(7500), 385-388; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317- Other NTTs that can be used are disclosed in 1322; their respective disclosures are incorporated herein by reference.

Orthogonal tRNA synthetase/tRNA pairs load unnatural amino acids into tRNAs and incorporate unnatural amino acids into polypeptide chains corresponding to codons. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii ( Mj - Tyr ) aaRS/tRNA pairs, E. coli TyrRS ( Ec-Tyr )/Bacillus stearothermophilus tRNA _CUA pairs, E. coli LeuRS ( Ec- Leu )/Bacillus stearothermophilus tRNA _CUA pair, and pyrrolidinyl-tRNA pair. Other aaRS-tRNA pairs that can be used in accordance with the present invention include those derived from M. mazei, such as Feldman et al., J Am Chem Soc., 2018 140:1447-1454; and Zhang et al. Proc Described in Natl Acad Sci USA, 2017, 114:1317-1322; the disclosures of each of which are incorporated herein by reference.

In some embodiments, methods are provided for making the IL-2 conjugates disclosed herein in cellular systems expressing NTT and tRNA synthetases. In some embodiments described herein, the NTT is selected from the group consisting of PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from the group consisting of Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Stearothermophilus Bacillus, and Methanosarcina mazei. In some embodiments, the NTT is PtNTT1 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT2 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT4 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT5 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei. In some embodiments, the NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS ( EC-Tyr )/Bacillus stearothermophilus, or Methanosarcina mazei.

、

、

、

、

、和

。 在一些具體例中，包含雙股寡核苷酸中的非天然鹼基對的第一和第二非天然核苷酸可衍生自

和

。 在一些具體例中，第一和第二非天然核苷酸的三磷酸包括

、

、和

、或其鹽。 在一些具體例中，第一和第二非天然核苷酸的三磷酸包括

、和

、或其鹽。 在一些具體例中，包含第一非天然核苷酸和第二非天然核苷酸之衍生自mRNA的雙股寡核苷酸可包含衍生自以下的非天然核苷酸的密碼子：

、

、和

。 在一些具體例中，馬氏甲烷八疊球菌tRNA可包含含有非天然核苷酸的反密碼子，其辨識包含mRNA的非天然核苷酸的密碼子。馬氏甲烷八疊球菌tRNA的反密碼子可包含衍生自

、

、

、

、

、和

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸。 在一些具體例中，tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸而tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸而tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸而tRNA包含衍生自

的非天然核苷酸。 在一些具體例中，mRNA包含衍生自

的非天然核苷酸而tRNA包含衍生自

的非天然核苷酸。 宿主細胞培養在含有適當營養物的培養基中，且補充有(a)去氧核糖核苷的三磷酸酯，其包含一或多種非天然鹼基，這些鹼基是編碼攜帶密碼子的細胞激素基因的質體複製所必需的；(b)核糖核苷的三磷酸酯，其包含一或多種轉錄(i)對應於細胞激素編碼序列且含有包含一或多個非天然鹼基的密碼子的mRNA；以及(ii)含有包含一或多個非天然鹼基的反密碼子的tRNA所必需的非天然鹼基；及(c)要被併入感興趣細胞激素的多肽序列中的非天然胺基酸。然後將宿主細胞維持在允許感興趣蛋白表現的條件下。 In some embodiments, the IL-2 conjugates disclosed herein can be prepared in cells, such as E. coli, comprising (a) the nucleotide triphosphate transporter Pt NTT2 (including truncated variants in which the full-length protein (b) a plastid comprising a double-stranded oligonucleotide encoding an IL-2 variant with the desired amino acid sequence and containing An unnatural base pair comprising a first unnatural nucleotide and a second unnatural nucleotide to provide a codon at the desired position to be incorporated into an unnatural amino acid such as N 6-((2-Azidoethoxy)-carbonyl)-L-lysine (AzK), (c) plastids encoding tRNA derived from M. mazei and comprising non-natural nucleosides acid to provide a recognized anticodon (relative to the codon of the IL-2 variant) to replace its native sequence, and (d) to encode a Methanosarcina pasteurii-derived pyrrolidinosyl-tRNA synthetase ( Mb PylRS), which is the same as or different from the plastid encoding the tRNA. In some embodiments, the cells are further supplemented with deoxyribose triphosphates comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with ribose triphosphates comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with one or more unnatural amino acids, such as N6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, at positions 34, 37, 40, 41, 42, 43, 44, 61, 64, 68, or 71 of, for example, a sequence encoding a protein having SEQ ID NO: 3, or at positions encoding a protein having SEQ ID NO: 3 Position 35, 38, 41, 42, 43, 45, 62, 65, 69, or 72 of the sequence of the protein of NO: 4, a double-stranded oligonucleotide encoding the amino acid sequence of the desired IL-2 variant Contains the codon AXC, where X is an unnatural nucleotide. In some embodiments, the cell further comprises a plastid, which may be a protein-expressing plastid, or another plastid, encoding an orthogonal tRNA gene from M. mazei, which contains an AXC-matched anticodon GYT in its place A native sequence, where Y is a complementary non-natural nucleotide and may or may not be the same as the non-natural nucleotide in the codon. In some embodiments, the unnatural nucleotides in the codons are different and complementary to the unnatural nucleotides in the anticodons. In some embodiments, the unnatural nucleotide in the codon is the same as the unnatural nucleotide in the anticodon. In some embodiments, the first and second unnatural nucleotides comprising unnatural base pairs in the double-stranded oligonucleotide can be derived from

,

,

,

,

,and

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

and

. In some embodiments, the triphosphates of the first and second unnatural nucleotides include

,

,and

, or its salt. In some embodiments, the triphosphates of the first and second unnatural nucleotides include

,and

, or its salt. In some embodiments, an mRNA-derived double-stranded oligonucleotide comprising a first non-natural nucleotide and a second non-natural nucleotide can comprise codons derived from the following non-natural nucleotides:

,

,and

. In some embodiments, the M. mazei tRNA can comprise anticodons containing non-natural nucleotides that recognize codons comprising non-natural nucleotides of the mRNA. The anticodon of M. mazei tRNA may comprise derived from

,

,

,

,

,and

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of unnatural nucleotides. In some embodiments, the tRNA comprises derived from

of unnatural nucleotides. In some embodiments, the tRNA comprises derived from

of unnatural nucleotides. In some embodiments, the tRNA comprises derived from

of unnatural nucleotides. In some embodiments, the tRNA comprises derived from

of unnatural nucleotides. In some embodiments, the tRNA comprises derived from

of unnatural nucleotides. In some embodiments, the tRNA comprises derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of non-natural nucleotides whereas tRNA contains derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of non-natural nucleotides whereas tRNA contains derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of non-natural nucleotides whereas tRNA contains derived from

of unnatural nucleotides. In some embodiments, the mRNA comprises derived from

of non-natural nucleotides whereas tRNA contains derived from

of unnatural nucleotides. Host cells are cultured in a medium containing appropriate nutrients and supplemented with (a) triphosphates of deoxyribonucleoside, which contain one or more unnatural bases encoding codon-carrying cytokine genes Necessary for plastid replication of ; (b) ribonucleoside triphosphates comprising one or more transcripts (i) mRNAs corresponding to cytokine coding sequences and comprising codons comprising one or more unnatural bases and (ii) unnatural bases necessary for tRNAs containing anticodons comprising one or more unnatural bases; and (c) unnatural amine groups to be incorporated into the polypeptide sequence of the cytokine of interest acid. The host cells are then maintained under conditions that allow expression of the protein of interest.

The resulting expressed protein containing AzK can be purified by methods known to those of ordinary skill in the art, and then can be reacted with alkynes (such as those containing alkynes as disclosed herein, under conditions known to those of ordinary skill in the art). DBCO) reaction of PEG chains of the desired average molecular weight to yield the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; Those disclosed in WO 2019014267; WO 2019028419; and WO2019/028425; the disclosures of each of which are incorporated herein by reference.

The resulting protein expressed comprising one or more non-natural amino acids (eg AzK) can be purified by methods known to those of ordinary skill in the art, and can then be subjected to conditions known to those of ordinary skill in the art. It is reacted with an alkyne, such as DBCO comprising PEG chains of desired average molecular weight as disclosed herein, to yield the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; Those disclosed in WO 2019014267; WO 2019028419; and WO2019/028425; the disclosures of which are incorporated herein by reference.

Alternatively, can be prepared by introducing into a host cell a nucleic acid construct described herein comprising a tRNA and an aminoacyl tRNA synthetase and comprising a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons IL-2 polypeptides comprising unnatural amino acids. Host cells are cultured in a medium containing appropriate nutrients and supplemented with (a) triphosphates of deoxyribonucleoside, which contain one or more unnatural bases that code for carrying new codons and anticodons. Necessary for the plastid replication of the cytokine gene of the codon; (b) the triphosphate of the ribonucleoside, which is transcriptionally corresponding to (i) the codon-containing cytokine sequence, and (ii) the anticodon-containing orthogonal Necessary for mRNA for tRNA; and (c) unnatural amino acids. The host cells are then maintained under conditions that allow expression of the protein of interest. Incorporation of unnatural amino acids into polypeptide chains corresponds to unnatural codons. For example, one or more unnatural amino acids are incorporated into an IL-2 polypeptide. Alternatively, two or more unnatural amino acids can be incorporated into the IL-2 polypeptide at two or more sites in the protein.

