Cancer therapy with IL-2 PEG conjugates

Abstract

Disclosed herein is a method for treating cancer in a subject in need, comprising administering an IL-2 conjugate (a) about once a week over a first set of weeks, and then (b) about once at least every two weeks over a second set of weeks. Also disclosed herein is a method for treating solid tumors in a subject in need, comprising administering an IL-2 conjugate (a) about once a week over a first set of weeks, and then (b) about once at least every two weeks over a second set of weeks. Also disclosed herein is a method for treating melanoma in a subject in need, comprising administering an IL-2 conjugate (a) about once a week over a first set of weeks, and then (b) about once at least every two weeks over a second set of weeks.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 453,370, filed on Mar. 20, 2023, the content of which is incorporated herein by reference for all purposes.

Background Art

[0002] Distinct populations of T cells regulate the immune system to maintain immune homeostasis and immune tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological autoreactivity, while cytotoxic T cells target and destroy infected cells and / or cancer cells. In some instances, modulating different populations of T cells provides options for the treatment of diseases or conditions.

[0003] Cytokines include a family of cell - signaling proteins such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors that play roles in the homeostasis of innate and adaptive immune cells. Cytokines are produced by immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, endothelial cells, fibroblasts, and different stromal cells. In some instances, cytokines regulate the balance between the humoral immune response and the cell - based immune response.

[0004] Interleukins are signaling proteins that regulate the development and differentiation of T lymphocytes, B lymphocytes, monocytic cells, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4 + T lymphocytes, B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue - resident cells.

[0005] In some instances, interleukin - 2 (IL - 2) signaling is used to regulate T - cell responses and subsequently for the treatment of cancer.

Summary of the Invention

Means for Solving the Problem

[0006] In this specification, a method for treating cancer in a subject who needs it, which includes administering an IL-2 conjugate to the subject, and the IL-2 conjugate has the non-natural amino acid residue described in this specification at position 64, and has an amino acid sequence that is at least about 90% (for example, including at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or up to 100%) identical to the amino acid sequence of SEQ ID NO: 1, is described. In some embodiments, an exemplary IL-2 conjugate includes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the method described in this specification is applicable to solid tumors. In some embodiments, the method described in this specification is applicable to blood cancers.

[0007] In this specification, a method for treating solid tumors in a subject who needs it, which includes administering an IL-2 conjugate to the subject, and the IL-2 conjugate has the non-natural amino acid residue described in this specification at position 64, and has an amino acid sequence that is at least about 90% (for example, including at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or up to 100%) identical to the amino acid sequence of SEQ ID NO: 1, is described. In some embodiments, an exemplary IL-2 conjugate includes the amino acid sequence of SEQ ID NO: 2.

[0008] This disclosure provides, in particular, administration regimens (e.g., combinations of dosage, frequency of administration, or both) and / or improvements in the treatment of cancer to improve the stimulation and / or expansion of specific immune cells (e.g., CD8+ T cells and / or natural killer (NK) cells) useful in the treatment of cancer. In some embodiments, such cancer is a solid tumor. In some embodiments, such cancer is a hematological cancer.

[0009] In one embodiment, the foregoing provides a method for treating cancer in a subject in need, comprising administering an IL-2 conjugate to the subject (a) about once per week over a first set of weeks, and then (b) about once at least once every two weeks over a second set of weeks. In another embodiment, the foregoing provides a method for treating a solid tumor in a subject, comprising administering an IL-2 conjugate (a) about once per week over a first set of weeks, and then (b) about once at least once every two weeks over a second set of weeks. In yet another embodiment, the foregoing provides a method for treating a melanoma in a subject, comprising administering an IL-2 conjugate (a) about once per week over a first set of weeks, and then (b) about once at least once every two weeks over a second set of weeks.

[0010] Examples of exemplary embodiments include the following:

[0011] Embodiment 1 is a method for treating cancer in a subject in need of such treatment, comprising administering an IL-2 conjugate to the subject. The IL-2 conjugate contains an amino acid sequence that is at least about 90% (e.g., including at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100%) identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is of formula (I): [ka] It is replaced by the structure, in the formula, Z is CH2, and Y is [ka] Is it, Y is CH2, and Z is, [ka] Is it, Z is CH2, and Y is [ka] is or Y is CH2, and Z is, [ka] And, W is a PEG group with an average molecular weight of approximately 25 kDa to approximately 35 kDa. q is 1, 2, or 3. X is structure: [ka] It is an L-amino acid that has, X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to the subsequent amino acid residue. The IL-2 conjugate is administered to subjects (a) approximately once a week over a first set of weeks, and then (b) approximately once every two weeks over a second set of weeks (for example, approximately once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more).

[0012] Embodiment 1.1 is a method for treating cancer in a subject in need of such treatment, comprising administering an IL-2 conjugate to the subject. The IL-2 conjugate contains the amino acid sequence of SEQ ID NO: 1, and the amino acid at position P64 is given by formula (I): [ka] It is replaced by the structure, in the formula, Z is CH2, and Y is [ka] Is it, Y is CH2, and Z is, [ka] Is it, Z is CH2, and Y is [ka] is or Y is CH2, and Z is, [ka] And, W is a PEG group with an average molecular weight of approximately 25 kDa to 35 kDa. q is 1, 2, or 3. X is structure: [ka] It is an L-amino acid that has, X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to the subsequent amino acid residue. The IL-2 conjugate is administered to the subjects (a) approximately once a week over a first set of weeks, and then (b) approximately once every two weeks over a second set of weeks.

[0013] Embodiment 2 is the method according to Embodiment 1 or 1.1, wherein the PEG group in the IL-2 conjugate has an average molecular weight of approximately 30 kDa.

[0014] Embodiment 3 is an IL-2 conjugate in which Z is CH2 and Y is [ka] This is the method according to Embodiment 1, 1.1, or 2.

[0015] Embodiment 4 is an IL-2 conjugate in which Y is CH2 and Z is [ka] This is the method according to Embodiment 1, 1.1, or 2.

[0016] Embodiment 5 is an IL-2 conjugate in which Z is CH2 and Y is [ka] This is the method according to Embodiment 1, 1.1, or 2.

[0017] Embodiment 6 is an IL-2 conjugate in which Y is CH2 and Z is [ka] This is the method according to Embodiment 1, 1.1, or 2.

[0018] Embodiment 7 has the structure of formula (I) as formula (IV) or formula (V): [ka] The method according to Embodiment 1, 1.1, or 2, having the structure of or being a mixture of formula (IV) and formula (V), wherein, q is 1, 2, or 3. X is structure: [ka] It is an L-amino acid that has, X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to the following amino acid residue.

[0019] Embodiment 8 is an embodiment in which the structure of formula (I) is that of formula (XII) or formula (XIII): [ka] The method according to Embodiment 1, 1.1, or 2, having the structure of or being a mixture of formula (XII) and formula (XIII), wherein, n is -(OCH2CH2) n -OCH3 is an integer such that it has a molecular weight of approximately 30 kDa. q is 1, 2 or 3, and The wavy lines indicate covalent bonds to unsubstituted amino acid residues within SEQ ID NO: 1.

[0020] Embodiment 9 is the method described in any one of Embodiments 1, 1.1 and 2 to 8, wherein q is 1.

[0021] Embodiment 10 is the method according to any one of Embodiments 1, 1.1 and 2 to 8, wherein q is 2.

[0022] Embodiment 11 is the method according to any one of Embodiments 1, 1.1 and 2 to 8, wherein q is 3.

[0023] Embodiment 12 is the method according to any one of Embodiments 1, 1.1 and 2 to 11, wherein the cancer is a solid tumor.

[0024] Embodiment 13 is the method of Embodiment 12, wherein the solid tumor is an advanced or metastatic solid tumor.

[0025] Embodiment 14 is the method of Embodiment 12, wherein the solid tumor is a recurrent or refractory solid tumor, or the solid tumor has recurred after one or more prior lines of systemic therapy for solid tumors.

[0026] Embodiment 15 is the method of any one of Embodiments 12 to 14, further comprising selecting a subject to be administered the IL-2 conjugate on at least partly the basis that the subject has received one or more prior lines of systemic therapy for a solid tumor.

[0027] Embodiment 16 is the method according to any one of Embodiments 12 to 15, wherein the subject is receiving one or more lines of systemic therapy for a solid tumor.

[0028] Embodiment 17 is the method of Embodiment 16, wherein the subject is receiving two or more lines of systemic therapy for a solid tumor.

[0029] Embodiment 18 is the method of Embodiment 17, wherein the subject is receiving three or more lines of systemic therapy for a solid tumor.

[0030] Embodiment 19 is the method of Embodiment 12, wherein the solid tumor is a melanoma.

[0031] Embodiment 20 is the method according to Embodiment 19, wherein the melanoma is metastatic melanoma.

[0032] Embodiment 21 is the method according to Embodiment 19, wherein the melanoma is recurrent or refractory melanoma, or the melanoma has recurred after one or more prior lines of systemic therapy for melanoma.

[0033] Embodiment 22 is the method according to any one of Embodiments 19 to 21, further comprising selecting a subject to be administered the IL-2 conjugate on at least partly the basis that the subject has received one or more prior lines of systemic therapy for melanoma.

[0034] Embodiment 23 is the method according to any one of Embodiments 19 to 22, wherein the subject is receiving one or more lines of systemic therapy for melanoma.

[0035] Embodiment 23.1 is the method according to any one of Embodiments 19 to 22, wherein the subject is receiving one or more lines of systemic therapy for a solid tumor.

[0036] Embodiment 24 is the method of Embodiment 23 or 23.1, wherein the subject is receiving two or more lines of systemic therapy for melanoma.

[0037] Embodiment 24.1 is the method of Embodiment 23 or 23.1, wherein the subject is receiving two or more lines of systemic therapy for a solid tumor.

[0038] Embodiment 25 is the method according to Embodiment 24 or 24.1, wherein the subject is receiving three or more lines of systemic therapy for melanoma.

[0039] Embodiment 25.1 is the method of Embodiment 24 or 24.1, wherein the subject is receiving three or more lines of systemic therapy for a solid tumor.

[0040] Embodiment 26 is the method according to any one of Embodiments 16-18 and 23-25, wherein one or more preceding systemic therapies for solid tumors or melanoma include an immune checkpoint inhibitor.

[0041] Embodiment 26.1 is the method according to any one of Embodiments 23, 23.1, 24, 24.1, 25, and 25.1, wherein one or more preceding lines of systemic therapy for melanoma include an immune checkpoint inhibitor.

[0042] Embodiment 27 is the method according to Embodiment 26 or 26.1, wherein the immune checkpoint inhibitor includes a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 inhibitor, or a combination thereof.

[0043] Embodiment 27.1 is the method according to Embodiment 26 or 26.1, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a LAG-3 inhibitor.

[0044] Embodiment 28 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, wherein a dose of IL-2 of approximately 8 μg / kg is administered to the subject as an IL-2 conjugate.

[0045] Embodiment 29 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, wherein a dose of IL-2 of approximately 16 μg / kg is administered to the subject as an IL-2 conjugate.

[0046] Embodiment 30 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, wherein a dose of IL-2 of approximately 24 μg / kg is administered to the subject as an IL-2 conjugate.

[0047] Embodiment 31 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, wherein a dose of IL-2 of approximately 32 μg / kg is administered to the subject as an IL-2 conjugate.

[0048] Embodiment 32 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, comprising administering approximately 8 μg / kg of IL-2 as an IL-2 conjugate about once a week over a first set of weeks.

[0049] Embodiment 33 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, comprising administering approximately 16 μg / kg of IL-2 as an IL-2 conjugate about once a week over a first set of weeks.

[0050] Embodiment 34 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, comprising administering approximately 24 μg / kg of IL-2 as an IL-2 conjugate about once a week over a first set of weeks.

[0051] Embodiment 35 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, and 27.1, comprising administering approximately 32 μg / kg of IL-2 as an IL-2 conjugate approximately once a week over a first set of weeks.

[0052] Embodiment 36 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 8 μg / kg of IL-2 as an IL-2 conjugate at least once every two weeks over a second set of weeks (for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more).

[0053] Embodiment 36.1 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 8 μg / kg of IL-2 as an IL-2 conjugate approximately once every two weeks over a second set of weeks.

[0054] Embodiment 37 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 16 μg / kg of IL-2 as an IL-2 conjugate at least once every two weeks over a second set of weeks (for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more).

[0055] Embodiment 37.1 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 16 μg / kg of IL-2 as an IL-2 conjugate approximately once every two weeks over a second set of weeks.

[0056] Embodiment 38 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 24 μg / kg of IL-2 as an IL-2 conjugate at least once every two weeks over a second set of weeks (for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more).

[0057] Embodiment 38.1 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 24 μg / kg of IL-2 as an IL-2 conjugate approximately once every two weeks over a second set of weeks.

[0058] Embodiment 39 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 32 μg / kg of IL-2 as an IL-2 conjugate at least once every two weeks over a second set of weeks (for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more).

[0059] Embodiment 39.1 is the method according to any one of Embodiments 32 to 35, comprising administering approximately 32 μg / kg of IL-2 as an IL-2 conjugate approximately once every two weeks over a second set of weeks.

[0060] Embodiment 40 is the method according to any one of Embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39 and 39.1, wherein the first set of weeks is approximately 5 weeks, approximately 6 weeks, or approximately 7 weeks.

[0061] Embodiment 41 is the method of Embodiment 40, wherein the first set of weeks is approximately six weeks.

[0062] Embodiment 42 is the method according to any one of Embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40, wherein the second set of weeks is at least about six weeks.

[0063] Embodiment 43 is the method according to any one of Embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40, wherein the second set of weeks is at least about 12 weeks.

[0064] Embodiment 44 is the method according to any one of Embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second set of weeks ranges from about 6 weeks to about 46 weeks.

[0065] Embodiment 45 is the method according to any one of Embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40, wherein the second set of weeks ranges from about 6 weeks to about 98 weeks.