Once the IL-2 polypeptide with the unnatural amino acid has been produced in the host cell, it can be extracted therefrom by a variety of techniques known in the art including enzymatic, chemical and/or osmolysis and physical disruption . IL-2 polypeptides can be purified by standard techniques known in the art such as preparative ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or any other suitable technique known to those of ordinary skill in the art .

Suitable host cells may include bacterial cells (eg, E. coli, BL21(DE3)), but most suitable host cells are eukaryotic cells, such as insect cells (eg, Drosophila, such as Drosophila melanogaster ), Yeast cells, nematodes (e.g. C. elegans ), mouse (e.g. mouse) or mammalian cells (e.g. Chinese hamster ovary (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells and mouse erythrocytic leukemia (MEL) cells) or human cells or other eukaryotic cells. Other suitable host cells are well known to those skilled in the art. Suitably, the host cells are mammalian cells, such as human cells or insect cells. In some embodiments, a suitable host cell comprises E. coli.

Other suitable host cells that can generally be used in embodiments of the invention are those mentioned in the Examples paragraph. Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of recognized techniques for introducing foreign nucleic acid molecules (eg, DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran Glycan-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

When creating cell lines, it is generally preferred to prepare stable cell lines. For example, in the case of stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells can incorporate foreign DNA into their genome. To identify and select for these ensembles, a gene encoding a selectable marker (eg, antibiotic resistance) is typically introduced into the host cell along with the gene of interest. Preferred selectable markers include those conferring drug resistance, such as G418, hygromycin or methotrexate. Nucleic acid molecules encoding the selectable marker can be introduced into the host cell on the same vector or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (eg, cells with the selectable marker gene will survive while other cells die).

In a specific example, the constructs described herein are incorporated into the genome of a host cell. One advantage of stable integration is that consistency between individual cells or clones is achieved. Another advantage is that the selection of the best producer can be carried out. Therefore, there is a need to create stable cell lines. In another specific example, the constructs described herein are transfected into host cells. One advantage of transfecting constructs into host cells is that protein yields can be maximized. In one aspect, cells comprising the nucleic acid constructs or vectors described herein are 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 inhibitor antibody is selected from the group consisting of cetuximab (Erbitux), panitumumab (Vectibix), nexituzumab (Portrazza), JNJ-61186372 (amivumab) , IMC-C225, ABX-EGF, ICR62, and EMD 55900. In some embodiments, the anti-EGFR inhibitor antibody is cetuximab (Erbitux). In some embodiments, the anti-EGFR inhibitor antibody is panitumumab (Vectibix). In some embodiments, the anti-EGFR inhibitor antibody is nexituzumab (Portrazza). In some embodiments, the anti-EGFR inhibitor antibody is JNJ-61186372 (amivumab). In some embodiments, the anti-EGFR inhibitor antibody is IMC-C225. In some embodiments, the anti-EGFR inhibitor antibody is ABX-EGF. In some embodiments, the anti-EGFR inhibitor antibody is ICR62. In some embodiments, the anti-EGFR inhibitor antibody is EMD 55900.x. treatment method

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

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

In another aspect, there is provided the use of an IL-2 conjugate as described herein for the manufacture of a medicament for use in the methods disclosed herein for treating cancer in an individual in need thereof. cancer type

In some embodiments, the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classic Hodgkin lymphoma (cHL), primary mediastinal B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable carcinoma, microsatellite stable carcinoma, gastric cancer, colon cancer, colorectal cancer (CRC), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophagus, 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) deficiency, bladder cancer, ovarian cancer, tumors with moderate to low mutational burden, cutaneous squamous cell carcinoma (CSCC) ), squamous cell skin cancer (SCSC), tumors with low to no expression of PD-L1, tumors with systemic diffuse to the liver and CNS beyond their primary anatomical origin, and diffuse large B-cell lymphoma.

In some embodiments, the individual's cancer 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 specific examples, the cancer is urothelial carcinoma. In some specific examples, 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. dosing

In some embodiments, the response is a complete response, a partial response, or a stable condition. In some embodiments, the IL-2 conjugate is administered by intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intraarticular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal Administration is administered to an individual. 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 individual by intravenous administration. In some embodiments, the IL-2 conjugate is administered to the individual by subcutaneous administration. In some embodiments, the IL-2 conjugate is administered to the individual 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 for up to another 24 months.

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

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

In some embodiments, the amount corresponding to this amount for a given agent will vary depending on a number of factors, such as the particular compound, the severity of the disease, the characteristics of the individual or host in need of treatment (eg, body weight), but still on a case-by-case basis The surrounding specific circumstances (including, for example, the particular agent administered, the route of administration, and the individual or host being treated) are routinely determined in a manner known in the art. In some cases, the desired dose is conveniently presented as a single dose or as divided doses administered simultaneously (or within a short period of time) or at appropriate intervals, for example as two, three, four or More sub-doses are available.

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 every 3 weeks at a dose as described herein.

In some embodiments, the anti-EGFR antibody can be administered according to a dose and dosing regimen determined to be safe and effective for the antibody alone or in combination with the IL-2 conjugate. In some embodiments, the anti-EGFR antibody is administered by intravenous infusion. In some specific examples, once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, Every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, every 19 weeks, Every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 25 weeks, every 26 weeks, every 27 weeks, or every 28 weeks to individuals in need Cetuximab (or another anti-EGFR antibody) is administered. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once a week. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered biweekly to an individual in need thereof. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every three weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need every 4 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need every 5 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need every 6 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to a subject in need every 7 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need every 8 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need every 9 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 10 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 11 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 12 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 13 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 14 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 15 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 16 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 17 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 18 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 19 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 20 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 21 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 22 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 23 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is administered to an individual in need thereof once every 24 weeks. In some embodiments, cetuximab (or other anti-EGFR antibodies).

In some embodiments, the anti-EGFR antibody is cetuximab. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 100 mg/m ² to about 500 mg/m ² . In any of the embodiments described herein, the loading dose of cetuximab is ^mg /m2 of the subject's body surface area. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 100 ^mg /m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 150 ^mg /m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about ²⁰⁰ mg/m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about ²⁵⁰ mg/m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 300 ^mg /m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 350 ^mg /m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 400 ^mg /m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 450 ^mg /m2. In some embodiments, cetuximab is administered by intravenous infusion at a loading dose of about 500 ^mg /m2. In some embodiments, the initial dose of cetuximab is administered by intravenous infusion at a loading dose of about 400 ^mg /m and is administered by intravenous infusion at a loading dose of about ²⁵⁰ mg/m All subsequent doses of cetuximab. In any of these embodiments, the cetuximab is infused over about 30-240 minutes. In some embodiments, the cetuximab is infused over about 30 minutes. In some embodiments, the cetuximab is infused over about 60 minutes. In some embodiments, the cetuximab is infused over about 90 minutes. In some embodiments, the cetuximab is infused over about 120 minutes. In some embodiments, the cetuximab is infused over about 150 minutes. In some embodiments, the cetuximab is infused over about 180 minutes. In some embodiments, the cetuximab is infused over about 210 minutes. In some embodiments, the cetuximab is infused over about 240 minutes. In any of these embodiments, at an infusion rate of about 1 mg/min to about 10 mg/min, such as 1 mg/min, 2 mg/min, 3 mg/min, 4 mg/min, 5 mg/min , 6 mg/min, 7 mg/min, 8 mg/min, 9 mg/min, or 10 mg/min administered cetuximab. 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 every 3 weeks at a dose as described herein. In some embodiments, cetuximab is administered every 2 weeks at a dose as described herein. In some embodiments, cetuximab is administered weekly at doses as described herein. Additional Pharmacy/Premedication

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

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

In some embodiments, any of the methods described herein further comprises administering a serotonin ₅ -HT3 receptor antagonist. In some embodiments, the serotonin ₅ -HT3 receptor antagonist is granisetron. In some embodiments, the serotonin ₅ -HT3 receptor antagonist is dolasetron. In some embodiments, the serotonin ₅ -HT3 receptor antagonist is tropisetron. In some embodiments, the serotonin ₅ -HT3 receptor antagonist is palonosetron. In some embodiments, the serotonin ₅ -HT3 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 further comprises administering an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine), an analgesic (eg, acetaminophen), and/or a serotonin ₅ -HT3 receptor antagonist (eg, granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, the method further comprises administering an antihistamine (eg, cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine) and an analgesic (eg, acetaminophen). In some embodiments, the method further comprises administering an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine) and a serotonin ₅ -HT3 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 -HT3 receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron) , or ondansetron). In some embodiments, any of the methods described herein further comprises administering an antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or diphenhydramine), an analgesic (such as acetaminophen), and serotonin ₅ -HT3 receptor antagonists (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron).