[0066] Embodiment 46 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, 27.1, 28-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40-45, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[0067] Embodiment 47 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, 27.1, 28-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40-46, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

[0068] Embodiment 48 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, 27.1, 28-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40-46, wherein the IL-2 conjugate is administered to the subject by subcutaneous injection.

[0069] Embodiment 49 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, 27.1, 28-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40-48, further comprising administering acetaminophen as the target.

[0070] Embodiment 50 is the method according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, 27.1, 28-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40-49, further comprising administering diphenhydramine to the target.

[0071] Embodiment 51 is the method according to Embodiment 49 or 50, wherein acetaminophen and / or diphenhydramine are administered to the subject before the administration of the IL-2 conjugate.

[0072] Embodiment 52 is an IL-2 conjugate for use in the method described in any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, 27.1, 28-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40-51.

[0073] Embodiment 53 is the use of IL-2 conjugate for manufacturing a pharmaceutical product according to any one of Embodiments 1, 1.1, 2-23, 23.1, 24, 24.1, 25, 25.1, 26, 26.1, 27, 27.1, 28-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1 and 40-51.

[0074] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and benefits of the present invention can be obtained by referring to the following detailed description and the appended drawings, which describe exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0075] [Figure 1] Lymphocyte expansion in cynomolgus monkeys treated with 0.1 mg / kg IL-2 conjugate at one of the following frequencies: a total of three times in Q1W, a total of three times in Q2W, a total of three times in Q3W, or a total of three times in Q4W. The number of days of administration for each treatment is indicated by the small arrow below the X axis. [Figure 2] This shows the change in peripheral CD8+ Teff cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 8 μg / kg [Q2W]. [Figure 3] This shows the change in peripheral NK cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 8 μg / kg [Q2W]. [Figure 4] This shows the change in peripheral CD4+ Treg cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 8 μg / kg [Q2W]. [Figure 5] This shows the change in peripheral lymphocyte cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 8 μg / kg [Q2W]. [Figure 6] This shows the change in peripheral eosinophil cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 8 μg / kg [Q2W]. [Figure 7A] The average concentrations of IL-2 conjugate administered at 8 μg / kg [Q2W] to the target subjects at specified time intervals after 1 and 2 cycles, respectively. [Figure 7B] The average concentrations of IL-2 conjugate administered at 8 μg / kg [Q2W] to the target subjects at specified time intervals after 1 and 2 cycles, respectively. [Figure 8] This shows the levels of IFN-γ, IL-6, and IL-5 in subjects treated with 8 μg / kg [Q2W] IL-2 conjugate at a specified time after administration of IL-2 conjugate. [Figure 9] This shows the change in peripheral CD8+ Teff cell count at a specified time after administration of IL-2 conjugate in eligible subjects treated with IL-2 conjugate 16 μg / kg [Q2W]. [Figure 10] This shows the change in peripheral NK cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 16 μg / kg [Q2W]. [Figure 11] This shows the change in peripheral CD4+ Treg cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 16 μg / kg [Q2W]. [Figure 12] This shows the change in peripheral eosinophil cell count at a specified time after administration of IL-2 conjugate in patients treated with IL-2 conjugate 16 μg / kg [Q2W]. [Figure 13] This shows the levels of IFN-γ, IL-6, and IL-5 in subjects treated with 16 μg / kg [Q2W] IL-2 conjugate at a specified time after administration of IL-2 conjugate. [Figure 14A] The average concentrations of IL-2 conjugate administered at 16 μg / kg [Q2W] to the target subjects at specified time intervals after 1 and 2 cycles, respectively. [Figure 14B] The average concentrations of IL-2 conjugate administered at 16 μg / kg [Q2W] to the target subjects at specified time intervals after 1 and 2 cycles, respectively. [Figure 15] The study design for Example 4 is shown. Abbreviations: C: cycle, D: day, DLT: dose-limiting toxicity, EOT: end of treatment, FU: follow-up, IMP: investigational drug, IV: intravenous administration, QW: once a week, Q2W: every two weeks, Q6W: every six weeks, Q8W: every eight weeks, Q12W: every twelve weeks. [Figure 16] The study design for Example 5 is shown. Abbreviations: C: cycle, D: day, DLT: EOT: end of treatment, FU: follow-up, IMP: investigational drug, IV: intravenous administration, QW: once a week, Q2W: every two weeks, Q6W: every six weeks, Q8W: every eight weeks, Q12W: every twelve weeks. [Figure 17A] The simulation values ​​(shaded areas and lines) and observed values ​​(dots) of CD8+ T cell counts are shown, along with their scaling changes. [Figure 17B] This shows the scaling changes of the simulated NK cell count (shaded area and line) and the observed value (dot). [Figure 18A] This shows the multiplier change in the number of CD8+ T cells in the blood of participants who were administered IL-2 conjugate at a dose of 16 μg / kg according to the prescribed administration schedule. [Figure 18B] This shows the multiplier change in the number of NK cells in the blood collected from participants who were administered IL-2 conjugate at a dose of 16 μg / kg according to the prescribed administration schedule. [Figure 19A] This shows the multiplier change in the number of Treg cells in the blood collected from participants who received IL-2 conjugate at concentrations of 8, 16, 24, or 32 μg / kg according to the Q2W schedule. [Figure 19B] This shows the multiplier change in the number of Treg cells in the blood collected from participants who received IL-2 conjugate at doses of 8, 16, 24, 32, or 40 μg / kg according to the Q3W schedule. [Figure 19C] This shows the multiplier change in Treg cell counts in blood samples taken from participants who received 16 or 24 μg / kg of IL-2 conjugate during the quarter week (QW), which is likely to be administered as part of the QW / Q2W schedule. [Modes for carrying out the invention]

[0076] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter pertains. It should be understood that the above general descriptions and the following detailed descriptions are illustrative and descriptive only and do not limit any claimed subject matter. To the extent that any substance incorporated herein as part of this specification conflicts with the expression of this disclosure, the expression shall prevail. In this application, unless otherwise specified, the use of the singular form includes the plural form. Where used herein and in the appended claims, unless the context explicitly indicates otherwise, the singular forms "a," "an," and "it" include multiple references. In this application, the use of "or" means "and / or" unless the context otherwise requires. Furthermore, the use of the term "includes" and other forms such as "include," "includes," and "includes" is not limited.

[0077] Any reference herein to “several embodiments,” “embodiment,” “one embodiment,” or “other embodiments” means that certain features, structures, or characteristics described in relation to an embodiment are not necessarily included in all embodiments of the present invention, although they may be present in at least some embodiments.

[0078] As used herein, ranges and quantities can be expressed as "approximately" a specific value or range. "Approximately" also includes exact quantities. Therefore, "approximately 5 μL" means "approximately 5 μL" and "5 μL". Generally, the term "approximately" includes quantities that are expected to be within an experimental error, such as 15%, 10%, or 5%.

[0079] The section headings used herein are for organizational purposes only and should never be construed as limiting the subject matter described herein.

[0080] As used herein, the terms “subject” and “patient” mean any mammal. In some embodiments, the mammal is human. In some embodiments, the mammal is non-human. Neither term requires, nor is it limited to, a situation characterized by the management (e.g., constant or intermittent) of a healthcare worker (e.g., physician, registered nurse, nurse practitioner, physician's assistant, nursing assistant, or hospice worker).

[0081] As used herein, the term “non-natural amino acids” refers to amino acids other than one of the 20 naturally occurring amino acids. Exemplary non-natural amino acids are described in Young et al., “Beyond the canonical 20 amino acids: expanding the genetic lexicon,” J. of Biological Chemistry 285(15):11039-11044 (2010), the disclosure of which is incorporated herein by reference.

[0082] In this specification, the term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0083] As used herein, “nucleotide” refers to a compound comprising a nucleoside moiety and a phosphate moiety. Exemplary natural nucleotides include 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), and deoxythymidine. Examples of natural deoxyribonucleotides containing deoxyribose as a sugar moiety include, but are not limited to, dATP, dTTP, dCTP, dGTP, dADP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Examples of natural ribonucleotides containing ribose as the sugar portion include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.

[0084] As used herein, “base” and “nucleic acid base” refer to at least the nucleic acid base portion of a nucleoside or nucleotide (nucleosides and nucleotides include ribovariants or deoxyribovariants), which in some cases may include further modifications to the sugar portion of the nucleoside or nucleotide. In some cases, “base” is also used to represent the entire nucleoside or nucleotide (for example, “base” may be incorporated into DNA by DNA polymerase or into RNA by RNA polymerase). However, the term “base” should not necessarily be interpreted as representing the entire nucleoside or nucleotide unless otherwise required by the context. In the chemical structures of bases or nucleic acid bases provided herein, only the base of the nucleoside or nucleotide is shown, and for clarity, the sugar portion and any optional phosphate residues are omitted. As used in the chemical structures of bases or nucleic acid bases provided herein, tildes represent binding to the nucleoside or nucleotide, and the sugar portion of the nucleoside or nucleotide may be further modified. In some embodiments, the wavy line represents the binding of a base or nucleic acid base to a sugar moiety such as a pentose of a nucleoside or nucleotide. In some embodiments, the pentose is ribose or deoxyribose.

[0085] In some embodiments, nucleic acid bases are generally the heterocyclic base portion of a nucleoside. Nucleic acid bases may be naturally occurring, modified, not necessarily similar to natural bases, and / or synthesized, for example, by organic synthesis. In certain embodiments, nucleic acid bases contain any atom or group of atoms in a nucleoside or nucleotide, and these atoms or groups of atoms can interact with bases of other nucleic acids, with or without the use of hydrogen bonds. In certain embodiments, non-natural nucleic acid bases are not derived from natural nucleic acid bases. It should be noted that non-natural nucleic acid bases are not necessarily basic, but are referred to as nucleic acid bases for simplicity. In some embodiments, when referring to nucleic acid bases, "(d)" indicates that the nucleic acid base may be bound to deoxyribose or ribose, while "d" without an indentation indicates that the nucleic acid base is bound to deoxyribose.

[0086] As used herein, "nucleoside" is a compound comprising a nucleic acid base and a sugar moiety. Examples of nucleosides include, but are not limited to, naturally occurring nucleosides (found in DNA and RNA), basic nucleosides, modified nucleosides, and nucleosides having mimetic bases and / or sugar groups. Examples of nucleosides include nucleosides containing any variety of substituents. A nucleoside may be a glycosidic compound formed by a glycosidic bond between a nucleic acid base and a sugar reducing group.

[0087] Where the term “analog” is used herein, a “analog” of a chemical structure refers to a chemical structure that retains substantial similarity to the parent structure, even if it cannot be readily derived synthetically from the parent structure. In some embodiments, the nucleotide analog is a non-natural nucleotide. In some embodiments, the nucleoside analog is a non-natural nucleotide. The related chemical structure that can be readily derived synthetically from the parent chemical structure is referred to as a “derivative.”

[0088] As used herein, “progressive solid tumor” refers to a malignant solid neoplasm that has metastasized extensively to other anatomical sites or is no longer responsive to treatment.

[0089] A "treatment line" or "therapy line" refers to a series of treatments or therapies administered sequentially as a patient's condition progresses. The first treatment (primary treatment) may be unsuccessful or cease to function after a certain period. After discontinuation of primary treatment, a second, different treatment (secondary treatment) may be administered. Subsequent treatment lines may be administered if secondary treatment is unsuccessful or ceases to function. Some patients may receive multiple treatment lines throughout the course of their disease.

[0090] As used herein, “severe cytokine release syndrome” refers to a level 4 or 5 cytokine release syndrome, as described in Teachey et al., Cancer Discov. 2016;6(6);664-79, which disclosure is incorporated herein as a part of this specification.

[0091] When used herein, the assignment of amino acids in the complementarity-determining region (CDR) to the heavy chain variable domain (VH) or light chain variable domain (VL) is as described in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991, IMGT.RTM. (International ImMunoGeneTics Information System, Lefranc et al, Dev.Comp.Immunol. 29:185-203; 2005), Kabat et al., AHo (Honegger and Pluckthun, J.Mol.Biol. 309(3):657-670; 2001), Chothia (Al-Lazikani et al., 1997 Journal of Molecular Biology 273:927-948) or Contact (Maccallum et al., 1996 Journal of We follow the definition in Molecular Biology 262:732-745.

[0092] As used herein, “percent identity” and related terms refer to a quantitative measurement of similarity between two polypeptides or two polynucleotide sequences. The percentage identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions shared between the two polypeptide sequences, taking into account the number of gaps that may need to be introduced to optimize the alignment of the two polypeptide sequences and the length of each gap. Similarly, the percentage identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions shared between the two polynucleotide sequences, taking into account the number of gaps that may need to be introduced to optimize the alignment of the two polynucleotide sequences and the length of each gap. Sequence comparison and determination of the percentage identity between two polypeptide sequences or two polynucleotide sequences can be achieved using mathematical algorithms. For example, the “percent identity” or “percent homology” of two polypeptides or two polynucleotide sequences can be determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) with its default parameters. Regarding the test sequence, expressions such as "contains a sequence that is at least X% identical to Y" mean that, when aligned with sequence Y as described above, the test sequence contains at least X% of the residues that are identical to Y.

[0093] While various features of the present invention can be described in relation to a single embodiment, the features may also be provided individually or in any suitable combination. Conversely, while the present invention may be described herein in relation to separate embodiments for clarity, the present invention may also be implemented in a single embodiment.

[0094] IL-2 Conjugate Interleukin 2 (IL-2) is a pleiotropic type 1 cytokine whose structure contains four α-helix bundles of 15.5 kDa. The precursor form of IL-2 is 153 amino acid residues in length, with the first 20 amino acids forming a signal peptide and residues 21 - 153 forming the mature form. IL-2 is mainly produced by CD4+ T cells after antigen stimulation, although production by CD8+ cells, natural killer (NK) cells and natural killer T (NKT) cells, activated dendritic cells (DC) and mast cells is lower. IL-2 signaling occurs through interaction with specific combinations of the IL-2 receptor (IL-2R) subunits, IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122) and IL-2Rγ (also known as CD132). The interaction between IL-2 and IL-2Rα forms a "low-affinity" IL-2 receptor complex with a K of approximately 10 -8 M. d The interaction between IL-2 and IL-2Rβ and IL-2Rγ forms an "intermediate-affinity" IL-2 receptor complex with a K of approximately 10 -9 M. d The interaction between IL-2 and all three subunits, IL-2Rα, IL-2Rβ and IL-2Rγ, forms a "high-affinity" IL-2 receptor with a K of approximately 10 -11 M or greater. d In some examples, IL-2 signaling by the "high-affinity" IL-2Rαβγ complex regulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4

[0095] In some examples, IL-2 signaling by the "high-affinity" IL-2Rαβγ complex regulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4 + CD25 + Foxp3 + Regulatory T (Treg) cells are CD4 + T cells, CD8 +They mediate the maintenance of immune homeostasis by suppressing effector cells such as T cells, B cells, NK cells, and NKT cells. In some cases, Treg cells are generated from the thymus (tTreg cells) or induced from peripheral naive T cells (pTreg cells). In some cases, Treg cells are thought to be mediators of peripheral tolerance. In fact, one study showed that CD25 depletion in peripheral CD4 + The introduction of T cells caused various autoimmune diseases in nude mice, but CD4 + CD25 + Simultaneous introduction of T cells suppressed the development of autoimmunity (Sakaguchi, et al. “Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25),” J.Immunol. 155(3):1151-1164 (1995), this disclosure is incorporated herein by reference as constituting a part of this specification). Increased Treg cell population downregulates the proliferation of effector T cells and suppresses autoimmunity and the antitumor response of T cells.

[0096] IL-2 signaling via the "intermediate affinity" IL-2Rβγ complex is controlled by CD8 + Regulates the activation and proliferation of effector T (Teff) cells, NK cells, and NKT cells. CD8 + Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, Tcon, or killer T cells) are T lymphocytes that recognize and kill damaged cells, cancer cells, and pathogen-infected cells. NK cells and NKT cells are CD8 + Teff cells, along with other lymphocytes, are a type of lymphocyte that targets cancer cells and pathogen-infected cells.

[0097] In some cases, IL-2 signaling is utilized to modulate specific immune cell responses (e.g., T cell responses and / or NK cell responses, though not limited to these) and consequently to be used in cancer treatment. For example, in some embodiments, IL-2 is administered in high-dose forms to induce an expansion of the Teff cell population in order to treat cancer. However, high-dose IL-2 also leads to co-stimulation of Treg cells, which reduces the anti-tumor immune response. High-dose IL-2 induces toxic adverse events mediated by the binding of vascular IL-2Rα chain-expressing cells, including type 2 innate immune cells (ILC-2), eosinophils, and endothelial cells. This results in eosinophilia, capillary leak syndrome, and vascular leak syndrome (VLS).

[0098] In one embodiment, a method is provided herein for stimulating and / or proliferating specific immune cells useful for the treatment of cancer (including, for example, Teff cells, NK cells, and / or NKT cells) in a subject in need, such as a subject having cancer, and such a method comprises administering the IL-2 conjugate described herein to the subject. In some embodiments, such cancer is a solid tumor. In some embodiments, such cancer is a hematological cancer. In another embodiment, provided herein is a method for treating a solid tumor, such as melanoma, in a subject in need, and comprises administering the IL-2 conjugate described herein to the subject.

[0099] In some embodiments, the IL-2 sequence included in the IL-2 conjugate described herein is the amino acid sequence of SEQ ID NO: 1: [ka] It contains an amino acid sequence that is identical to at least approximately 90% (for example, including at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or up to 100%), The amino acid at position P64 listed in Sequence ID No. 1 is given by formula (I): [ka] It is replaced by the structure, in the formula, Z is CH2, and Y is [ka] Is it, Y is CH2, and Z is, [ka] Is it, Z is CH2, and Y is [ka] is or Y is CH2, and Z is, [ka] And, W is a PEG group with an average molecular weight of approximately 25 kDa to 35 kDa. q is 1, 2, or 3. X is structure: [ka] It is an L-amino acid that has, X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to a subsequent amino acid residue. In some embodiments, the IL-2 sequence contained in the IL-2 conjugate described herein includes the sequence of SEQ ID NO: 1, and the amino acid at position P64 shown in SEQ ID NO: 1 is substituted by the structure of formula (I) described herein.

[0100] In any embodiment or variation of formula (I) described herein and in pharmaceutical compositions comprising the same, the average molecular weight includes both the weight-average molecular weight and the number-average molecular weight. In other words, for example, both the number-average molecular weight of 30 kDa and the weight-average molecular weight of 30 kDa are eligible as a molecular weight of 30 kDa. In some embodiments, the average molecular weight is the weight-average molecular weight. In other embodiments, the average molecular weight is the number-average molecular weight. In the methods provided herein, it is understood that administering the IL-2 conjugate described herein to a subject involves administering more than one molecule of the IL-2 conjugate. Therefore, the use of the term “average” to describe the molecular weight of the PEG group is understood to mean the average molecular weight of the PEG group of the IL-2 conjugate molecule at the dose administered to the subject.

[0101] In any embodiment or variation of formula (I) described herein, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt. In some embodiments, the IL-2 conjugate is a solvate. In some embodiments, the IL-2 conjugate is a hydrate.

[0102] In some embodiments of formula (I), Z is CH2, and Y is [ka] In some embodiments of formula (I), Y is CH2, and Z is [ka] In some embodiments of formula (I), Z is CH2, and Y is [ka] In some embodiments of formula (I), Y is CH2, and Z is [ka] That is the case.

[0103] In some embodiments of equation (I), q is 1. In some embodiments of equation (I), q is 2. In some embodiments of equation (I), q is 3.

[0104] In some embodiments of formula (I), W is a PEG group having an average molecular weight of about 25 kDa. In some embodiments of formula (I), W is a PEG group having an average molecular weight of about 30 kDa. In some embodiments of formula (I), W is a PEG group having an average molecular weight of about 35 kDa.

[0105] In some embodiments of equation (I), q is 1, and the structure of equation (I) is equation (Ia): [ka] This is the structure, and in the formula, Z is CH2, and Y is [ka] Is it, Y is CH2, and Z is, [ka] Is it, Z is CH2, and Y is [ka] is or Y is CH2, and Z is, [ka] And, W is a PEG group with an average molecular weight of approximately 25 kDa to approximately 35 kDa. X is structure: [ka] It is an L-amino acid that has, X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to the following amino acid residue.

[0106] In some embodiments of formula (Ia), Z is CH2 and Y is [ka] In some embodiments of equation (Ia), Y is CH2, and Z is [ka] In other embodiments of equation (Ia), Z is CH2, and Y is [ka] In some embodiments of equation (Ia), Y is CH2, and Z is [ka] That is the case.

[0107] In some embodiments of formula (Ia), the PEG group has an average molecular weight of about 30 kDa.

[0108] In some embodiments, the IL-2 conjugate is represented by Sequence ID No. 2: [ka] The formula includes the sequence, where [AzK_L1_PEG30kD] is N6-((2-azidoethoxy)-carbonyl)-L-lysine, which is stably conjugated to PEG via DBCO-mediated click chemistry to form a compound having the structure of formula (IV) or formula (V), where q is 1 (e.g., formula (IVa) or formula (Va)), and the PEG group has an average molecular weight of about 25-35 kDa (e.g., about 30 kDa) capped with a methoxy group. The term "DBCO" refers to the chemical moiety containing a dibenzocyclooctin group, including the mPEG-DBCO compounds shown, for example, in schemes 1 and 2 of Example 1.

[0109] The ratio of positional isomers produced from the click reaction is approximately 1:1 or greater than 1:1.

[0110] PEG typically contains several (OCH2CH2) monomers (or CH2CH2O monomers, depending on how PEG is defined). In some embodiments, the number of (OCH2CH2) monomers (or (CH2CH2O) monomers) is such that the average molecular weight of the PEG group is approximately 30 kDa.

[0111] In some cases, PEG is an end-cap polymer, i.e., a relatively inert group, such as lower C. 1~6 The polymer has at least one end capped with an alkoxy or hydroxyl group. In some embodiments, the PEG group is methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG in which one end of the polymer is a methoxy(-OCH3) group and the other end is a hydroxyl or other functional group that may be chemically modified at will.

[0112] In some embodiments, the PEG group is a linear or branched PEG group. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. For example, IL-2 conjugates containing PEG groups having molecular weights of 30,000 Da ± 3,000 Da, 30,000 Da ± 4,500 Da, or 30,000 Da ± 5,000 Da are included within the scope of this disclosure.

[0113] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1 by at least about 90% (e.g., including at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100%), wherein amino acid residue P64 is of formula (IV) or formula (V): [ka] The structure of is replaced by a mixture of formula (IV) and formula (V), in which, W is a PEG group with an average molecular weight of approximately 25 kDa to 35 kDa. q is 1, 2 or 3, and X is structure: [ka] It has, X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to a subsequent amino acid residue. In some embodiments, the IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue P64 is structure (IV) or (V), or a mixture of (IV) and (V).

[0114] In some embodiments of formula (IV) or formula (V) or a mixture of formula (IV) or formula (V), q is 1. In some embodiments of formula (IV) or formula (V) or a mixture of formula (IV) or formula (V), q is 2. In some embodiments of formula (IV) or formula (V) or a mixture of formula (IV) or formula (V), q is 3.

[0115] In some embodiments of formula (IV) or formula (V) or a mixture of formula (IV) or formula (V), W is a PEG group having an average molecular weight of about 25 kDa. In some embodiments of formula (IV) or formula (V) or a mixture of formula (IV) or formula (V), W is a PEG group having an average molecular weight of about 30 kDa. In some embodiments of formula (IV) or formula (V) or a mixture of formula (IV) or formula (V), W is a PEG group having an average molecular weight of about 35 kDa.

[0116] In any embodiment described herein, the structure of formula (I) has the structure of formula (IV) or formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the structure of formula (I) has the structure of formula (IV). In some embodiments, the structure of formula (I) has the structure of formula (V). In some embodiments, the structure of formula (I) is a mixture of formula (IV) and formula (V).

[0117] In some embodiments of formula (IV) or formula (V) or a mixture of formula (IV) and formula (V), q is 1, the structure of formula (IV) is the structure of formula (IVa), and the structure of formula (V) is the structure of formula (Va): [ka] This is the structure, and in the formula, W is a PEG group having an average molecular weight of approximately 25 kDa to approximately 35 kDa, and X is structure: [ka] It has, X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to the following amino acid residue.

[0118] In some embodiments of formula (IVa) or formula (Va) or a mixture of formula (IVa) and formula (Va), the PEG group has an average molecular weight of approximately 30 kDa.

[0119] In any embodiment described herein, the structure of formula (I) has the structure of formula (IVa) or formula (Va), or is a mixture of formula (IVa) and formula (Va). In some embodiments, the structure of formula (I) has the structure of formula (IVa). In some embodiments, the structure of formula (I) has the structure of formula (Va). In some embodiments, the structure of formula (I) is a mixture of formula (IVa) and formula (Va).

[0120] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (e.g., including at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100%) identical to the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue P64 is of formula (XII) or formula (XIII): [ka] The structure of or a mixture of formula (XII) and formula (XIII) is substituted, in the formula, n is -(OCH2CH2) n -OCH3 is an integer such that its molecular weight is approximately 25kDa to approximately 35kDa. q is 1, 2 or 3, and The wavy lines indicate covalent bonds to unsubstituted amino acid residues in SEQ ID NO: 1. In some embodiments, the IL-2 conjugates described herein comprise the amino acid sequence of SEQ ID NO: 1, where amino acid residue P64 is structure (XII) or (XIII) or a mixture of (XII) and (XIII).

[0121] In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII), q is 1. In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII), q is 2. In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII), q is 3.

[0122] In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII), n is -(OCH2CH2) n -OCH3 is an integer such that it has a molecular weight of approximately 30 kDa.

[0123] In any embodiment described herein, the structure of formula (I) has the structure of formula (XII) or formula (XIII), or is a mixture of formula (XII) and formula (XIII). In some embodiments, the structure of formula (I) has the structure of formula (XII). In some embodiments, the structure of formula (I) has the structure of formula (XIII). In some embodiments, the structure of formula (I) is a mixture of formula (XII) and formula (XIII).

[0124] In some embodiments of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII), q is 1, the structure of formula (XII) is the structure of formula (XIIa), and the structure of formula (XIII) is the structure of formula (XIIIa): [ka] This is the structure, and in the formula, n is -(OCH2CH2) n-OCH3 is an integer such that it has a molecular weight of approximately 25 kDa to approximately 35 kDa, and The wavy lines indicate covalent bonds to unsubstituted amino acid residues within SEQ ID NO: 1.

[0125] In some embodiments of formula (XIIa) or formula (XIIIa) or a mixture of formula (XIIa) and formula (XIIIa), n is -(OCH2CH2) n -OCH3 is an integer such that it has a molecular weight of approximately 30 kDa.

[0126] In any embodiment described herein, the structure of formula (I) has the structure of formula (XIIa) or formula (XIIIa), or is a mixture of formula (XIIa) and formula (XIIIa). In some embodiments, the structure of formula (I) has the structure of formula (XIIa). In some embodiments, the structure of formula (I) has the structure of formula (XIIIa). In some embodiments, the structure of formula (I) is a mixture of formula (XIIa) and formula (XIIIa).

[0127] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 1 by at least about 90% (e.g., including at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100%), wherein amino acid residue P64 is of formula (XIV) or formula (XV): [ka] The structure of is replaced by a mixture of formula (XIV) and formula (XV), in which, m is an integer between 0 and 20. p is an integer between 0 and 20. n is an integer such that the PEG group has an average molecular weight of approximately 25 kDa to approximately 35 kDa, and The wavy lines indicate covalent bonds to unsubstituted amino acid residues in SEQ ID NO: 1. In some embodiments, the IL-2 conjugates described herein comprise the amino acid sequence of SEQ ID NO: 1, where amino acid residue P64 is the structure of formula (XIV) or formula (XV) or a mixture of formulas (XIV) and (XV).