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

In some embodiments, the pre-administration of the IL-2 conjugate is different from the pre-administration of an anti-EGFR antibody, such as cetuximab. In some embodiments, the pre-administration of the IL-2 conjugate is the same as the pre-administration of an anti-EGFR antibody (eg, cetuximab). In some cases where the IL-2 conjugate and the pre-administration of an anti-EGFR antibody (such as cetuximab) are the same, only a single dose of the pre-administration is administered. In other instances where the IL-2 conjugate and the pre-administration of an anti-EGFR antibody (such as cetuximab) are the same, multiple doses of pre-administration are administered. In some embodiments, pre-administration is administered for all doses of IL-2 conjugate administered. In some embodiments, the pre-administration is for the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of the IL-2 conjugate but not for any subsequent doses of the IL-2 conjugate medicine. In some embodiments, the pre-dose is administered for the first 4 doses of the IL-2 conjugate rather than any subsequent doses of the IL-2 conjugate. In some embodiments, the pre-administration is administered for all doses of an anti-EGFR antibody (such as cetuximab) administered. In some embodiments, the pre-dose is administered for the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of an anti-EGFR antibody (such as cetuximab), but not for any Followed by several doses of anti-EGFR antibodies. In some embodiments, the pre-dose is administered to the first dose of the anti-EGFR antibody (such as cetuximab) rather than to any subsequent doses of the anti-EGFR antibody.

In some embodiments, any of the methods described herein further comprises administering a pre-administration prior to administering the IL-2 conjugate. In some embodiments, the pre-administration of the IL-2 conjugate is an antihistamine, such as cetirizine, promethazine, dextrochlorpheniramine, 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 pre-administration of the IL-2 conjugate is a serotonin ₅ -HT3 receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or Dansetron). In some embodiments, the serotonin ₅ -HT3 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 pre-administration of the IL-2 conjugate is an analgesic (such as acetaminophen). In some embodiments, acetaminophen is administered orally in a dose of about 650 to 1000 mg.

In some embodiments, any of the methods described herein further comprises administering a pre-administration prior to administering an anti-EGFR antibody, such as cetuximab. In some embodiments, the anti-EGFR antibody pre-administration 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 pre-administration of cetuximab is a serotonin ₅ -HT3 receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or Dansetron). In some embodiments, the serotonin ₅ -HT3 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 pre-administration is an analgesic (such as acetaminophen). In some embodiments, acetaminophen is administered orally in a dose of about 650 to 1000 mg.

In some embodiments, any of the methods described herein further comprises administering a first dose of pre-administration prior to administering the IL-2 conjugate, and administering an anti-EGFR antibody, such as cetuximab ) before the second dose of pre-dose. In some embodiments, the pre-administration of the IL-2 conjugate is the same as the pre-administration of an anti-EGFR antibody, such as cetuximab. In some embodiments, the pre-administration of the IL-2 conjugate is different from the pre-administration of an anti-EGFR antibody, such as cetuximab. In some embodiments, the pre-administration is an antihistamine, such as cetirizine, promethazine, dexpheniramine, 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 pre-administration is a serotonin ₅ -HT3 receptor antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or ondansetron). In some embodiments, the serotonin ₅ -HT3 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 pre-administration is an analgesic (such as acetaminophen). In some embodiments, acetaminophen is administered orally in a dose of about 650 to 1000 mg. In some embodiments, the pre-administration comprises an antihistamine and a serotonin ₅ -HT3 receptor antagonist. In some embodiments, the pre-administration includes an antihistamine and an analgesic. In some embodiments, the pre-administration comprises a serotonin ₅ -HT3 receptor antagonist and an analgesic. In some embodiments, the pre-administration comprises an antihistamine, a serotonin ₅ -HT3 receptor antagonist, and an analgesic. In some cases where the IL-2 conjugate and the pre-administration of the anti-EGFR antibody (such as cetuximab) (such as diphenhydramine) are the same, only a single dose of the pre-administration is administered. In other cases where the IL-2 conjugate and the pre-administration of an anti-EGFR antibody, such as cetuximab, are the same, multiple doses of pre-administration are administered.

In some embodiments of the methods described herein, the sequence of administration is as follows: (i) pre-administration of an anti-EGFR antibody (such as cetuximab); (ii) an anti-EGFR antibody (such as cetuximab) ; (iii) pre-administration of IL-2 conjugates; and (iv) IL-2 conjugates. In some variations where pre-administration of an anti-EGFR antibody (such as cetuximab) is the same as pre-administration of an IL-2 conjugate (such as diphenhydramine), administration of the IL-2 conjugate may be omitted Pre-administration of substances. In some embodiments, the sequence of administration is as follows: (i) pre-administration of IL-2 conjugate; (ii) IL-2 conjugate; (iii) pre-administration of anti-EGFR antibody (such as cetuximab) and (iv) an anti-EGFR antibody (such as cetuximab). In some variations where pre-administration of an anti-EGFR antibody (such as cetuximab) is the same as pre-administration of an IL-2 conjugate (such as diphenhydramine), the administration of the anti-EGFR antibody may be omitted. Pre-administration. individual

In some embodiments, an effective amount of the IL-2 conjugate and the anti-EGFR antibody is administered to an adult. In some specific instances, the adult is male. In other specific examples, the adult is female. In some embodiments, adulthood 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 infant, child, or adolescent is administered an effective amount of the IL-2 conjugate and the anti-EGFR antibody. In some embodiments, the individual is at least 1 month, 2 months, 3 months, 6 months, 9 months, or 12 months old. In some embodiments, the individual 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 individual has measurable disease (ie, cancer) as determined according to RECIST v1.1. In some embodiments, the individual has been adjudged to have an East Coast Cancer Clinical Research Collaborative (ECOG) performance status of 0 or 1. In some embodiments, the individual has adequate cardiovascular, hematological, hepatic, and renal function as determined by a clinician. In some embodiments, the individual has been determined (eg, by a clinician) to have a life expectancy greater than or equal to 12 weeks.

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

In some embodiments, the individual has a histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors, where the location of at least one tumor lesion can be safely produced based on clinical judgment (ie, as determined by a clinician). check. In some embodiments, the individual has received prior anticancer therapy prior to administration of the first therapeutic dose. In some embodiments, treatment-related toxicity of prior anticancer therapy has been eliminated to an appropriate degree.

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

In some embodiments, the individual is a male who has not been surgically sterilized and is using a medically acceptable method 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 or store sperm during the treatment period and continues for at least 3 months after the last treatment dose is administered.

In some embodiments, the individual has not received radiation therapy within 14 days of administration of the first therapeutic dose. In some embodiments, the individual has not received palliative radiation or stereotactic radiosurgery within 7 days of administration of the first therapeutic dose.

In some embodiments, the subject has not received systemic anticancer therapy or treatment with an investigational anticancer agent within 2 weeks of administration of the first therapeutic dose. In some embodiments, the subject has not been treated with immunotherapy or tyrosine kinase inhibitor therapy within 4 weeks of administration of the first therapeutic dose.

In some embodiments, the individual has not undergone major surgery within 30 days of administration of the first therapeutic dose. In some embodiments, the subject has not undergone major surgery more than 30 days prior to administration of the first therapeutic dose and has recovered to at least Grade 1 from any adverse effects associated with the procedure. In some specific instances, the individual is not expected to require major surgery during the course of treatment.

In some embodiments, the individual does not have an active autoimmune disease requiring systemic therapy within 3 months prior to administration of the first therapeutic dose. In some embodiments, the subject does not have a documented history of clinically severe autoimmune disease requiring systemic steroids or immunosuppressants prior to administration of the first therapeutic dose.

In some embodiments, the individual does not have a primary central nervous system (CNS) disease or leptomeningeal disease. In some embodiments, the individual has known CNS metastases, but has received appropriate treatment and is asymptomatic, has no evidence of radiographic progression for at least 8 weeks prior to administration of the first treatment dose, and No steroid or enzyme-inducing anticonvulsants are required within 14 days prior to dose.

In some specific instances, within 6 months of administration of the first therapeutic dose, the subject is free of lung function abnormalities, including pneumonia, active pneumonia, interstitial lung disease requiring steroid use, idiopathic pulmonary fibrosis, confirmed Hydropleural effusion, and severe dyspnea at rest or need for supplemental oxygen therapy.

In some embodiments, the subject is not taking parenteral antibiotics within 14 days of administration of the first therapeutic dose.

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

In some embodiments, the subject has no clinically significant bleeding (such as gastrointestinal bleeding, intracranial bleeding) within 2 weeks prior to administration of the first therapeutic dose. In some embodiments, the subject does not have a previously diagnosed deep vein embolism or pulmonary embolism within 3 months of administration of the first therapeutic dose.

In some embodiments, the subject has not had a severe or unstable cardiac condition (such as congestive heart failure (New York Heart Association Class III or IV), cardiac Bypass surgery or coronary stenting, angioplasty, cardiac ejection rate below the lower limit of normal, unstable angina, medically uncontrolled hypertension (eg, ≥160 mm Hg systolic or ≥100 mm Hg diastolic) ), uncontrolled arrhythmia requiring medication (≥ grade 2, according to NCI CTCAE V5.0), or myocardial infarction).

In some embodiments, the individual has no history of non-pharmacologically induced prolongation of the corrected QT interval, as determined using Fridericia's formula (QTcF) >450 milliseconds (msec) in males or >450 milliseconds (msec) in females 470 msec.

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

In some embodiments, the individual does not have an active second malignancy. In some specific instances, the individual has no history of prior malignancy. In some embodiments, the individual already has a non-melanoma skin cancer or cervical cancer that has been curatively removed by surgery prior to administration of the first therapeutic dose.

In some embodiments, the individual does not have any serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other apparent or unstable condition that would prevent or render treatment inappropriate Combined medical conditions.