[0128] In some embodiments of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV), n is an integer such that the PEG group has an average molecular weight of about 30 kDa.

[0129] In some embodiments, m is an integer from 0 to 15. In some embodiments, m is an integer from 0 to 10. In some embodiments, m is an integer from 0 to 5. In some embodiments, m is an integer from 1 to 5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

[0130] In some embodiments, p is an integer between 0 and 15. In some embodiments, p is an integer between 0 and 10. In some embodiments, p is an integer between 0 and 5. In some embodiments, p is an integer between 1 and 5. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5.

[0131] In some embodiments, m and p are each 2.

[0132] In any embodiment described herein, the structure of formula (I) has the structure of formula (XIV) or formula (XV), or is a mixture of formula (XIV) and formula (XV). In some embodiments, the structure of formula (I) has the structure of formula (XIV). In some embodiments, the structure of formula (I) has the structure of formula (XV). In some embodiments, the structure of formula (I) is a mixture of formula (XIV) and formula (XV).

[0133] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (e.g., including at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or up to 100%) identical to the amino acid sequence of SEQ ID NO: 1, wherein amino acid residue P64 is of formula (XVI) or formula (XVII): [ka] The structure of is replaced by a mixture of formulas (XVI) and (XVII), in which, m is an integer between 0 and 20. n is an integer such that the PEG group has an average molecular weight of approximately 25 kDa to 35 kDa, and The wavy lines indicate covalent bonds to unsubstituted amino acid residues in SEQ ID NO: 1. In some embodiments, the IL-2 conjugates described herein comprise the amino acid sequence of SEQ ID NO: 1, where amino acid residue P64 is the structure of formula (XVI) or formula (XVII) or a mixture of formulas (XVI) and (XVII).

[0134] In some embodiments of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII), n is an integer such that the PEG group has an average molecular weight of about 30 kDa.

[0135] In some embodiments, m is an integer from 0 to 15. In some embodiments, m is an integer from 0 to 10. In some embodiments, m is an integer from 0 to 5. In some embodiments, m is an integer from 1 to 5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

[0136] In any embodiment described herein, the structure of formula (I) has the structure of formula (XVI) or formula (XVII), or is a mixture of formula (XVI) and formula (XVII). In some embodiments, the structure of formula (I) has the structure of formula (XVI). In some embodiments, the structure of formula (I) has the structure of formula (XVII). In some embodiments, the structure of formula (I) is a mixture of formula (XVI) and formula (XVII).

[0137] Conjugation Chemistry In some embodiments, the IL-2 conjugates described herein can be prepared by conjugation reactions including 1,3-dipolar cycloaddition reactions. In some embodiments, the 1,3-dipolar cycloaddition reaction includes a reaction between an azide and an alkyne ("click" reaction). In some embodiments, the conjugation reaction described herein includes the reaction outlined in Scheme I, where X is the unnatural amino acid at position P64 of SEQ ID NO: 1. [ka]

[0138] In some embodiments, the conjugate moiety comprises a PEG group as described herein. In some embodiments, the reactive group comprises an alkyne or an azide.

[0139] In some embodiments, the conjugation reactions described herein include the reaction outlined in Scheme II, where X is the unnatural amino acid at position P64 of SEQ ID NO: 1. [Chemistry]

[0140] In some embodiments, the conjugation reaction described herein includes the reaction outlined in Scheme III, and X is a non-natural amino acid at position P64 of SEQ ID NO: 1. [Chemistry]

[0141] In some embodiments, the conjugation reaction described herein includes the reaction outlined in Scheme IV, and X is a non-natural amino acid at position P64 of SEQ ID NO: 1. [Chemistry]

[0142] In some embodiments, the conjugation reaction described herein includes a cycloaddition reaction between an azide moiety, such as those contained in a protein containing an amino acid residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as those derived from dibenzocyclooctyne (DBCO), a chemical moiety containing a dibenzocyclooctyne group. A PEG group containing a DBCO moiety can be commercially available or can be prepared by methods known to those skilled in the art. Exemplary reactions are shown in Schemes V and VI. [Chemistry] [Chemistry]

[0143] The conjugation reactions, such as click reactions, described herein may produce a single positional isomer or a mixture of positional isomers. In some cases, the ratio of positional isomers is about 1:1. In some cases, the ratio of positional isomers is about 2:1. In some cases, the ratio of positional isomers is about 1.5:1. In some cases, the ratio of positional isomers is about 1.2:1. In some cases, the ratio of positional isomers is about 1.1:1. In some cases, the ratio of positional isomers is greater than 1:1.

[0144] Production of IL-2 polypeptide In some cases, the IL-2 conjugates described herein, which contain either native or non-native amino acid mutations, are produced by recombinant or chemically synthesized. In some cases, the IL-2 conjugates described herein are produced recombinantly, for example, by either a host cell line or a cell-free line.

[0145] In some cases, IL-2 conjugates are recombinantly produced via a host cell line. In some cases, the host cell is a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell, or plant cell) or a prokaryotic cell (e.g., Gram-positive or Gram-negative bacteria). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is a stable cell line or a cell line that has incorporated the genetic material of interest into its own genome and is capable of expressing the product of the genetic material after several generations of cell division. In other cases, the mammalian host cell is a transient cell line or a cell line that has not incorporated the genetic material of interest into its own genome and is not capable of expressing the product of the genetic material after several generations of cell division.

[0146] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F(TM) cells, Flp-In(TM) T-REx(TM) 293 cell line, Flp-In(TM)-293 cell line, Flp-In(TM)-3T3 cell line, Flp-In(TM)-BHK cells strain, Flp-In(TM)-CHO cell line, Flp-In(TM)-CV-1 cell line, Flp-In(TM)-Jurkat cell line, FreeStyle(TM) 293-F cells, FreeStyle(TM) CHO-S cells, GripTite(TM) 293 MSR cell line, GS-CHO cell line, HepaRG™ cell line, 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.

[0147] 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.

[0148] 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.

[0149] In some embodiments, the eukaryotic host cell is a plant host cell. In some examples, the plant cell includes cells derived from algae. Exemplary plant cell lines include strains derived from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0150] In some embodiments, the host cell is a prokaryotic host cell. Examples of prokaryotic host cells include BL21, Mach1(trademark), DH10B(trademark), TOP10, DH5α, DH10Bac(trademark), OmniMax(trademark), MegaX(trademark), DH12S(trademark), INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2(trademark), Stbl3(trademark), or Stbl4(trademark).

[0151] In some examples, suitable polynucleic acid molecules or vectors for producing the IL-2 polypeptide described herein include any suitable vector derived from either a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors derived from bacteria (e.g., Escherichia coli), insects, yeasts (e.g., Pichia pastoris, K. phaffii), algae, or mammalian sources. Examples of bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0152] Examples of insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBac M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0153] Examples of yeast vectors include Gateway(registered trademark) pDEST(trademark) 14 vector, Gateway(registered trademark) pDEST(trademark) 15 vector, Gateway(registered trademark) pDEST(trademark) 17 vector, Gateway(registered trademark) pDEST(trademark) 24 vector, Gateway(registered trademark) pYES-DEST52 vector, pBAD-DEST49 Gateway(registered trademark) destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris (K. phaffii) vector, pGAPZ A, B&C Pichia pastoris (K. phaffii) vector, pPIC3.5K Pichia vector, and pPIC6 Examples include A, B&C Pichia vectors, pPIC9K Pichia vectors, pTEF1 / Zeo, pYES2 yeast vectors, pYES2 / CT yeast vectors, pYES2 / NT A, B&C yeast vectors, or pYES3 / CT yeast vectors.

[0154] Examples of algal vectors include the pChlamy-4 vector or the MCS vector.

[0155] Examples of mammalian vectors include transient expression vectors and 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. Examples of 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.

[0156] In some cases, cell-free systems are used for the production of IL-2 polypeptides as described herein. In some cases, cell-free systems comprise a mixture of cytoplasmic and / or nuclear components derived from cells and are suitable for in vitro nucleic acid synthesis. In some cases, cell-free systems utilize prokaryotic cell components. In other cases, cell-free systems utilize eukaryotic cell components. Nucleic acid synthesis is obtained in cell-free systems based, for example, Drosophila cells, Xenopus eggs, archaea, or HeLa cells. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.

[0157] Cell-free translation systems include a variety of components such as plasmids, mRNA, DNA, tRNA, synthases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and / or non-natural amino acids, and / or other components used for protein expression. Such components are optionally modified to improve yield, increase synthesis rate, enhance fidelity of protein products, or incorporate non-natural amino acids. In some embodiments, the cytokines described herein are synthesized using cell-free translation systems described in U.S. Patent No. 8,778,631, U.S. Patent Application Publication No. 2017 / 0283469, U.S. Patent Application Publication No. 2018 / 0051065, U.S. Patent Application Publication No. 2014 / 0315245, or U.S. Patent No. 8,778,631 (each of which disclosures is incorporated herein by reference). In some embodiments, the cell-free translation system includes modified release factors or even the removal of one or more release factors from the system. In some embodiments, the cell-free translation system includes a reduced protease concentration. In some embodiments, the cell-free translation system includes modified tRNA having reassigned codons used to encode non-natural amino acids. In some embodiments, the synthetic enzymes described herein for incorporating non-natural amino acids are used in the cell-free translation system. In some embodiments, the tRNA is preloaded with non-natural amino acids using enzymatic or chemical methods before being added to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from modified organisms such as modified bacteria, yeast, or other organisms.

[0158] In some embodiments, the IL-2 polypeptide is generated in a cyclically substituted form via either an expression host system or a cell-free system.

[0159] Production of cytokine polypeptides containing unnatural amino acids In the present disclosure, one or more specific codons present in the nucleic acid sequence of an IL-2 polypeptide are assigned to encode non-natural amino acids, whereby an orthogonal or expanded genetic code can be used to genetically incorporate them into IL-2 by using an orthogonal tRNA synthetase / tRNA pair. The orthogonal tRNA synthetase / tRNA pair can add a non-natural amino acid to the tRNA and incorporate that non-natural amino acid into the polypeptide chain in response to the codon.

[0160] In some examples, the codon is a codon amber, ochre, opal or quadruplet codon. In some cases, the codon corresponds to an orthogonal tRNA used to carry the non-natural amino acid. In some cases, the codon is amber. In other cases, the codon is an orthogonal codon.

[0161] In some examples, the codon is a quadruplet codon that can be decoded by an orthogonal ribosome ribo-Q1. In some cases, the quadruplet codon is as shown in Neumann, et al., “Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome,” Nature, 464(7287):441-444(2010), the disclosure of which is incorporated herein by reference.

[0162] In some cases, the codons used in this disclosure are rare codons that have been replaced with recorded codons, such as synonymous or surrogate codons. In some cases, the recorded 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 recorded codons are as described in Ostrov et al., “Design, synthesis, and testing toward a 57-codon genome,” Science 353(6301):819-822 (2016), the disclosure of which is incorporated herein by reference.

[0163] In some cases, non-natural nucleic acids are utilized, with one or more non-natural amino acids incorporated into IL-2. Exemplary non-natural nucleic acids include uracil-5-yl, hypoxanthin-9-yl(I), 2-aminoadenine-9-yl, 5-methylcytosine(5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, and 6-azoura. Examples include, but are not limited to, syl, cytosine and thymine, 5-uracil (psouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain 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, universal nucleic acids, hydrophobic nucleic acids, indiscriminate nucleic acids, size-enhancing nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. Purines, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, adenine and other alkyl derivatives of guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiuracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl(-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azocymine,5-uracil (pseudracil), 4-thiuracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazadenine, tricyclic pyrimidine, phenoxazine cytidine ([5,4-b][l,4 ]benzoxazine-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][l,4]benzothiadin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazine-2(3H)-one, carbazole cytidine (2H-pyrimido[4,5-b]indole-2-one), pyridoindole cytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one), purine or pyrimidine bases placed in other heterocycles Substitutes, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodoracyl, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-iodracil, 2-aminoadenine, 6-thio-guanine, 2-thio Orthymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and U.S. Patent Nos. 3,687,808, 4,845,205, 4,910,300, 4,948,882, 5,093,232, 5,130,302, 5,134,066, and 5,175,273,Specification No. 5,367,066, Specification No. 5,432,272, Specification No. 5,457,187, Specification No. 5,459,255, Specification No. 5,484,908, Specification No. 5,502,177, Specification No. 5,525,711, Specification No. 5,552,540, Specification No. 5,587,469, Specification No. 5,594,121, Specifications No. 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653, and 6,005,096; International Publication No. 99 / 62923; Kandimalla et al., (2001) Bioorg. This includes, but is not limited to, the works described in 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 U.S. Patent No. 3,687,808, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993, the respective disclosures of which are incorporated herein by reference.

[0164] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acids may, in some cases, contain one or more heterocyclic bases other than the five main base components of naturally occurring nucleic acids. For example, heterocyclic bases include, in some cases, uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4-aminopyrrolo[2,3-d]pyrimidine-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidine-5-yl, and 2-amino-4-oxopyrrolo[2,3-d]pyrimidine-3-yl groups, where purines are attached to the sugar moiety of the nucleic acid via position 9, pyrimidines via position 1, pyrrolopyrimidines via position 7, and pyrazolopyrimidines via position 1.

[0165] In some embodiments, nucleotide analogs are also modified with phosphate moieties. Modified phosphate moieties include, but are not limited to, those having modifications to the bond between two nucleotides, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and 3'-alkylene phosphonates and other alkyl phosphonates including chiral phosphonates, phosphinates, 3'-aminophosphoramides and phosphoramides, such as aminoalkyl phosphoramides, thionophosphoramides, thionoalkyl phosphonates, thionoalkyl phosphotriesters and boranophosphates. These phosphate bonds or modified phosphate bonds between two nucleotides are mediated via 3'-5' or 2'-5' bonds, and the bonds are understood to include reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Numerous U.S. patents teach methods for the preparation and use of nucleotides containing modified phosphates, including Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, and 5,405,9 Examples include, but are not limited to, Specifications 39, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,625,050, the disclosures of which are incorporated herein by reference.

[0166] In some embodiments, non-natural nucleic acids include 2',3'-dideoxy-2',3'-didehydronucleosides (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, and Eppacher et al., Helvetica Chimica). Examples include 5'-substituted monomers prepared as monophosphates having a modified base (Acta, 2004, 87, 3004-3020, PCT / JP2000 / 004720, PCT / JP2003 / 002342, PCT / JP2004 / 013216, PCT / JP2005 / 020435, PCT / JP2006 / 315479, PCT / JP2006 / 324484, PCT / JP2009 / 056718, PCT / JP2010 / 067560) or a modified base (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23(1&2), 317-337), the disclosures of each thereof are incorporated herein by reference.

[0167] In some embodiments, non-natural nucleic acids include modifications at the 5' and 2' positions of a sugar ring, such as 5'-CH2-substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924) (PCT / US94 / 02993). In some cases, non-natural nucleic acids include amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, in which the 3'-linked nucleosides in the dimer (5'~3') contain 2'-OCH3 and 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids may include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Non-natural nucleic acids may include 5'-methylenephosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids may include 5'-phosphonate monomers having a 2' substitution (U.S. Patent Application Publication 2006 / 0074035) and other modified 5'-phosphonate monomers (International Publication 1997 / 35869). Non-natural nucleic acids may include 5'-modified methylene phosphonate monomers (European Patent No. 614907 and European Patent No. 629633).Non-natural nucleic acids may include analogues of 5' or 6'-phosphonate ribbonucleosides 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'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids may include nucleosides having a 6'-phosphonate group that is unsubstituted at the 5' and / or 6' positions, or substituted with a thio-tert-butyl group (SC(CH3)3) (and its analogues), a methyleneamino group (CH2NH2) (and its analogues), or a cyano group (CN) (and its analogues) (Fairhurst et al., Synlett, 2001, 4, 467-472, Kappler et al., J.Med.Chem., 1986, 29, 1030-1038, Kappler et al., J.Med.Chem., 1982, 25, 1179-1184, Vrudhula et al., J.Med.Chem., 1987, 30, 888-894, Hampton et al. (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 paragraph are incorporated herein by reference.

[0168] In some embodiments, non-natural nucleic acids also include modifications of the sugar moiety. In some cases, nucleic acids comprise one or more nucleosides with modified sugar groups. Such sugar-modified nucleosides may confer improved nuclease stability, increased binding affinity, or some other beneficial biological properties. In certain embodiments, nucleic acids comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include substituents (including 5' and / or 2' substituents), bridging of two ring atoms to form a bicyclic nucleic acid (BNA), substitution of the ribosyl ring oxygen atom with S, N(R) or C(R1)(R2) (R=H, C1~C 12 Examples of chemically modified sugars include, but are not limited to, alkyl groups or protecting groups and combinations thereof. Examples of chemically modified sugars can be found in International Publication No. 2008 / 101157, U.S. Patent Application Publication No. 2005 / 0130923, and International Publication No. 2007 / 134181, the disclosures of which are incorporated herein by reference.

[0169] In some examples, modified nucleic acids include modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety may be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or sugar "analog" cyclopentyl group. The sugar may be in the form of pyranosyl or furanosyl. The sugar moiety may be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar may be attached to the respective heterocyclic base in either an [α] or [β] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications may include 2'-O-methyluridine or 2'-O-methylcytidine. Examples of sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs, and the respective "nucleosides" in which such sugars or analogs are bonded to heterocyclic bases (nucleic acid bases), is known. Sugar modifications can be constructed independently or in combination with other modifications.

[0170] Modifications to the sugar moiety include natural and unnatural modifications of ribose and deoxyribose. Sugar modifications include the following modifications at the 2' position: OH, F, O-, S- or N-alkyl, O-, S- or N-alkenyl, O-, S- or N-alkynyl, or O-alkyl-O-alkyl (where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C). 10 , alkyl or C2~C 10 Examples of 2' sugar modifications include, but are not limited to, alkenyl and alkynyl compounds. n O] m CH3, -O(CH2) n OCH3, O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2 and -O(CH2) nON[(CH2nCH3)]2 is also included, but is not limited to these, and n and m range from 1 to approximately 10.

[0171] Other modifiers at the 2' position include C1~C 10Examples of modified sugars include, but are not limited to, lower alkyl groups, substituted lower alkyl groups, alkaryl groups, aralkyl groups, O-alkaryl groups, O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties of oligonucleotides or groups that improve the pharmacodynamic properties of oligonucleotides, and other substituents having similar properties. Similar modifications can also be made at other positions of the sugar, particularly at the 3' position of the sugar in the 3'-terminal nucleotide or the 3' position of the sugar in 2'-5' linked oligonucleotides and at the 5' position of the 5'-terminal nucleotide. Modified sugars may also include those with modifications to the cross-linking ring oxygen such as CH2 and S. Nucleotide sugar analogs may also have sugar mimes such as cyclobutyl moieties instead of pentofuranosyl sugars.There are many U.S. patents that teach the preparation of such modified sugar structures and detail and describe the scope of base modification, for example, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, and 5,5 Specification No. 14,785, Specification No. 5,519,134, Specification No. 5,567,811, Specification No. 5,576,427, Specification No. 5,591,722, Specification No. 5,597,9 Specification No. 09, Specification No. 5,610,300, Specification No. 5,627,053, Specification No. 5,639,873, Specification No. 5,646,265, Specification No. 5,658,873 5,670,633, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,175,273, Specification No. 5,367,066, Specification No. 5,432,272, Specification No. 5,457,187, Specification No. 5,459,255, Specification No. 5,484,908, Specification No. 5,502 These include 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 which are incorporated herein by reference.

[0172] Examples of nucleic acids containing modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl(R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position may be allyl, amino, azide, thio, O-allyl, or O-(C1~C 1O Alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ) and O-CH2-C(=O)-N(R m )(R n ) can also be selected from each R mand R n These are, independently, H or substituted or unsubstituted C1-C 10 It is alkyl.

[0173] In certain embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between 4' and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids in which the bridge comprises a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include the following formulas: 4'-(CH2)-O-2'(LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2'(ENA), 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2', and their analogues (see U.S. Patent No. 7,399,845), 4'-C(CH3)(CH3)-O-2' and its analogues (see International Publication No. 2009 / 006478, International Publication No. 2008 / 150729, U.S. Patent Application Publication No. 2004 / 0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al. One example is, but is not limited to, al., J. Org. Chem., 209, 74, 118-134 and International Publication No. 2008 / 154401. For example, Singh et al.,Chem.Commun.,1998,4,455-456, Koshkin et al.,Tetrahedron,1998,54,3607-3630, Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638, Kumar et al. 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. 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, 5,118,802, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461 and 7,399,845, International Publication No. 2004 / 106356, International Publication No. 1994 / 14226, International Publication No. 2005 / 02157 Brochure No. 0, International Publication Brochure No. 2007 / 090071 and International Publication Brochure No. 2007 / 134181, US Patent Application Publication No. 2004 / 0171570, No. 2007 / 0287831 and No. 2008 / 0039618, US Provisional Patent Application No. 60 / 989,574, No. 61 / 026,995, No. 61 / 026,998, No. 61 / 056,564, No. 61 / 086,231, No. 61 / 097,787 and No. 61 / 099,844 and International Application PCT / US2008 / 064591, PCT See also US2008 / 066154, PCT US2008 / 068922, and PCT / DK98 / 00393. The disclosures of each reference listed in this paragraph are incorporated herein by reference.

[0174] In certain embodiments, nucleic acids include linked nucleic acids. Nucleic acids can be linked together using any internucleotide linkage. Two main classes of internucleotide linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleotide linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleotide linkage groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-), siloxanes (-O-Si(H)2-O-), and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleotide linkages having a chiral atom can be prepared as separate enantiomers, such as alkylphosphonates and phosphorothioates, or as a racemic mixture. Non-natural nucleic acids may contain a single modification. Non-natural nucleic acids may contain multiple modifications within a single part or between different parts.

[0175] Examples of phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (crosslinked or uncrosslinked), phosphotryesters, phosphorodithioates, phosphodithioates, and boranophosphates, and can be used in any combination. Other non-phosphate crosslinks can also be used.

[0176] In some embodiments, skeletal modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate nucleotide linkages) can confer immunomodulatory activity to modified nucleic acids and / or enhance their stability in vivo.

[0177] In some cases, the phosphorus derivative (or modified phosphate group) is attached to a sugar or sugar analog moiety and may be monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, etc. Exemplary polynucleotides containing modified phosphate crosslinks or non-phosphate crosslinks include: Peyrottes et al., 1996, Nucleic Acids Res. 24:1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview”, Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, “Oligonucleoside Phosphorothioates”, Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp.165-190; Miller et al., 1971, JACS These disclosures can be found in 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, each of which is incorporated herein by reference.

[0178] In some cases, skeletal modifications involve substituting phosphodiester bonds with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include anionic nucleoside bonds, N3'-P5' phosphoramidate modifications, boranophosphate DNA, prooligonucleotides, neutral nucleoside bonds such as methylphosphonates, amide-bonded DNA, methylene (methylimino) bonds, formal bonds and thioformal bonds, skeletons containing sulfonyl groups, morpholino oligos, peptide nucleic acids (PNAs), and positively charged deoxyribonuclear guanidine (DNG) oligos (the disclosure of which is incorporated herein by reference Micklefield, 2001, Current Medicinal Chemistry 8:1157-1179). Modified nucleic acids may contain chimeric or mixed skeletons comprising one or more modifications, such as combinations of phosphate crosslinks, such as combinations of phosphodiester and phosphorothioate crosslinks.

[0179] Examples of phosphate substitutions include short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar portion of a nucleoside), siloxane skeletons, sulfides, sulfoxides and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl backbones, alkene-containing skeletons, sulfamic acid skeletons, methyleneimino and methylenehydrazino skeletons, sulfonic acid and sulfonamide skeletons, amide skeletons, and others containing a mixture of N, O, S and CH2 components. Numerous U.S. patents disclose how to manufacture and use these types of phosphate substitutions, including U.S. Patents No. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, and 5,489,6 Examples include, but are not limited to, Specification No. 77, Specification No. 5,541,307, Specification No. 5,561,225, Specification No. 5,596,086, Specification No. 5,602,240, Specification No. 5,610,289, Specification No. 5,602,240, Specification No. 5,608,046, Specification No. 5,610,289, Specification No. 5,618,704, Specification No. 5,623,070, Specification No. 5,663,312, Specification No. 5,633,360, Specification No. 5,677,437 and Specification No. 5,677,439. It is also understood that in nucleotide substitutions, both the sugar and phosphate portions of the nucleotide can be replaced, for example, by an amide-type bond (aminoethylglycine) (PNA).U.S. Patents No. 5,539,082, No. 5,714,331, and No. 5,719,262 teach methods for the preparation and use of PNA molecules, which are incorporated herein by reference, respectively. See also Nielsen et al., Science, 1991, 254, 1497-1500. Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs.Such conjugates include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309, Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654, Shea et al., Nucl. Acids Examples include, but are not limited to, Res., 1990, 18, 3777-3783, polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973 or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et 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).Numerous U.S. patents teach the preparation of such conjugates, including U.S. Patents Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,580,731, 5,591,584, 5,109,124, 5,118,802, and 5,138 ,045 specification, 5,414,077 specification, 5,486,603 specification, 5,512,439 specification, 5,5 Specification No. 78,718, Specification No. 5,608,046, Specification No. 4,587,044, Specification No. 4,605,735, Specification No. 4 , 667,025, 4,762,779, 4,789,737, 4,824,941, 4,824,941, Specification No. 4,835,263, Specification No. 4,876,335, Specification No. 4,904,582, Specification No. 4,958,013, Specification No. 5,082,830, Specification No. 5,112,963, Specification No. 5,214,136, Specification No. 5,082,830 5,112,963, 5,214,136, 5,245,022, 5,254,469 Specification, Specification No. 5,258,506, Specification No. 5,262,536, Specification No. 5,272,250, Specification No. 5,292,87 Specification No. 3, Specification No. 5,317,098, Specification No. 5,371,241, Specification No. 5,391,723, Specification No. 5,416,2 Examples include, but are not limited to, Specification No. 03, Specification No. 5,451,463, Specification No. 5,510,475, Specification No. 5,512,667, Specification No. 5,514,785, Specification No. 5,565,552, Specification No. 5,567,810, Specification No. 5,574,142, Specification No. 5,585,481, Specification No. 5,587,371, Specification No. 5,595,726, Specification No. 5,597,696, Specification No. 5,599,923, Specification No. 5,599,928 and Specification No. 5,688,941.The disclosures of each reference listed in this paragraph are incorporated herein by reference.

[0180] In some cases, non-natural nucleic acids further form non-natural base pairs. Exemplary non-natural nucleotides that can form non-natural 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: [ka] Examples 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.

[0181] Other examples of non-natural nucleotides that can form non-natural UBPs that can be used to prepare the IL-2 conjugates disclosed herein include 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 These can be found in Soc. 2013, 135:5408-5419 and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010, the respective disclosures of which are incorporated herein by reference. In some embodiments, non-natural nucleotides include: [ka] These are some examples.

[0182] In some embodiments, the non-natural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein are of the following formula: [ka] It can be derived from the compound, In the formula, R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano and azide, and The wavy lines indicate binding to ribosyl or 2'-deoxyribosyl, where the 5'-hydroxyl group of the ribosyl or 2'-deoxyribosyl moiety is either free, bound to a monophosphate, diphosphate, tripphosphate, α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or contained within RNA, DNA, RNA analogues, or DNA analogues.