In some specific instances, the subject is not pregnant or breastfeeding. In some embodiments, the subject is not expected to become pregnant or have a child during the course of treatment and is followed up to 3 months after the last treatment dose is administered.

In some embodiments, the subject did not receive any concomitant treatment with study agents, vaccines, or devices during the course of treatment. In some embodiments, the subject is receiving concomitant therapy with an investigational agent, vaccine, or device during the course of treatment after physician approval. drug efficacy

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

In some embodiments, treatment is discontinued based on a clinician's review of the radiographic images taken for the individual.

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

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

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

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

In some embodiments, administering an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not cause hypotension and reduce organ perfusion in the individual.

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

In some embodiments, administering an effective amount of an IL-2 conjugate and an anti-EGFR antibody to an individual is not associated with an increased risk of disseminated infection in the individual. In some embodiments, the disseminated infection is sepsis or bacterial endocarditis. In some specific cases, 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 IL-2 conjugate and anti-EGFR antibody are administered prior to administration of a drug selected from the group consisting of oxacillin, nafcillin, ciprofloxacin, and vancomycin. ) antibacterial agents to treat individuals.

In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not exacerbate a pre-existing or initially manifested autoimmune disease or inflammatory disorder in the individual. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not exacerbate a pre-existing or initially manifested autoimmune disease in the individual. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not exacerbate a pre-existing or initially manifested inflammatory disorder in the individual. In some embodiments, the subject's autoimmune disease or inflammatory disorder is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes, ocular myasthenia gravis, crescentic IgA glomerulonephritis , cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid. In some embodiments, the individual's autoimmune disease or inflammatory disorder is Crohn's disease. In some embodiments, the subject's autoimmune disease or inflammatory disorder is scleroderma. In some embodiments, the subject's autoimmune disease or inflammatory disorder is thyroiditis. In some embodiments, the subject's autoimmune disease or inflammatory disorder is inflammatory arthritis. In some embodiments, the individual's autoimmune disease or inflammatory disorder is diabetes. In some embodiments, the subject's autoimmune disease or inflammatory disorder is ocular myasthenia gravis. In some embodiments, the individual's autoimmune disease or inflammatory disorder is crescent IgA glomerulonephritis. In some embodiments, the subject's autoimmune disease or inflammatory disorder is cholecystitis. In some embodiments, the subject's autoimmune disease or inflammatory disorder is cerebral vasculitis. In some embodiments, the individual's autoimmune disease or inflammatory disorder is Stevens-Johnson syndrome. In some embodiments, the individual's autoimmune disease or inflammatory disorder is bullous pemphigoid.

In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not cause altered mental status, speech difficulties, cortical blindness, limb or gait ataxia, hallucinations, agitation in the individual , dullness, or coma. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not cause seizures in the individual. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to an individual is not contraindicated in an individual with a known epilepsy disorder.

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

In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not result in a decrease in the individual's mean arterial blood pressure following administration of the IL-2 conjugate to the individual. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to an individual does cause hypotension in the individual. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not result in the individual experiencing a systolic blood pressure below 90 mm Hg or a 20 mm Hg drop relative to baseline systolic blood pressure.

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

In some embodiments, administering an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not cause hypereosinophilia in the individual. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not result in an eosinophil count in the peripheral blood of the individual exceeding 500/μL. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not result in a peripheral blood eosinophil count in the individual exceeding 500/μL to 1500/μL. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not result in a peripheral blood eosinophil count in the individual exceeding 1500/μL to 5000/μL. In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not result in a peripheral blood eosinophil count of more than 5000/μL in the individual. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to an individual is not contraindicated in an individual using an existing psychotropic medication regimen.

In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to a subject in an existing regimen of nephrotoxic, myelotoxic, cardiotoxic or hepatotoxic drugs is not contraindicated. In some embodiments, in an individual using a current regimen of aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase, the individual is administered an effective amount of IL- 2 Conjugates and anti-EGFR antibodies are not contraindicated. In some embodiments, administration of an effective amount of an IL-2 conjugate and an anti-EGFR antibody to an individual receiving a combination regimen containing an antineoplastic agent is not contraindicated. In some embodiments, the anti-tumor agent is selected from the group consisting of dacarbazine, cisplatin, tamoxifen, and interferon-alpha.

In some embodiments, administration of an effective amount of the IL-2 conjugate and the anti-EGFR antibody to the individual does not cause one or more grade 4 adverse events in the individual following administration of the IL-2 conjugate to the individual. In some specific examples, the grade 4 adverse event is selected from the group consisting of hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; AV block II; 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 reactions; convulsions; grand mal convulsions; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; 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 population of individuals does not cause one or more Grade 4 in more than 1% of individuals following administration of the IL-2 conjugate to the individuals adverse events. In some specific examples, the grade 4 adverse event is selected from the group consisting of hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; AV block II; 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 reactions; convulsions; grand mal convulsions; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; 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 population of individuals does not cause one or more adverse events in more than 1% of individuals following administration of the IL-2 conjugate to the individuals , wherein one or more adverse events are selected from 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 population of individuals does not cause one or more adverse events in more than 1% of the individuals following administration, wherein the one or more adverse events is selected from malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary embolism; stroke; intestinal perforation; hepatic or renal failure; major depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure.

In some embodiments, administering an IL-2 conjugate and an anti-EGFR antibody to an individual increases the number of peripheral CD8+ T cells and NK cells in the individual without increasing the number of peripheral CD4+ regulatory T cells in the individual. In some embodiments, administering an IL-2 conjugate and an anti-EGFR antibody to an individual increases the number of peripheral CD8+ T and NK cells in the individual without increasing the number of peripheral eosinophils in the individual. In some embodiments, administering an IL-2 conjugate and an anti-EGFR antibody to an individual increases the number of peripheral CD8+ T and NK cells in the individual without increasing the number of intratumoral CD8+ T and NK cells in the individual and without increasing the individual The number of CD4+ regulatory T cells in the tumor.

In some embodiments, administering an effective amount of an IL-2 conjugate and an anti-EGFR antibody to an individual does not require the availability of an intensive care facility or a skilled specialist in cardiopulmonary or critical care medicine. In some embodiments, administering an effective amount of an IL-2 conjugate to an individual does not require the availability of an intensive care facility or a skilled specialist in cardiopulmonary or critical care medicine. In some embodiments, the availability of an intensive care facility is not required to administer an effective amount of the IL-2 conjugate to the individual. In some embodiments, administering an effective amount of an IL-2 conjugate to an individual does not require the availability of a skilled specialist in cardiopulmonary or critical care medicine.

In some embodiments, administration of the IL-2 conjugate and anti-EGFR antibody combination therapy improves ADCC responses to cancer, eg, by improving ADCC function of the anti-EGFR antibody. In some embodiments, administration of an IL-2 conjugate and an anti-EGFR antibody in 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 an IL-2 conjugate and an anti-EGFR antibody results in a synergistic improvement in the anticancer activity of the combination of the two agents when compared to the anticancer activity of either agent alone. In some embodiments, administration of the IL-2 conjugate increases the number and activation of NK cells, which enhances ADCC triggered by anti-EGFR antibodies. extra potion

In some embodiments, in addition to the anti-EGFR antibody, the methods further comprise administering to the individual a therapeutically effective amount of one or more chemotherapeutic agents. In some embodiments, the one or more chemotherapeutic agents comprise one or more platinum-based chemotherapeutic agents. In some embodiments, the one or more chemotherapeutic agents comprise carboplatin and pemetrexed. In some embodiments, the one or more chemotherapeutic agents comprise carboplatin and nab-paclitaxel. In some embodiments, the one or more chemotherapeutic agents comprise carboplatin and docetaxel. In some embodiments, the individual's cancer is non-small cell lung cancer (NSCLC). Kits/Products

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

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

In one specific example, the label is on or associated with the container. In one particular example, a label is attached to a container when the letters, numbers or other characters forming the label are affixed, moulded or inscribed into the container itself, and a label is attached to a container when the label is present in a rack or shelf that holds the container. Associated with the container, for example as a package insert. In one specific example, the label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates instructions for using the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms containing a compound provided herein. For example, the package contains metal or plastic foil, such as a blister pack. In one embodiment, instructions for administration are accompanied by the pack or dispenser device. In one specific example, the package or dispenser is further accompanied by a notice associated with the container, in a form prescribed by a governmental agency regulating the manufacture, use or sale of a drug product, the notice reflecting the agency's approval of the following drug form for human use or veterinary administration. Examples of such notifications are FDA-approved drug labels or approved product inserts. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in a suitable container, and labeled for treatment of an indicated condition. Illustrative concrete example

The present invention will be further explained by the following specific examples. Where appropriate and practical, the features of each specific example may be combined with any other specific example.

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；或 Y是CH ₂且Z是

； W是具有平均分子量選自約5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa、50 kDa、和60 kDa的PEG基團；且 X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點； 其中式(I)結構在IL-2接合物的胺基酸序列中的位置是選自K34、F41、F43、K42、E61、P64、R37、T40、E67、Y44、V68、和L71， 或其醫藥上可接受的鹽、溶劑合物，或水合物。 Specific Example P1. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody, wherein the IL-2 conjugate Comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is substituted with a structure of formula (I):

Formula (I); wherein: Z is _CH and Y is

; Y is _CH and Z is

; Z is _CH and Y is

; or Y is _CH and Z is

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

X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue; wherein the structure of formula (I) is at the amine of the IL-2 conjugate The position in the amino acid sequence is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, or a pharmaceutically acceptable salt, solvate, or hydrate thereof .