[0183] In some embodiments, the non-natural nucleotides that may be used to prepare the IL-2 conjugate disclosed herein are of the following formula: [ka] It can be derived from the compound, in which, Each X is independently either carbon or nitrogen. R2 is not present when X is nitrogen, but is present when X is carbon, and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azide. Y is sulfur, oxygen, selenium, or a secondary amine. E is oxygen, sulfur, or selenium, and The wavy lines indicate binding sites to ribosyl, deoxyribosyl, or dideoxyribosyl moieties or analogs thereof, where the ribosyl, deoxyribosyl, or dideoxyribosyl moieties or analogs are either free, bound to a mono-phosphate, diphosphate, tripphosphate, α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or contained within RNA, DNA, RNA analogues, or DNA analogues.

[0184] In some embodiments, each X is carbon. In some embodiments, at least one X is carbon. In some embodiments, one X is carbon. In some embodiments, at least two X are carbon. In some embodiments, two X are carbon. In some embodiments, at least one X is nitrogen. In some embodiments, one X is nitrogen. In some embodiments, at least two X are nitrogen. In some embodiments, two X are nitrogen.

[0185] In some embodiments, Y is sulfur. In some embodiments, Y is oxygen. In some embodiments, Y is selenium. In some embodiments, Y is a secondary amine.

[0186] In some embodiments, E is sulfur. In some embodiments, E is oxygen. In some embodiments, E is selenium.

[0187] In some embodiments, R2 is present when X is carbon. In some embodiments, when X is nitrogen, R 2 It does not exist. In some embodiments, each R2, if present, is hydrogen. In some embodiments, R2 is alkyl, e.g., methyl, ethyl, or propyl. In some embodiments, R2 is alkenyl, e.g., -CH2=CH2. In some embodiments, R2 is alkynyl, e.g., ethynyl. In some embodiments, R2 is methoxy. In some embodiments, R2 is methanethiol. In some embodiments, R2 is methaneseleno. In some embodiments, R2 is halogen, e.g., chloro, bromo, or fluoro. In some embodiments, R2 is cyano. In some embodiments, R2 is azide.

[0188] In some embodiments, E is sulfur, Y is sulfur, and each X is independently carbon or nitrogen.

[0189] In some embodiments, non-natural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein are [ka] It can be derived from the compound. In some embodiments, non-natural nucleotides that can be used to prepare the IL-2 conjugate disclosed herein include: [ka] [ka] Or, its salt.

[0190] In some embodiments, non-natural base pairs generate non-natural amino acids as described in Dumas et al., “Designing logical codon reassignment—Expanding the chemistry in biology,” Chemical Science, 6:50-69 (2015), which is incorporated herein by reference.

[0191] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL polypeptides) by synthetic codons containing non-natural nucleic acids. In some examples, non-natural amino acids are incorporated into cytokines by orthogonal synthase / tRNA pairs. Such orthogonal pairs include non-natural synthases that can charge non-natural tRNA with non-natural amino acids while minimizing a) charging of non-natural tRNA with other endogenous amino acids and b) charging of other endogenous tRNA with non-natural amino acids. Such orthogonal pairs include tRNA that can be charged by non-natural synthases while avoiding charging of other endogenous amino acids by endogenous synthases. In some embodiments, such pairs are identified from various organisms such as bacteria, yeast, archaea, or human sources. In some embodiments, the orthogonal synthase / tRNA pair includes components derived from a single organism. In some embodiments, the orthogonal synthase / tRNA pair includes components derived from two different organisms. In some embodiments, the orthogonal synthase / tRNA pair includes components that facilitate the translation of two different amino acids before 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 asparagine 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 glutamine synthase. In some embodiments, the orthogonal synthase is a modified glutamate synthase. In some embodiments, the orthogonal synthase is a modified alanin lysine synthase. 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 glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine lysine. 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.

[0192] In some embodiments, non-natural amino acids are incorporated into cytokines (e.g., IL polypeptides) by aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pairs. Exemplary aaRS-tRNA pairs include the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair and the Escherichia coli (E. coli) TyrRS (Ec-Tyr) / B. stearothermophilus tRNA pair. CUA vs. *E. coli* LeuRS (Ec-Leu) / *B. stearothermophilus* tRNA CUA Examples include, but are not limited to, Mj-TyrRS / tRNA pairs and pyrrolyl-tRNA pairs. In some cases, non-natural amino acids are incorporated into cytokines (e.g., IL polypeptides) by Mj-TyrRS / tRNA pairs. Exemplary UAAs that can be incorporated by Mj-TyrRS / tRNA pairs include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine, meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine and 3-iodotyrosine, phenylselenocysteine, p-boronophenylalanine and o-nitrobenzyltyrosine.

[0193] In some cases, non-natural amino acids are Ec-Tyr / tRNA CUA or Ec-Leu / tRNA CUA Ec-Tyr / tRNA is incorporated into cytokines (e.g., IL polypeptides) through pairing. CUA or Ec-Leu / tRNA CUA Examples of UAAs that can be incorporated by pair include, but are not limited to, benzophenone, ketone, phenylalanine derivatives containing iodide or azide substituents, O-propargyltyrosine, α-aminocaprylic acid, O-methyltyrosine, O-nitrobenzylcysteine, and 3-(naphthalene-2-ylamino)-2-aminopropanoic acid.

[0194] In some cases, non-natural amino acids are incorporated into cytokines (e.g., IL polypeptides) by pyrrolidine-tRNA pairs. In some cases, PylRS are obtained from archaea, e.g., methanogenic archaea. In some cases, PylRS are obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated by pyrrolidine-tRNA pairs include amide and carbamate-substituted lysines, e.g., 2-amino-6-((R)-tetrahydrofuran-2-carboxamide)hexanoic acid, N-ε- D - Prolil - L -Lysine and N-ε-cyclopentyloxycarbonyl- L -Lysine, N-ε-Acryloyl- L -Lysine, N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]- L Examples include, but are not limited to, lysine and N-ε-(1-methylcyclopropyl-2-encarboxamide)lysine. In some embodiments, the IL-2 conjugates disclosed herein may be prepared by using M. mazei tRNA selectively charged with a non-natural amino acid such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (Azk) by M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS). Other methods, for example, those disclosed in Zhang et al., Nature 2017, 551(7682):644-647, are known to those skilled in the art and are incorporated herein by reference.

[0195] In some cases, non-natural amino acids are incorporated into cytokines (e.g., IL polypeptides) as described herein by synthases disclosed in U.S. Patent No. 9,988,619 and U.S. Patent No. 9,938,516, respectively, each of which is incorporated herein by reference.

[0196] The host cells into which the constructs or vectors disclosed herein are introduced are cultured or maintained in a suitable medium so as to produce tRNA, tRNA synthetase, and the protein of interest. The medium also contains non-natural amino acids so that the protein of interest incorporates non-natural amino acids. In some embodiments, nucleoside triphosphate transporters (NTTs) derived from bacteria, plants, or algae are also present in the host cells. In some embodiments, the IL-2 conjugates disclosed herein are prepared by using host cells expressing NTTs. In some embodiments, the nucleotide nucleoside triphosphate transporters used in host cells are TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis). NTT can be selected from trachomatis), PamNTT1, PamNTT2 (Protochlamia amoebophila), CcNTT (Caedibacter caryophilus), and RpNTT1 (Rickettsia prowazekii). In some embodiments, NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, NTT is PtNTT1. In some embodiments, NTT is PtNTT2. In some embodiments, NTT is PtNTT3. In some embodiments, NTT is PtNTT4. In some embodiments, NTT is PtNTT5. In some embodiments, NTT is PtNTT6.Other NTTs that may be used are disclosed in Zhang et al., Nature 2017, 551(7682):644-647, Malyshev et al. Nature 2014(509(7500), 385-388, and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322.

[0197] Orthogonal tRNA synthetase / tRNA pairs charge tRNA with non-natural amino acids and incorporate these non-natural amino acids into the polypeptide chain in response to codons. Exemplary aaRS-tRNA pairs include the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair and the E. coli (Ec-Tyr) / B. stearothermophilus tRNA pair. CUA vs. *E. coli* LeuRS (Ec-Leu) / *B. stearothermophilus* tRNA CUA Examples include, but are not limited to, aaRS-tRNA pairs and pyrrolyl-tRNA pairs. Other aaRS-tRNA pairs that may be used in accordance with this disclosure include those obtained from M. mazei as described in Feldman et al., J Am Chem Soc., 2018 140:1447-1454 and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322, the respective disclosures of which are incorporated herein by reference.

[0198] In some embodiments, methods are provided for preparing the IL-2 conjugate disclosed herein in cell lines expressing NTT and tRNA synthetase. In some embodiments described herein, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5 and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii, Escherichia coli TyrRS (Ec-Tyr) / B. stearothermophilus and M. mazei. In some embodiments, NTT is PtNTT1, and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS (Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, NTT is PtNTT2, and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS (Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, NTT is PtNTT3, and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS (Ec-Tyr) / B. stearothermophilus, or M. mazei.In some embodiments, NTT is PtNTT4, and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS (Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, NTT is PtNTT5, and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS (Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, NTT is PtNTT6, and the tRNA synthetase is derived from Methanococcus jannaschii, Escherichia coli TyrRS (Ec-Tyr) / B. stearothermophilus, or M. mazei.

[0199] In some embodiments, the IL-2 conjugates disclosed herein may be prepared in cells such as Escherichia coli, comprising a plasmid containing (a) a nucleotide triphosphate transporter PtNTT2 (including a cleaved variant in which the first 65 amino acid residues of the full-length protein are deleted), (b) a plasmid containing a double-stranded oligonucleotide containing a first non-natural nucleotide and a second non-natural nucleotide for providing a codon at a desired position in which a non-natural amino acid such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (Azk) is incorporated, (c) a plasmid containing a non-natural nucleotide for providing an anticodon (for the codon of the IL-2 variant) recognized in place of its natural sequence, or (d) a plasmid containing pyrrolidine-tRNA synthetase (Mb pylrs) from M. barkeri, which may be the same plasmid as the one encoding the tRNA or a different plasmid. In some embodiments, the cells are further supplemented with a deoxyribotriptyphosphate containing one or more non-natural bases. In some embodiments, the cells are further supplemented with a ribbonotriptyphosphate containing one or more non-natural bases. In some embodiments, the cells are further supplemented with one or more non-natural amino acids, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, a double-stranded oligonucleotide encoding the amino acid sequence of a desired IL-2 variant includes the codon AXC at position 64 of the sequence encoding the protein having SEQ ID NO: 1, where X is a non-natural nucleotide. In some embodiments, the cells further include a plasmid, which may be a protein expression plasmid or another plasmid, encoding an orthogonal tRNA gene derived from M. mazei, which includes the AXC-matching anticodon GYT instead of its natural sequence, where Y is a complementary non-natural nucleotide that may be the same as or different from the non-natural nucleotide in the codon. In some embodiments, the non-natural nucleotide in the codon is complementary to it, unlike the non-natural nucleotide in the anticodon.In some embodiments, the non-natural nucleotide in the codon is the same as the non-natural nucleotide in the anticodon. In some embodiments, the first and second non-natural nucleotides containing non-natural base pairs in the double-stranded oligonucleotide are... [ka] It may originate from the following. In some embodiments, the first and second unnatural nucleotides containing unnatural base pairs in the double-stranded oligonucleotide are [ka] It may be derived from the following. In some embodiments, the triphosphates of the first and second unnatural nucleotides are: [ka] Or salts thereof. In some embodiments, the triphosphates of the first and second unnatural nucleotides are: [ka] Or a salt thereof. In some embodiments, mRNA derived from a double-stranded oligonucleotide comprising a first non-natural nucleotide and a second non-natural nucleotide is [ka] It may contain a codon containing a non-natural nucleotide obtained from. In some embodiments, M. mazei tRNA may contain an anticodon containing a non-natural nucleotide that recognizes a codon containing a non-natural nucleotide in mRNA. The anticodon of M. mazei tRNA is [ka] It may contain non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, mRNA is [ka] It contains non-natural nucleotides obtained from. In some embodiments, tRNA is [ka] The culture medium contains non-natural nucleotides obtained from [source]. Host cells are cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more non-natural bases necessary for replication of a plasmid encoding a cytokine gene having a codon, (b) mRNA containing one or more non-natural bases necessary for transcription of (i) mRNA containing a codon corresponding to the coding sequence of the cytokine and containing one or more non-natural bases, and (ii) ribonucleoside triphosphates containing one or more non-natural bases necessary for transcription of tRNA containing an anticodon containing one or more non-natural bases, and (c) non-natural amino acids incorporated into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that allow for the expression of the protein of interest.

[0200] The resulting AzK-containing protein can be purified by methods known to those skilled in the art and then reacted with an alkyne such as DBCO containing a PEG chain having a desired average molecular weight as disclosed herein, under conditions known to those skilled in the art, to obtain the IL-2 conjugate disclosed herein. Other methods, such as those disclosed in Zhang et al., Nature 2017, 551(7682):644-647, International Publication No. 2015157555, International Publication No. 2015021432, International Publication No. 2016115168, International Publication No. 2017106767, International Publication No. 2017223528, International Publication No. 2019014262, International Publication No. 2019014267, International Publication No. 2019028419 and International Publication No. 2019 / 028425, are known to those skilled in the art, and each of those disclosures is incorporated herein by reference.

[0201] The resulting protein, containing one or more expressed non-natural amino acids, such as Azk, can be purified by methods known to those skilled in the art and then reacted under conditions known to those skilled in the art with an alkyne, such as DBCO containing a PEG chain having a desired average molecular weight as disclosed herein, to obtain the IL-2 conjugate disclosed herein. Other methods, such as those disclosed in Zhang et al., Nature 2017, 551(7682):644-647, International Publication No. 2015157555, International Publication No. 2015021432, International Publication No. 2016115168, International Publication No. 2017106767, International Publication No. 2017223528, International Publication No. 2019014262, International Publication No. 2019014267, International Publication No. 2019028419 and International Publication No. 2019 / 028425, are known to those skilled in the art, and each of those disclosures is incorporated herein by reference.