。 Example P2. The method of Example P1, wherein in the IL-2 conjugate, Z is _CH and Y is

.

。 Example P3. The method of Example P1, wherein in the IL-2 conjugate, Y is _CH and Z is

.

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

Example P5. The method of Example 4, wherein in the IL-2 conjugate, the PEG group has an average molecular weight of 30 kDa.

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

式(XII)；

式(XIII)； 其中： n是約2至約5000範圍內的整數；且 波浪線表示與SEQ ID NO：3內未被取代的胺基酸殘基的共價鍵， 或其醫藥上可接受的鹽、溶劑合物，或水合物。 Specific example P7. The method of specific example P1, wherein the structure of formula (I) has the structure of formula (XII) or formula (XIII), or is a mixture of structures of formula (XII) and formula (XIII):

Formula (XII);

Formula (XIII); wherein: n is an integer in the range of about 2 to about 5000; and the wavy line represents a covalent bond to the unsubstituted amino acid residue within SEQ ID NO: 3, or a pharmaceutically acceptable salts, solvates, or hydrates.

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

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

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

式(IV)；

式(V)； 其中： W是具有平均分子量選自5 kDa、10 kDa、15 kDa、20 kDa、25 kDa、30 kDa、35 kDa、40 kDa、45 kDa、50 kDa、和60 kDa的PEG基團； X具有以下結構：

； X-1表示與前一個胺基酸殘基的附接點；以及 X+1表示與下一個胺基酸殘基的附接點； 或其醫藥上可接受的鹽、溶劑合物，或水合物。 Specific example P11. A method of treating cancer in an individual in need, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody, wherein the IL-2 conjugate An amino acid sequence comprising SEQ ID NO: 50, wherein [AzK_L1_PEG30kD] has a structure of formula (IV) or formula (V), or a mixture of structures of formula (IV) and formula (V):

formula (IV);

Formula (V); wherein: W is a PEG group having an average molecular weight selected from the group consisting of 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa group; X has the following structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue; or a pharmaceutically acceptable salt, solvate thereof, or Hydrate.

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.

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

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

式(XII)；

式(XIII)； 其中： n是一個使得-(OCH ₂CH ₂) _n-OCH ₃具有約30 kDa的分子量的整數；且 波浪線表示與SEQ ID NO：50內未被取代的胺基酸殘基的共價鍵， 或其醫藥上可接受的鹽、溶劑合物、或水合物。 Specific example P15. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) cetuximab, wherein the IL-2 The conjugate comprises the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG30kD] has a structure of formula (XII) or formula (XIII), or a mixture of structures of formula (XII) and formula (XIII):

Formula (XII);

Formula (XIII); wherein: n is an integer such that -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 30 kDa; and the wavy line represents the unsubstituted amino acid residue in SEQ ID NO: 50 A covalent bond of the base, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Specific example P16. The method of any one of specific examples P1-15, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), typical Hodge Gold lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable carcinoma, microsatellite stable carcinoma, gastric cancer, colon cancer, colorectal cancer (CRC), sub- Cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophagus, 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) deficiency, bladder cancer, ovarian cancer, moderate to low mutation tumor burden, cutaneous squamous cell carcinoma (CSCC), squamous cell skin carcinoma (SCSC), tumors with low to no expression of PD-L1, tumors with systemic diffuse to the liver and CNS beyond their primary anatomical origin, and diffuse large B-cell lymphoma.

Specific example P17. The method of any one of specific examples P1-16, wherein once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, and once every 7 weeks The IL2 conjugate is administered to the individual once, or once every 8 weeks.

Embodiment P18. The method of any one of Embodiments P1-17, wherein the IL-2 conjugate is administered to the subject by intravenous administration. Example

These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. Example 1. Preparation of IL-2 conjugate IL-2_P65_[AzK_L1_PEG30kD]-1.

IL-2 for bioconjugation was expressed as inclusion bodies in E. coli using the methods disclosed herein using (a) expression plasmids encoding: (i) a protein with the desired amino acid sequence whose gene Contains the first unnatural base pair to provide at the desired position to incorporate the unnatural amino acid N6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK) codons, and (ii) a tRNA derived from M. pasteurii Pyl whose gene contains a second non-natural nucleotide to provide a matching anti-codon in place of its native sequence; (b) coding derived from Plastids of Methanosarcina pyrrolidinyl-tRNA synthetase ( Mb PylRS); (c) AZK; and (d) variants of the truncated nucleotide triphosphate transporter PtNTT2 in which the full-length protein The first 65 amino acid residues are missing. The double-stranded oligonucleotide encoding the amino acid sequence of the IL-2 variant contains codon AXC as codon 64 encoding the sequence of the protein having SEQ ID NO: 3, wherein P64 is replaced by the unnatural amino group described herein Acid substitution. The plastid encoding the orthogonal tRNA gene from M. mazei contains an anticodon GYT matching AXC in place of its native sequence, where Y is a non-natural nucleotide disclosed herein. X and Y are selected from the non-natural nucleotides dTPT3 and dNaM as disclosed herein. The expressed protein was extracted from the inclusion bodies and refolded using standard procedures, and the AzK-containing IL-2 product was site-specifically pegylated using DBCO-mediated copper-free click chemistry to convert the stable co- A valent mPEG 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, wherein the proline at position 64 is replaced by AzK_L1_PEG30kD, wherein AzK_L1_PEG30kD is defined as the structure of formula (IV) or formula (V) , or a mixture of formula (IV) and formula (V), and a linear mPEG chain of 30 kDa. The IL-2 conjugate "IL-2_P65_[AzK_L1_PEG30kD]-1" is also defined as a compound comprising SEQ ID NO: 3, wherein the proline residue (P64) at position 64 is represented by 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" is also defined as a compound comprising SEQ ID NO: 3 wherein the proline residue at position 64 (P64) is replaced by 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 cellular cytotoxicity (ADCC) assay using IL-2_P65_[AzK_L1_PEG30kD]-1 and cetuximab

Calcein-Acetyloxymethyl (calcein-AM; Invitrogen) release assay was used to examine the efficacy of IL-2_P65_[AzK_L1_PEG30kD]-1 on the ADCC function of cetuximab.

Material.

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

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

program.

Human primary NK cells were negatively selected from PBMCs using the RoboSep™ instrument according to the manufacturer's recommended protocol. Purified NK cells were treated with IL-2_P65_[AzK_L1_PEG30kD]-1 at different concentrations (0.1 μg/mL, 0.01 μg/mL, 0.001 μg/mL, and 0 μg/mL) in RPMI 1640 medium (supplemented with 1% low IgG). FBS) for 18 h at 37 °C in a humidified incubator with 5% _CO . These cultured cells serve as effector cells. Human EGFR-positive cancer cell lines (A431) were labeled with Calcein-AM for 30 minutes (50 μg diluted in 25 μL DMSO to prepare a stock solution, then 10 μL of the calcein stock solution was added to 4 mL RPMI 1640 + 1% low IgG FBS + 1% Prosulfone 4 x 10 ⁶ cells were stained for suture), then washed and plated in 96-well round dishes at a density of 5 x 10 ³ cells/well. Cetuximab and isotype human IgG1 antibodies were added at various concentrations (from 10 μg/mL to 1 pg/mL) for 30 minutes to allow for opsonization prior to the addition of NK cells. IL-2_P65_[AzK_L1_PEG30kD]-1 activated NK cells were collected and added as effector cells at an E:T ratio of 3:1 ( ⁶ x 104 NK cells for 2 x ¹⁰⁴ target cells). Plates were then incubated in a humidified incubator at 37°C, 5% CO for ₁ hour, 90 μL of supernatant was harvested and transferred to light-tight 96-well microplates for fluorometry on an Envision 2104 plate reader. (Excitation: 492 nm; Emission: 515 nm).

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

The cytotoxicity and ADCC percentages for each plate (duplicate) were calculated using the following formula: Cytotoxicity (%)=(A-B)/(C-B)×100 ADCC (%) = cytotoxicity (%, with antibody) - cytotoxicity (%, without antibody) For each experiment, measurements were performed in triplicate using three replicate wells. Each experiment was repeated at least 3 times. Half-maximal effective concentration (EC50) values were calculated by fitting the data points to a 4-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA).

result.

In the absence of activation by IL-2_P65_[AzK_L1_PEG30kD]-1, human NK cells treated with cetuximab exhibited cytotoxicity against EGFR-expressing cancer cell lines (A431 and A549), but not against EGFR null Expressive cells (NCI-H69). Cetuximab-treated human NK cells exhibited enhanced cytotoxicity against EGFR-expressing cancer cell lines (A431 and A549) after activation by IL-2_P65_[AzK_L1_PEG30kD]-1. Example 3. In vitro studies of IL-2 conjugates and cetuximab (PBMC ADCC assay).

Studies were conducted to investigate the effect of antibody-dependent cellular cytotoxicity (ADCC) by the IL-2 conjugate of Example 1 and with cetuximab using human PBMCs with calcein-labeled cancer cell lines (CAL27 and A431). co-culture. CAL27 cells.

reagents.

Bioassay buffer: 1% ultra-low IgG FBS was added to phenol red free RPMI. Complete Assay Buffer: Add 450 μL probenecid to 45 mL bioassay buffer for a final probenecid concentration of 77 μg/mL. Calcein-acetoxymethyl ester (Calcein-AM): 50 μg in 25 μL DMSO. Calcein-AM staining buffer: Add 10 μL of Calcein-AM to 4 mL of Complete Assay Buffer (final concentration of Calcein-AM is 5 μg/mL). Triton-X-100 Lysis Buffer: Add 20 μL Triton-X-100 to 4 mL Complete Assay Buffer (0.5% final concentration).

program.

On day 1, 6-point, 1-to-5 serial dilutions (in PBS) of the IL-2 conjugate were prepared. IL-2 conjugate concentrations were 2, 0.4, 0.08, 0.016, 0.0032, and 0 μg/mL. PBMCs were collected by centrifugation at 200 xg 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 aliquots of multiwell reservoirs to which a series of IL-2 conjugate dilutions were added. The PBMCs were well mixed with the IL-2 conjugate by pipetting up and down, and 50 μL were transferred into a round bottom 96-well plate using a multichannel pipette (final number of PBMCs per well was 1 million). Six empty wells were reserved for controls to be added 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 expressing dissociation buffer and harvested by centrifugation at 200 x g for 5 minutes. Cells were counted and 5 million cells were resuspended in 4 mL of Calcein-AM staining buffer and incubated at 37°C in the presence of 5% carbon dioxide for 30 minutes. 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 0.4 million cells/mL for a final target cell count of 20,000 cells/well.

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

50 μL of calcein-AM-stained CAL27 cells without PBMCs treated with complete assay buffer (background signal), or 50 μL of Triton-X-100-treated stained CAL27 (maximum signal after lysis) control wells, both made up to a final volume of 150 μL with complete assay buffer and prepared in triplicate. The disks were centrifuged at 200 x g for 1 minute and then incubated at 37°C in the presence of 5% carbon dioxide for 60 minutes. After incubation, the plates were briefly centrifuged again, then 90 μL of the supernatant was transferred to a fresh black transparent dish and the fluorescent signal was read on an Envision 2104 plate reader (excitation: 492 nm; emission: 515 nm).

Cytotoxicity was calculated using the following formula: Cytotoxicity (%)=(A-B)/(C-B)×100 where A is the fluorescence value of treated cells; B is the background from target cells only; C is the maximum release value obtained from Triton-X-100 treatment.

Data represent percent cytotoxicity of IL-2 conjugate-treated human PBMCs against target cancer cells in the presence of cetuximab. The average percentage of technical replicates was converted to a scale. Analyses were performed using a two-way generalized linear mixed model (GLMM) with factors for IL-2 conjugate, cetuximab, and their interaction, together with random donor effects, treating proportions as pseudo-binomial variables. A post hoc assay (adjusted using Dunnett-Hsu) was then performed to compare the IL-2 conjugate treated group with the control group. Statistical analysis was performed using SAS (1) version 9.4 software. A probability of less than 5% (p<0.05) was considered significant.

result.

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

Fixed effects assays in the GLMM model showed that the factors IL-2 conjugates, cetuximab and their interaction had significant effects on cytotoxicity, that is, for different cetuximab concentrations, the IL-2 conjugate group had a significant effect on cytotoxicity. The differences are significantly different. Pairwise comparisons were made between the indicated IL-2 conjugate 2 mg/mL group and the control group at a cetuximab concentration of 0.001 mg/mL (p=0.0001), and IL-2 conjugate 0.4 mg/mL There was a significant difference between the mL group and the control group (p=0.0001). Pairwise comparisons also indicated IL-2 conjugate 0.4 mg/mL between IL-2 conjugate 2 mg/mL and the control group at a cetuximab concentration of 0.01 mg/mL (p<0.0001) There was a significant control between the group and the control group (p<0.0001), and between the IL-2 conjugate 0.08 mg/mL group and the control group (p=0.0003). In addition, pair-wise comparisons indicated between the IL-2 conjugate 2 mg/mL group and the control group at a cetuximab concentration of 0.1 mg/mL (p<0.0001), IL-2 conjugate 0.4 mg There were significant differences between the /mL group and the control group (p<0.0001), and between the IL-2 conjugate 0.08 mg/mL group and the control group (p<0.0001). Finally, pair-wise comparisons at a cetuximab concentration of 1 mg/mL indicated that between the IL-2 conjugate 2 mg/mL group and the control group (p<0.0001), the IL-2 conjugate 0.4 mg There were significant differences between the /mL group and the control group (p<0.0001), and between the IL-2 conjugate 0.08 mg/mL group and the control group (p<0.0001).

The data demonstrate that the IL-2 conjugate enhances the ADCC function of cetuximab against EGFR-expressing CAL27 cancer cells. No significant differences were observed with the IL-2 conjugate and with the isotype control. A431 cells

The study was performed using EGFR expressing A431 cells (epidermoid carcinoma) following the procedure outlined above for CAL27 cells. Figure 2B shows that cetuximab improves ADCC function against EGFR expressing A431 cells (PBMC to A431 ratio 50:1). The data demonstrate that the IL-2 conjugate enhances the ADCC function of cetuximab against EGFR expressing A431 cancer cells. Example 4. ADCC analysis using the engineered cell line NK-92.CD16 V as effector cells.

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

Material.

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

The following reagents were used: cetuximab antibody (Eli Lilly &Co.); human isotype IgG1 antibody (Biolegend); calcein-acetoxymethyl (calcein-AM; Invitrogen C3100MP) and probenecid ( Invitrogen; P36400). 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 Cat# 05150) supplemented with IL-2 (100 U/mL) and hydrocortisone (Sigma H6909; 10 mL at 50 µM) for NK-92.CD16 V cell culture.

program.

IL-2 supplements were retrieved from NK-92.CD16 V cell cultures, which were incubated overnight before starting the analysis. The following day, IL-2_P65_[AzK_L1_PEG30kD at different concentrations (0.1 μg/mL, 0.01 μg/mL, 0.001 μg/mL, and 0 μg/mL) in phenol red-free RPMI 1640 medium (supplemented with 1% low IgG FBS) ]-1, cells were plated in 96-well round dishes (60,000 cells were plated at a 3:1 ratio of effector to target cells) at 37°C in a 5% _CO humidified incubator for 18 hours . These cells serve 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 diluted in 25 μL DMSO to prepare a stock solution, and then 10 μL of the calcein stock solution was added to the cells). 4 ml RPMI 1640 containing 1% low IgG FBS with 1% probenecid for staining ⁵ x 106 cells), followed by washing. Cells were split into several labeled tubes for incubation with different concentrations of cetuximab or isotype controls. Cetuximab and isotype human IgG1 antibody were added at 3X concentrations (10 μg/mL to 1 pg/mL final assay concentration), while labeled target cells and antibody were mixed and allowed to stand for 30 minutes to allow for opsonization effect. After this incubation, target cells (20,000) and antibody were added on top of NK-92.CD16 V cells in 100 μL. The disks were briefly centrifuged at 1100 rpm for 1 min, then incubated at 37 °C and 5% _CO for 1 h. After incubation, the plates were briefly centrifuged again as before and 90 μL of supernatant was transferred from each well to a black plate with a clear bottom without disturbing the cells. Fluorescence signals were read using an Envision 2104 (excitation: 492 nm; emission: 515 nm).

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

The cytotoxicity and ADCC percentages per plate (duplicate) were calculated using the following formulas: Cytotoxicity (%)=(A-B)/(C-B)×100 ADCC (%) = cytotoxicity (%, with antibody) - cytotoxicity (%, without antibody) For each experiment, measurements were performed in triplicate using three replicate wells. Each experiment was repeated at least 3 times. Half-maximal effective concentration (EC50) values were calculated by fitting the data points to a 4-parameter equation using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA).

result.

EGFR-expressing A431 (epidermoid carcinoma) (NK92 vs. A431 vs. 3:1), DLD-1 (adenocarcinoma, colorectal) (NK92 vs. DLD-1 vs. 3:1 vs. 3:1), FaDu (epithelial squamous cell carcinoma) (NK92 to FaDu ratio 3:1), and CAL27 (epithelial squamous cell carcinoma) (NK92 to CAL27 ratio 3:1) cells, cytotoxicity data analyzed using the NK92 cell line ADCC are shown in Figures 3A-D, respectively. The data demonstrate that the IL-2 conjugate enhances the ADCC function of cetuximab against EGFR-expressing cancer cells. Example 5. Clinical study of combination therapy with IL-2 conjugate and cetuximab.

Overview. Monotherapy using the IL-2 conjugate of Example 1 has been shown to promote an increase in NK cell numbers at the periphery, which mediates antibody-dependent cellular cytotoxicity (ADCC) for IgG1 antibodies such as cetuximab. ) important effector cells.