[0202] Alternatively, IL-2 polypeptides containing non-natural amino acids are prepared by introducing a nucleic acid construct described herein into host cells, which includes a nucleic acid sequence of interest comprising tRNA and aminoacyl-tRNA synthetase and having one or more in-frame orthogonal (stop) codons. The host cells are cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more non-natural bases necessary for replication of a cytokine gene encoding a novel codon and anticodon, (b) ribonucleoside triphosphates necessary for transcription of mRNA corresponding to the orthogonal tRNA containing (i) a codon and (ii) an anticodon, and (c) non-natural amino acids. The host cells are then maintained under conditions that allow for the expression of the protein of interest. The non-natural amino acids are incorporated into the polypeptide chain in response to the non-natural codon. For example, one or more non-natural amino acids are incorporated into the IL-2 polypeptide. Alternatively, two or more non-natural amino acids can be incorporated into the IL-2 polypeptide at two or more sites in the protein.

[0203] Once IL-2 polypeptides incorporating non-natural amino acids are produced in host cells, they can be extracted therefrom by a variety of techniques known in the art, including enzymatic, chemical, and / or osmotic lysis 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 techniques known to those skilled in the art.

[0204] Suitable host cells include bacterial cells (e.g., Escherichia coli, BL21(DE3)), but the most suitable host cells are eukaryotic cells, such as insect cells (e.g., Drosophila melanogaster and other Drosophila species), yeast cells, nematodes (e.g., C. elegans), mice (e.g., Mus musculus), or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells), or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Preferably, the host cell is a human cell or a mammalian cell such as an insect cell. In some embodiments, suitable host cells include Escherichia coli.

[0205] Other suitable host cells that may be commonly used in embodiments of the present invention are mentioned in the Examples section. Vector DNA can be introduced into host cells by conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of well-known techniques for introducing foreign nucleic acid molecules (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

[0206] When constructing cell lines, it is generally preferable to prepare stable cell lines. For example, for stable transfection of mammalian cells, it is known that only a small fraction of cells can incorporate foreign DNA into their genomes, depending on the expression vector and transfection technique used. To identify and select these integrants, genes encoding selection markers (e.g., for antibiotic resistance) are generally introduced into host cells along with the gene of interest. Preferred selection markers include those that confer resistance to drugs such as G418, hygromycin, or methotrexate. The nucleic acid molecule encoding the selection marker can be introduced into host cells on the same vector or on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells incorporating the selection marker gene survive, while other cells are killed).

[0207] In one embodiment, the construct described herein is incorporated into the genome of a host cell. The advantage of stable incorporation is that uniformity between individual cells or clones is achieved. Another advantage is that the selection of the best producer can be carried out. Therefore, it is desirable to create a stable cell line. In another embodiment, the construct described herein is transfected into a host cell. The advantage of transfecting the construct into a host cell is that protein yield can be maximized. In one embodiment, a cell containing the nucleic acid construct or vector described herein is described.

[0208] Treatment method In one embodiment, methods are provided herein for stimulating and / or proliferating specific immune cells useful for the treatment of cancer, such as Teff cells, NK cells and / or NKT cells, in a subject in need, such as a subject having cancer, the methods comprising administering to the subject a first dose (a) about once a week over a first number of weeks, and then a second dose (b) about once every two weeks at least over a second number of weeks (e.g., once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more). In some embodiments, such cancer is a solid tumor. In some embodiments, such cancer is a hematological cancer.

[0209] In another embodiment, a method for treating cancer in a person in need thereof, comprising administering the IL-2 conjugate described herein at a first dose (a) about once a week over a first number of weeks, and then at a second dose (b) at least once every two weeks (for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more) over a second number of weeks as described herein.

[0210] In some embodiments of the various embodiments described herein, the first dose and / or the second dose are the same. In some embodiments of the various embodiments described herein, the first dose and / or the second dose are different. In some embodiments of the various embodiments described herein, the second dose is lower than the first dose. In some embodiments of the various embodiments described herein, the second dose is higher than the first dose.

[0211] In some embodiments of the various embodiments described herein, the first dose and / or second dose is about 8 μg / kg of IL-2 as the IL-2 conjugate described herein. In some embodiments of the various embodiments described herein, the first dose and / or second dose is about 16 μg / kg of IL-2 as the IL-2 conjugate described herein. In some embodiments of the various embodiments described herein, the first dose and / or second dose is about 24 μg / kg of IL-2 as the IL-2 conjugate described herein. In some embodiments of the various embodiments described herein, the first dose and / or second dose is about 32 μg / kg of IL-2 as the IL-2 conjugate described herein.

[0212] Types of PHP In some embodiments, cancer is a solid tumor as described herein. In some embodiments, cancer is a hematological cancer, including, but not limited to, leukemia, lymphoma, and multiple myeloma. In some embodiments, cancers are selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), urothelial carcinoma, microsatellite unstable cancer, microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer (CRC), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal 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 loading, cutaneous squamous cell carcinoma (CSCC), PD-L1 low-to-non-expressing tumors, PD-L1 expressing tumors, and tumors that have disseminated systemically to the liver and CNS beyond the primary anatomical site of origin.

[0213] In one embodiment, cancer is a solid tumor. In one embodiment, cancer is melanoma. In one embodiment, cancer is renal cell carcinoma (RCC). In one embodiment, cancer is cancer such as ovarian cancer, colorectal cancer, pancreatic cancer, or hepatocellular carcinoma.

[0214] In one embodiment, a method is provided herein for treating cancer (e.g., solid tumors or hematological cancers) in a subject in need thereof, comprising administering the IL-2 conjugate described herein to the subject. In some embodiments, a method is provided herein for treating cancer (e.g., solid tumors or hematological cancers) in a subject in need thereof, comprising administering about 8 μg / kg of IL-2 as the IL-2 conjugate described herein to the subject. In some embodiments, a method is provided herein for treating cancer (e.g., solid tumors or hematological cancers) in a subject in need thereof, comprising administering about 16 μg / kg of IL-2 as the IL-2 conjugate described herein to the subject. In some embodiments, a method is provided herein for treating cancer (e.g., solid tumors or hematological cancers) in need thereof, comprising administering about 24 μg / kg of IL-2 as the IL-2 conjugate described herein to the subject. In some embodiments, methods are provided herein for treating cancer (e.g., solid tumors or hematological cancers) in subjects requiring such treatment, the methods comprising administering approximately 32 μg / kg of IL-2 as an IL-2 conjugate described herein to the subject.

[0215] In a further embodiment, the use of IL-2 conjugate for manufacturing a pharmaceutically acceptable substance for a method disclosed herein, for treating a solid tumor, such as cancer including melanoma or renal cell carcinoma (RCC), in a subject in need thereof.

[0216] The embodiments described in the following sections apply to any of the embodiments described above.

[0217] Administration In some embodiments, the IL-2 conjugate is administered as at least a second or subsequent line of treatment. In some embodiments, the IL-2 conjugate is administered as at least a third or subsequent line of treatment. In some embodiments, the IL-2 conjugate is administered as at least a fourth or subsequent line of treatment. In some embodiments, the IL-2 conjugate is administered as at least a fifth or subsequent line of treatment.

[0218] In some embodiments, the pre-treatment line includes an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor includes a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a LAG-3 inhibitor.

[0219] In some embodiments, the IL-2 conjugate is administered to the subject by intravenous injection, subcutaneous injection, intramuscular injection, intracerebral injection, intranasal injection, intra-arterial injection, intradermal injection, intravitreous injection, intraosseous injection, intraperitoneal injection, or intrathecal injection. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, or intramuscular injection. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous injection. In some embodiments, the IL-2 conjugate is administered to the subject by subcutaneous injection. In some embodiments, the IL-2 conjugate is administered to the subject by intramuscular injection. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous injection.

[0220] The IL-2 conjugate may be administered two or more times, for example, two, three, four, five or more times. In some embodiments, the treatment period is up to 24 months, such as one month, two months, three months, six months, nine months, twelve months, fifteen months, eighteen months, twenty-one months, or twenty-four months. In some embodiments, the treatment period may be further extended by up to an additional 24 months.

[0221] In some embodiments, the IL-2 conjugate is administered to subjects requiring it approximately once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the IL-2 conjugate is administered to subjects requiring it once a week. In some embodiments, the IL-2 conjugate is administered to subjects requiring it once every two weeks. In some embodiments, the IL-2 conjugate is administered to subjects requiring it once every three weeks. In some embodiments, the IL-2 conjugate is administered to subjects requiring it once every four weeks. In some embodiments, the IL-2 conjugate is administered approximately every 14, 15, 16, 17, 18, 19, 20, or 21 days.

[0222] Administration can be carried out, for example, once, multiple times, and / or over a long period of time. In some embodiments, the IL-2 conjugate is administered to a subject in need about once a week over a first number of weeks (induction period), and then administered at least once every two weeks over a second number of weeks (e.g., once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more) (maintenance period). In some embodiments, the number of weeks in the induction period is about three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks. In some embodiments, the maintenance period of multiple weeks is approximately 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 30 weeks, 40 weeks, 50 weeks, 60 weeks, 70 weeks, 80 weeks, 90 weeks, or 100 weeks. In some embodiments, the maintenance period of multiple weeks is in the range of approximately 4 weeks to 100 weeks, or approximately 6 weeks to 100 weeks, or approximately 6 weeks to 98 weeks, or approximately 6 weeks to 46 weeks. In some embodiments, the maintenance period of several weeks is at least about 4 weeks, at least about 6 weeks, at least about 8 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 18 weeks, at least about 20 weeks, at least about 30 weeks, at least about 40 weeks, at least about 46 weeks, at least about 50 weeks, at least about 60 weeks, at least about 70 weeks, at least about 80 weeks, at least about 90 weeks, at least about 98 weeks, or at least about 100 weeks.

[0223] In some embodiments, the IL-2 conjugate is administered to subjects requiring it once weekly for about six weeks, and then administered at least once every two weeks (for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more) over several weeks (maintenance periods). In some embodiments, the several weeks of the maintenance period may be about six weeks, about twelve weeks, 18 weeks, 24 weeks, about 30 weeks, about 36 weeks, about 42 weeks, about 48 weeks, about 54 weeks, about 60 weeks or more.

[0224] In some embodiments, the IL-2 conjugate is administered to subjects requiring it at doses of approximately 8 μg / kg of IL-2 as an IL-2 conjugate, approximately 16 μg / kg of IL-2 as an IL-2 conjugate, approximately 24 μg / kg of IL-2 as an IL-2 conjugate, and approximately 32 μg / kg of IL-2 as an IL-2 conjugate, approximately once a week over a first set of weeks.

[0225] In some embodiments, the IL-2 conjugate is administered to subjects in need at least once every two weeks (for example, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks or more for a second set of weeks).

[0226] In some embodiments, the IL-2 conjugate is administered to subjects requiring it on days 1, 8, 15, 22, 29 and 36±1 of the first 6-week cycle, and then on days 1, 15 and 29±1 of each subsequent 6-week cycle. In some embodiments, the number of subsequent 6-week cycles is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0227] In some cases, the desired dose is presented, for convenience, as a single dose or as divided doses administered simultaneously (or over a short period) or at appropriate intervals, for example, as two, three, four or more partial doses per day.

[0228] In some embodiments, the methods described herein further include administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents include one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent includes pemetrexed. In some embodiments, the chemotherapeutic agent includes a platinum-based agent such as carboplatin. In some embodiments, the chemotherapeutic agent includes cisplatin. In some embodiments, the chemotherapeutic agent includes nab-paclitaxel. In some embodiments, the chemotherapeutic agent includes pemetrexed and a platinum-based agent such as carboplatin. In some embodiments, the chemotherapeutic agent includes pemetrexed and cisplatin. In some embodiments, the additional therapeutic agent includes one or more immunotherapeutic agents. In some embodiments, the additional therapeutic agent includes an antihistamine such as diphenhydramine.

[0229] In some embodiments, the additional therapeutic agent includes a chemotherapeutic agent and an antihistamine, such as diphenhydramine. In some embodiments, the additional therapeutic agent includes one of the aforementioned chemotherapeutic agents and an antihistamine such as diphenhydramine. In some embodiments, the additional therapeutic agent includes an analgesic such as acetaminophen. In some embodiments, the further therapeutic agent includes a chemotherapeutic agent and an analgesic, such as acetaminophen. In some embodiments, the additional therapeutic agent includes one of the aforementioned chemotherapeutic agents and an analgesic such as acetaminophen.

[0230] In some embodiments, the additional therapeutic agent comprises one or more vitamins, such as folic acid and / or vitamin B12. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent and one or more vitamins, such as folic acid and / or vitamin B12. In some embodiments, the additional therapeutic agent comprises one of the aforementioned chemotherapeutic agents and one or more vitamins, such as folic acid and / or vitamin B12.

[0231] In some embodiments, the additional therapeutic agent comprises an antihistamine and an analgesic, for example, diphenhydramine and acetaminophen. In some embodiments, the additional therapeutic agent comprises an antihistamine and one or more vitamins, for example, diphenhydramine and one or both of folic acid and vitamin B12. In some embodiments, the additional therapeutic agent comprises an analgesic and one or more vitamins, for example, acetaminophen and one or both of folic acid and vitamin B12. In some embodiments, the additional therapeutic agent comprises an antihistamine, an analgesic and one or more vitamins, for example, diphenhydramine, acetaminophen and one or both of folic acid and vitamin B12. In any of the embodiments described above, the additional therapeutic agent may further comprise a chemotherapeutic agent, such as one of the chemotherapeutic agents described above.

[0232] subject In some embodiments, the administration of IL-2 conjugate is administered to adult subjects. In some embodiments, the adult subjects are male. In other embodiments, the adult subjects are female. In some embodiments, the adult subjects are at least 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age. In some embodiments, the adult subjects have recurrent or refractory solid tumors or recurrent or refractory cancers such as recurrent or refractory hematological malignancies. In some embodiments, the subjects have recurrent or refractory melanoma or recurrent or refractory renal cell carcinoma (RCC). In some embodiments, the subjects have refractory melanoma. In some embodiments, the subjects have recurrent melanoma. In some embodiments, the subjects have advanced solid tumors. In some embodiments, the subjects have metastatic solid tumors. In some embodiments, the metastatic solid tumors are metastatic melanoma or metastatic renal cell carcinoma (RCC).