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

Participants received IL-2 conjugate (16 or 24 μg/kg dose) by IV infusion every 3 weeks. Here and throughout this discussion of this population, the mass of drug per kilogram of subject (eg, 16 μg/kg) refers to the mass of IL-2 excluding the mass of PEG and linker. Cetuximab was administered as an initial loading dose of 400 mg/m2 infused over ¹²⁰ minutes (maximum infusion rate 10 mg/min) on Day 1 of Cycle 1, followed by all subsequent doses starting on Day 8 of Cycle 1 A dose of ²⁵⁰ mg/m2 was infused over 60 minutes (maximum infusion rate 10 mg/min). Cetuximab was administered on days 1, 8, and 15 of each 21-day cycle. The infusion time of the IL-2 conjugate was approximately 30 minutes each. For each treatment cycle, prior to IL-2 conjugate administration, all participants received IL-2 conjugate pre-administration 30 to 60 minutes prior to IL-2 conjugate infusion to prevent or reduce infusion Acute effects of associated reaction (IAR) or flu-like symptoms. The IL-2 conjugate was pre-administered as follows: antipyretic, oral, and antihistamine (H1 blocker). Antiemetics were provided at the discretion of the supervising clinician. All participants were pre-administered with diphenhydramine (approximately 25 to 50 mg, intravenously) prior to the first dose of cetuximab. Pre-administration of subsequent doses of cetuximab is optional based on the assessment of the supervising clinician. When the IL-2 conjugate and cetuximab were administered on the same day, participants taking diphenhydramine as a pre-administration of cetuximab may have omitted diphenhydramine as a pre-IL-2 conjugate Dosing. The dosing sequence was as follows: (i) pre-administration of cetuximab (30 to 60 minutes prior to start of cetuximab infusion); (ii) cetuximab; (ii) IL-2 engagement pre-administration of the IL-2 conjugate (administered 30 to 60 minutes before the start of the IL-2 conjugate infusion); and (iv) the IL-2 conjugate. Treatment was repeated up to a total of 35 cycles or a duration of up to 735 days.

The following biomarkers were used as surrogate predictors of safety and/or efficacy: Eosinophilia (increased peripheral eosinophil count): a cellular surrogate marker for IL-2-induced proliferation of cells (eosinophils) associated with vascular leak syndrome (VLS); Interleukin 5 (IL-5): IL-2-induced activation of type 2 innate lymphocytes and the release of this cytokine surrogate marker for chemoattractant eosinophilia and potential VLS; Interleukin 6 (IL-6): a cytokine surrogate marker for IL-2-induced cytokine release syndrome (CRS); and Interferon gamma (IFN-γ): a cytokine surrogate marker for IL-2-induced activation of CD8+ cytotoxic T lymphocytes.

The following biomarkers were used as surrogate predictors of antitumor immune activity: Peripheral CD8+ effector cells: a marker of IL-2-induced proliferation of these target cells in the periphery, which upon infiltration becomes a surrogate marker for inducing a possible latent therapeutic response; Peripheral CD8+ memory cells: a marker of IL-2-induced proliferation of these target cells in the periphery, which upon infiltration becomes a surrogate marker for inducing populations that may sustain latent therapy and maintain memory; Peripheral NK cells: a marker of IL-2-induced proliferation of these target cells in the periphery, which upon infiltration becomes a surrogate marker for inducing a potentially rapid therapeutic response; and Peripheral CD4+ regulatory cells: IL-2-induced marker of proliferation of these target cells in the periphery, which upon infiltration becomes a surrogate marker for inducing immunosuppressive TME and counteracting the effects of effector-like therapeutics. First population using IL-2 conjugate at a dose of 16 μg/kg

result. Five subjects (including two human males and three females, with a median age of 69 years (range 65-72 years)). All subjects had an East Coast Cancer Clinical Research Collaborative (ECOG) performance status of 0 or 1 and had received 1 to 4 prior lines of systemic therapy. Cancers were anal carcinoma (1 subject), colon adenocarcinoma (1 subject), adrenocortical carcinoma (1 subject), lung squamous cell carcinoma (1 subject), and small bowel cancer (1 subject). All five subjects had metastatic disease.

Subjects received IL-2 conjugate (16 μg/kg) and cetuximab combination therapy for 2 to 7 cycles (2-7 doses of IL-2 conjugate). Two subjects (one with anal cancer and one with metastatic adrenocortical cancer) had demonstrated progressive disease (PD) after 2 cycles of combination therapy leading to IL-2 conjugate and cetuximab Anti-combination therapy was discontinued. One subject with colon cancer had disease progression after 5 cycles. Two subjects are still ongoing: one (lung squamous cell carcinoma) for 3 cycles and one (small bowel cancer) for 7 cycles.

Peripheral CD8+ _Teff cell counts were measured (FIG. 4A-B). In some subjects, prolonged CD8+ expansion over baseline (eg, greater than or equal to 2-fold change) was observed 3 weeks after the previous dose.

Peripheral NK cell counts are shown in Figure 5A-B. Increased NK cell counts were observed in each subject. Subjects typically exhibited elevated NK cell counts above baseline at 8 days and 3 weeks after the previous dose.

Peripheral CD4+ T _reg counts are shown in Figures 6A-B.

Eosinophil counts were measured (Figure 7A-B). Measurements did not increase more than fourfold, and as reported in Pisani et al., Blood 1991 Sep 15;78(6):1538-44, in patients with IL-2-induced eosinophilia, the same Below the range of 2328-15958 eosinophils/μL. Lymphocyte counts were also measured (Figure 8A-B).

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

An adverse event (AE) is any inappropriate medical event, regardless of causality, that occurs in a clinical research subject administered a medicinal product. Dose-limiting toxicity was defined as an AE that occurred within ±1 days from Day 1 to Day 29 (inclusive) of a treatment cycle that was not uniquely or indisputably related to an extrinsic cause and that met at least one of the following criteria : • Grade 3 neutropenia (absolute neutropenia <1000/mm ³ > 500/mm ³ ) for ≥ 7 days, or Grade 4 neutropenia of any duration • Grade 3 or higher Febrile neutropenia • Grade 4 or higher thrombocytopenia (platelet count 25,000/mm ³ ) • Grade 3 or higher thrombocytopenia (platelet count < 50,000-25,000/mm ³ ) for ≥ 5 days, or with clinical Significant bleeding or need for platelet transfusion Failure to meet recovery criteria of absolute neutrophil count of at least 1,000 cells/ ^mm3 and platelet count of at least 75,000 cells/ ^mm3 within 10 days Any other grade 4 or higher hematology Toxicity persisting for ≥5 days Grade 3 or higher ALT or AST with bilirubin >2 times ULN, no evidence of cholestasis or other causes (such as viral infection or other drugs) (ie, Hy's law) Before Grade 3 infusion-related reactions that occurred with drug placement; Grade 4 infusion-related reactions • Grade 3 Vascular Leak Syndrome, defined as hypotension associated with fluid retention and pulmonary edema • Grade 3 hypersensitivity reactions • Grade 3 or higher hypotension • 3 Grade ≥ AEs that do not resolve to <Grade 2 within Initiating Acceptable Care Medical Management Criteria 7 • Grade 3 or greater cytokine release syndrome The following exceptions apply to non-hematological AEs: • Grade 3 fatigue, nausea, vomiting or diarrhea, use Best care medical management does not resolve to <Grade 2 within ≤ 3 days Grade 3 fever (defined as >40°C ≤ 24 hours) Grade 3 infusion-related reactions that occur without predose; subsequent doses should be With pre-dose and DLT if reaction recurs Grade 3 arthralgia or rash, resolves to ≤ 2 within 7 days after acceptable standard of care medical management (eg, systemic corticosteroid therapy) if subject A grade 1 or 2 ALT or AST elevation at baseline is considered to occur after liver metastases, and a grade 3 elevation must also be >3 times baseline and persist for >7 days.

A serious AE is defined as any AE that results in any of the following outcomes: death; life-threatening AE; hospitalization or prolongation of pre-existing hospitalization; persistent or significant incapacity or substantial disruption of the ability to carry out normal life functions ; or congenital anomalies/birth defects. A major medical event that may not result in death, life-threatening disease, or require hospitalization, may be considered serious when, based on proper medical judgment, it may endanger the subject and may require medical or surgical intervention to prevent one of the above outcomes. Examples of such medical events include allergic bronchospasm requiring intensive treatment in the emergency room or at home, blood dyscrasias or convulsions that do not lead to hospitalization, or the development of drug dependence or drug abuse.

IL-5 was not significantly elevated. No cumulative toxicity. No end-organ toxicity. There was no QTc prolongation or other cardiotoxicity. Overall, the IL-2 conjugates were considered to be well tolerated.

Four of the five subjects had at least one treatment-emergent AE (TEAE). Most TEAEs were grade 1-2, with one subject having at least one grade 3 TEAE and one subject having at least one grade 4 TEAE. Four subjects had treatment-related AEs. These included: one grade 1 infusion reaction; one grade 1 nausea; one grade 1 fatigue; one grade 2 diarrhea; and one grade 4 decreased lymphocyte count. Two subjects had 3 unrelated SAEs: one with dysphagia and spinal cord compression; and one with hydropleural effusion. The TEAE did not result in any drug discontinuation, dose reduction, DLT, and no anaphylaxis or CRS. Treatment-related AEs were resolved using acceptable standards of care. TEAEs are detailed in Table 2, while treatment-related adverse events are summarized in Table 3. Table 2. Treatment-emergent adverse events: n=5. System Organ Class Level 1 Level 2 Level 3 Level 4 Level 5 General Diseases and Administration Site Conditions 2/5 (40%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Gastrointestinal disease 2/5 (40%) 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%) Research 0/5 (0%) 0/5 (0%) 0/5 (0%) 1/5 (20%) 0/5 (0%) infection and infestation 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Injuries, surgical complications 2/5 (40%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Nervous system disease 0/5 (0%) 0/5 (0%) 2/5 (40%) 0/5 (0%) 0/5 (0%) Skin and subcutaneous tissue disorders 3/5 (60%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Blood and Lymphatic System Disorders 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) eye disease 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Metabolic and Nutritional Diseases 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Respiratory, Thoracic and Mediastinal Disorders 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) endocrine disease 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Musculoskeletal and connective tissue disorders 1/5 (20%) 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) mental illness 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Reproductive and Breast Disorders 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Kidney and Urinary Diseases 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Table 3. Treatment-related adverse events: n=5. System Organ Class Level 1 Level 2 Level 3 Level 4 Level 5 endocrine disease 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) General Diseases and Administration Site Conditions 2/5 (40%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Gastrointestinal disease 1/5 (20%) 1/5 (205%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Research 0/5 (0%) 0/5 (0%) 0/5 (0%) 1/5 (20%) 0/5 (0%) Injuries, surgical complications 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%) Nervous system disease 0/5 (0%) 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%) Second population using IL-2 conjugate at 24 μg/kg dose

result. Three subjects were human males with a median age of 71 years (range 65 to 75 years). All subjects had an East Coast Cancer Clinical Research Collaborative (ECOG) performance status of 1. One subject had received 1 frontline therapy, while the second subject had received 4 previous lines of therapy. Cancers were gastric cancer (1 subject), head and neck squamous cell carcinoma (HNSCC) (1 subject), and colon cancer (1 subject). All subjects had metastatic disease. Subjects received a combination of IL-2 conjugate (24 µg/kg) and cetuximab for 1 cycle (1 dose of IL-2 conjugate). One subject exhibited progressive disease (PD) after the first cycle of combination therapy, precluding administration of further therapeutic doses of the IL-2 conjugate and cetuximab combination therapy.

Two subjects experienced at least one TEAE. One of the subjects experienced at least one grade 3 treatment-related AE, grade 3 chills. No subjects had associated SAEs. No DLTs were observed, and there were no discontinuations due to TEAEs. TEAEs are detailed in Table 4, while treatment-related adverse events are summarized in Table 5. Table 4. Treatment-emergent adverse events: n=3. System Organ Class Level 1 Level 2 Level 3 Level 4 Level 5 Gastrointestinal disease 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) General Diseases and Administration Site Conditions 1/3 (33.3%) 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) infection and infestation 0/3 (0%) 2/3 (66.7%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Metabolic and Nutritional Diseases 0/3 (0%) 2/3 (66.7%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Musculoskeletal and connective tissue disorders 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Nervous system disease 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Kidney and Urinary Diseases 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Respiratory and Mediastinal Disorders 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Skin and subcutaneous tissue disorders 1/3 (33.3%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Table 5. Treatment-related adverse events: n=3. System Organ Class Level 1 Level 2 Level 3 Level 4 Level 5 Gastrointestinal disease 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) General Diseases and Administration Site Conditions 1/3 (33.3%) 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) infection and infestation 0/3 (0%) 2/3 (66.7%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Metabolic and Nutritional Diseases 0/3 (0%) 2/3 (66.7%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Nervous system disease 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Respiratory and Mediastinal Disorders 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) Skin and subcutaneous tissue disorders 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%)

While the foregoing invention has been described in detail by way of illustration and illustration for the purpose of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. Numerous variations, changes and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the specific examples of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and to thereby cover methods and structures within the scope of these claims and their equivalents. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety. To the extent that any material incorporated herein by reference is inconsistent with the statement of the present invention, the statement shall control.

none

The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be more fully understood with reference to the following description, which sets forth illustrative examples in which the principles of the invention are utilized, and the accompanying drawings, wherein:

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

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

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

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

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

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

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

Figure 4A shows peripheral CD8+ T _eff cell counts at indicated times following administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab.

Figure 4B shows changes in peripheral CD8+ T _eff cell counts at indicated times after administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab. Data were normalized to pre-treatment (C1D1) CD8+ T _eff cell counts.

Figure 5A shows peripheral NK cell counts at indicated times after administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab.

Figure 5B shows changes in peripheral NK cell counts at indicated times following administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab. Data were normalized to pre-treatment (C1D1) NK cell counts.

Figure 6A shows peripheral CD4+ T _reg cell counts at indicated times after administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab.

Figure 6B shows changes in peripheral CD4+ T _reg cell counts at indicated times after administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab. Data were normalized to pre-treatment (C1D1) CD4+ T _reg cell counts.

Figure 7A shows eosinophil counts at indicated times after administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab.

Figure 7B shows changes in eosinophil counts at indicated times following administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab. Data were normalized to pre-treatment (C1D1) eosinophil counts.

Figure 8A shows lymphocyte counts at indicated times following administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab.

Figure 8B shows changes in lymphocyte counts at indicated times following administration of IL-2 conjugate in indicated individuals treated with 16 μg/kg IL-2 conjugate and cetuximab. Data were normalized to pre-treatment (C1D1) lymphocyte counts.

none

Claims (29)

A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) an anti-EGFR antibody, wherein the IL-2 conjugate comprises SEQ ID NO : the amino acid sequence of 3, wherein at least one amino acid residue in the IL-2 conjugate is substituted with a structure of formula (I):

Formula (I); wherein: Z is _CH and Y is

; Y is _CH and Z is

; Z is _CH and Y is

; or Y is _CH and Z 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; and X is a PEG group with L-amino acids of the following structures:

X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue; wherein the structure of formula (I) is in the IL-2 conjugate The positions in the amino acid sequence are selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. The method of claim 1, wherein in the IL-2 conjugate, Z is _CH and Y is

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

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

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

. The method of any one of claims 1-5, wherein in the IL-2 conjugate, the PEG group has an average molecular weight of about 25 kDa, 30 kDa, or 35 kDa. The method of claim 6, wherein in the IL-2 conjugate, the PEG group has an average molecular weight of about 30 kDa. The method of any one of claims 1-7, 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 a structure of formula (IV) or formula (V), or is a mixture of structures of formula (IV) and formula (V):

formula (IV);

Formula (V); wherein: 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 is an L-amino acid having the structure:

; X-1 represents the point of attachment to the previous amino acid residue; and X+1 represents the point of attachment to the next amino acid residue. 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. The method of any one of claims 1-10, wherein the anti-EGFR antibody is cetuximab. A method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (a) an IL-2 conjugate, and (b) cetuximab, wherein the IL-2 conjugate comprises SEQ The amino acid sequence of ID NO: 50, wherein [AzK_L1_PEG30kD] has a structure of formula (XII) or formula (XIII), or a mixture of structures of formula (XII) and formula (XIII):

Formula (XII);

Formula (XIII); wherein: 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; and the wavy line indicates within SEQ ID NO: 50 Covalent bonding of unsubstituted amino acid residues. A method as in any of claims 1-12, wherein q is one. A method as in any of claims 1-12, wherein q is 2. A method as in any of claims 1-12, wherein q is 3. The method of any one of claims 1-15, wherein the average molecular weight is a number average molecular weight. The method of any one of claims 1-15, wherein the average molecular weight is a weight average molecular weight. The method of any one of claims 1-17, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. The method of any one of claims 1-18, wherein the IL-2 conjugate is administered to the individual about once every two weeks, about once every three weeks, or about once every four weeks. The method of any one of claims 1-19, wherein the anti-EGFR antibody is administered to the individual about once a week, about once every two weeks, about once every three weeks, or about once every four weeks. The method of any one of claims 1-20, wherein the IL-2 conjugate is administered to the individual by intravenous administration. The method of any one of claims 1-21, wherein the IL-2 conjugate and the anti-EGFR antibody are administered separately. The method of claim 23, wherein the IL-2 conjugate and the anti-EGFR antibody are administered sequentially. The method of any one of claims 1-23, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classic Hodgkin lymphoma ( cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable carcinoma, microsatellite stable carcinoma, gastric cancer, colon cancer, colorectal cancer (CRC), cervical cancer, liver cancer cell carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophagus, 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) deficiency, bladder cancer, ovarian cancer, tumors with moderate to low mutational burden, Cutaneous squamous cell carcinoma (CSCC), squamous cell skin carcinoma (SCSC), tumors with low to no expression of PD-L1, tumors with systemic diffuse to the liver and CNS beyond their primary anatomical origin, and diffuse large tumors B-cell lymphoma. The method of any one of claims 1-24, comprising administering to the individual about 16 μg/kg of the IL-2 conjugate. The method of any one of claims 1-24, comprising administering to the individual about 24 μg/kg of the IL-2 conjugate. The method of any one of claims 1-10 or 12-26, wherein the anti-EGFR antibody is selected from panitumumab (Vectibix), necitumumab (Portrazza), JNJ - 61186372 (Amivantamab), IMC-C225, ABX-EGF, ICR62, and EMD 55900. An IL-2 conjugate for use in the method of any one of claims 1-27. Use of an IL-2 conjugate for the manufacture of a medicament for use in the method of any one of claims 1-27.

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