[0233] In some embodiments, subjects are 18 years of age or older. In some embodiments, subjects have previously received anticancer therapy (e.g., radiotherapy, chemotherapy, surgery, targeted therapy), including immunotherapy. In some embodiments, subjects have previously received anticancer therapy and any treatment-related toxicity has improved (1) to grade 0 or 1 (excluding alopecia) according to NCI CTCAE v5.0, or (2) to at least grade 2 according to NCI CTCAE v5.0. In some embodiments, subjects have a performance status of 0 or 1 in the East Coast Cancer Clinical Group (ECOG). In some embodiments, subjects have been determined (in some embodiments, e.g., by a physician) to have a mean life expectancy of 12 weeks or more. In some embodiments, subjects have a diagnosis of histologically or cytologically confirmed advanced and / or metastatic solid tumor and have at least one tumor lesion in a location where it can be safely biopsied, as determined by a physician. In some embodiments, subjects have received at least one prior therapy line for metastatic melanoma and / or have no standard of care (SoC) treatment options, or the participant refuses or cannot tolerate SoC treatment. In some embodiments, subjects have advanced or metastatic solid tumors and have refused standard of care, or there is no reasonable standard of care that provides a clinical benefit, or standard of care is tolerable, ineffective, or unavailable. In some embodiments, subjects have a measurable disease and at least one measurable lesion according to RECIST v1.1. In some embodiments, subjects have: (1) an absolute lymphocyte count of 0.5 times or more below the lower limit of normal, and (2) a platelet count of 100 × 10⁶ 9 (3) Hemoglobin of 9.0 g / dL or higher (no growth factors or transfection within the past two weeks, and a one-week washout for erythropoiesis-stimulating agents (ESAs) and colony-stimulating factors (CSFs) is sufficient), (4) Absolute neutrophil count of 1.5 × 10⁻¹⁴ 9The subject has appropriate experimental parameters, including (5) prothrombin time (PT) and partial thromboplastin time (PTT) being 1.5 times or less of ULN, (6) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) being 2.5 times or less of ULN, except in the case of liver metastases, and (7) total bilirubin being 1.5 × ULN or less. In some embodiments, the subject satisfies each of the above criteria.

[0234] In some embodiments, subjects had not received radiotherapy within 14 days prior to the first dose of IL-2 conjugate, or had not received palliative radiotherapy or stereotactic radiosurgery within 7 days prior to the first dose of IL-2 conjugate. In some embodiments, subjects had not received systemic anticancer therapy or investigational drugs within 2 weeks prior to the first dose of IL-2 conjugate or within 4 weeks for immunotherapy and tyrosine kinase inhibitor therapy. In some embodiments, subjects had not experienced grade 3 or higher immuno-related toxicity from previous immuno-oncology therapy. In some embodiments, subjects had not undergone major surgery within 30 days prior to the first dose of IL-2 conjugate, or had not recovered to at least grade 1 from adverse effects of such procedure, or were expected to require major surgery during treatment with IL-2 conjugate. In some embodiments, subjects have not had an active autoimmune disease requiring systemic treatment within the past three months, nor a documented history of a clinically severe autoimmune disease requiring systemic steroids or immunosuppressants, nor have they received corticosteroids (excluding inhaled, intranasal, intraocular, and topical steroids for disease evaluation or steroids for prophylactic use against contrast reactions) at doses exceeding 10 mg per day of prednisone or its equivalent. In some embodiments, subjects have not had primary central nervous system (CNS) disease or leptomeningeal disease, known CNS metastases, except in cases where the subject was treated, asymptomatic, showed no radiological progression for at least eight weeks, and did not require steroids or enzyme-induced anticonvulsants in the 14 days prior to screening. In some embodiments, subjects have not had pneumonia, active pneumonia, interstitial lung disease requiring steroid use, idiopathic pulmonary fibrosis, confirmed pleural effusion, severe dyspnea at rest, or pulmonary dysfunction requiring oxygen therapy within the past six months. In some embodiments, subjects have not had a history of allogeneic or solid organ transplantation. In some embodiments, the subjects do not have, in the physician's opinion, uncontrolled diabetes or other uncontrolled immune-related endocrine disorders.In some embodiments, subjects have not received parenteral antibiotics within 14 days prior to the first dose of the IL-2 conjugate, and do not have a severe systemic fungal, bacterial, viral, or other infection that is uncontrolled or requires intravenous antibiotics. In some embodiments, subjects do not have a known human immunodeficiency virus (HIV) infection, active hepatitis C infection, or a known uncontrolled hepatitis B virus (HBV) infection (unless the subject has received anti-HBV therapy before the initiation of the first dose of the IL-2 conjugate and has an HBV viral load <2000 IU / mL (10⁴ copies / mL), or has an HBV viral load that is anti-HBc positive, anti-HBs positive, HBsAg negative, and has not received HBV therapy). In some embodiments, subjects have not received live virus or live attenuated virus vaccination (excluding seasonal influenza vaccines or SARS-CoV-2 vaccines that do not contain live virus) within 14 days prior to treatment. In some embodiments, subjects had not experienced clinically significant bleeding (e.g., gastrointestinal bleeding, intracranial hemorrhage) within two weeks prior to the first administration of IL-2 conjugate. In some embodiments, subjects had not been diagnosed with deep vein thrombosis or pulmonary embolism within three months of screening. In some embodiments, subjects had not had any severe or unstable cardiac conditions within six months prior to the first administration of IL-2 conjugate, such as congestive heart failure (New York Cardiology Class III or IV), cardiac bypass surgery or coronary stenting, angioplasty, normal or unstable angina, medically uncontrolled hypertension (e.g., systolic ≥ 160 mmHg or diastolic ≥ 100 mmHg), poorly controlled cardiac arrhythmias requiring medication (grade 2 or higher, according to NCI CTCAE v5.0), or myocardial infarction. In some embodiments, subjects had no history of non-pharmacologically induced corrected QT interval prolongation (greater than 450 milliseconds (msec) in male subjects and greater than 470 msec in female subjects) as determined using Fridericia's formula (QTcF). In some embodiments, subjects had no known hypersensitivity or contraindications to any component of IL-2 conjugate, PEG, PEGylated drugs, or Escherichia coli (E. coli)-derived proteins.In some embodiments, subjects had no history of active secondary malignancies or prior malignancies that would affect the evaluation of any of the study endpoints (excluding subjects with curatively surgically excised non-melanoma skin cancer or cervical cancer). In some embodiments, subjects did not have any serious medical conditions (including pre-existing autoimmune or inflammatory disorders), laboratory abnormalities, psychiatric conditions, or any other significant or unstable concurrent medical conditions that, in the physician's opinion, would interfere with protocol therapy or make the subject unsuitable for the study, such as subjects with clinically significant symptomatic active SARS-CoV-2 (COVID-19) infection. In some embodiments, subjects were not pregnant or breastfeeding, or were not expected to become pregnant or father, during the expected study period, starting from the screening visit and for at least 7 days for female subjects and at least 3 days after the last dose of IL-2 conjugate for male subjects. In some embodiments, subjects did not receive combination therapy with other investigational drugs, vaccines, or devices (excluding concurrent participation in observational studies). In some embodiments, subjects' baseline oxygen saturation was less than 92%. In some embodiments, the subject does not have a uvea or eye or fibrous metastatic melanoma. In some embodiments, the subject does not have any of the features listed in this paragraph. In some embodiments, the subject does not have any of the features listed in this paragraph and satisfies each of the criteria in the previous paragraph.

[0235] In some embodiments, the subjects are women of childbearing age who (1) agree to use a medically acceptable method of contraception during treatment and for at least 7 days after the last dose of IL-2 conjugate, and (2) have a negative serum pregnancy test within 7 days prior to the first dose of IL-2 conjugate. In some embodiments, the subjects are men who are not surgically infertile, and (1) agree to use a medically approved method of contraception during treatment and for at least 3 days after the last dose of IL-2 conjugate, and (2) agree to refrain from donating or storing sperm during treatment and for at least 3 days after the last dose of IL-2 conjugate.

[0236] In some embodiments, the subject does not have any known hypersensitivity or contraindications to the administered IL-2 conjugate or PEG.

[0237] In some embodiments, the subject does not have a serious medical condition (including a pre-existing autoimmune disease or inflammatory disorder), laboratory abnormalities, a psychiatric condition, or any other serious or unstable concurrent medical condition that interferes with or makes treatment inappropriate.

[0238] In some embodiments, the subjects are neither pregnant nor breastfeeding. In some embodiments, the subjects do not expect to become pregnant or give birth to a child during the course of treatment or up to 1, 2, 3, 4, 5, 6, or 7 months after the administration of the final therapeutic dose.

[0239] In some embodiments, subjects do not receive any combination therapy with investigational drugs, vaccines, or devices during the course of treatment. In some embodiments, subjects receive combination therapy during the course of treatment with the IL-2 conjugate described herein. In some embodiments, subjects receive combination therapy with investigational drugs, vaccines, or devices during the course of treatment with the IL-2 conjugate described herein, after physician approval.

[0240] Effects of administration according to the method described herein In some embodiments, administration of IL-2 conjugate provides complete response, partial response, or stable disease.

[0241] In some embodiments, after administration of the IL-2 conjugate, the subjects experience a complete response (CR) as defined by the Solid Tumor Response Criteria (RECIST) version 1.1. In some embodiments, after administration of the IL-2 conjugate, the subjects experience a partial response (PR) as defined by the Solid Tumor Response Criteria (RECIST) version 1.1. In some embodiments, after administration of the IL-2 conjugate, the subjects experience disease stability (SD) as defined by the Solid Tumor Response Criteria (RECIST) version 1.1.

[0242] In some embodiments, administration of IL-2 conjugate to a subject does not cause vasoleap syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 2, grade 3, or grade 4 vasoleap syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 2 vasoleap syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 3 vasoleap syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 4 vasoleap syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause loss of vascular tone in the subject.

[0243] In some embodiments, administration of an IL-2 conjugate to a subject does not cause extravasation of plasma proteins and fluids into the extravascular space of the subject.

[0244] In some embodiments, administration of IL-2 conjugate to subjects does not cause hypotension and decreased organ perfusion in the subjects.

[0245] In some embodiments, administration of IL-2 conjugates to subjects does not cause neutrophil dysfunction in the subjects. In some embodiments, administration of IL-2 conjugates to subjects does not cause a decrease in chemotaxis in the subjects.

[0246] In some embodiments, administration of IL-2 conjugate to a subject is not associated with an increased risk of disseminated infection in the subject. In some embodiments, the disseminated infection is sepsis or bacterial endocarditis. In some embodiments, the disseminated infection is sepsis. In some embodiments, the disseminated infection is bacterial endocarditis. In some embodiments, the subject is treated for a pre-existing bacterial infection prior to administration of IL-2 conjugate. In some embodiments, the subject is treated with an antimicrobial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of IL-2 conjugate.

[0247] In some embodiments, administration of IL-2 conjugate to a subject does not exacerbate any existing or early-onset autoimmune disease or inflammatory disorder in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not exacerbate any existing or early-onset autoimmune disease in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not exacerbate any existing or early-onset inflammatory disorder in the subject. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, severe myasthenia oculi, crescent IgA glomerulonephritis, cholecystitis, cerebrovascular vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is Crohn's disease. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is scleroderma. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is thyroiditis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is inflammatory arthritis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is diabetes mellitus. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is ocular myasthenia gravis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is crescent-type IgA glomerulonephritis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cholecystitis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cerebrovascular vasculitis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is Stevens-Johnson syndrome. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is bullous pemphigoid.

[0248] In some embodiments, administration of IL-2 conjugates to subjects does not cause changes in the subjects' mental state, speech disorders, cortical blindness, limb or gait ataxia, hallucinations, agitation, blunting, or coma. In some embodiments, administration of IL-2 conjugates to subjects does not cause seizures. In some embodiments, administration of IL-2 conjugates to subjects is not contraindicated in subjects with known paroxysmal disorders.

[0249] In some embodiments, administration of IL-2 conjugate to a subject does not cause capillary leak syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 2, grade 3, or grade 4 capillary leak syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 2 capillary leak syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 3 capillary leak syndrome in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause grade 4 capillary leak syndrome in the subject.

[0250] In some embodiments, administration of IL-2 conjugate to a subject does not cause a decrease in the subject's mean arterial pressure after administration. In some embodiments, administration of IL-2 conjugate to a subject causes hypotension in the subject. In some embodiments, administration of IL-2 conjugate to a subject does not cause the subject to experience a systolic blood pressure of less than 90 mmHg or a decrease of 20 mmHg from baseline systolic blood pressure.

[0251] In some embodiments, administration of IL-2 conjugates to subjects does not cause edema or impaired renal or hepatic function.

[0252] In some embodiments, administration of IL-2 conjugates to subjects does not cause eosinophilia in the subjects. In some embodiments, administration of IL-2 conjugates to subjects does not cause the number of eosinophils in the subjects' peripheral blood to exceed 500 / μL. In some embodiments, administration of IL-2 conjugates to subjects does not cause the number of eosinophils in the subjects' peripheral blood to exceed 500 / μL to 1,500 / μL. In some embodiments, administration of IL-2 conjugates to subjects does not cause the number of eosinophils in the subjects' peripheral blood to exceed 1,500 / μL to 5,000 / μL. In some embodiments, administration of IL-2 conjugates to subjects does not cause the number of eosinophils in the subjects' peripheral blood to exceed 5,000 / μL. In some embodiments, administration of IL-2 conjugates to subjects is not contraindicated to subjects with existing psychotropic drug regimens.

[0253] In some embodiments, administration of IL-2 conjugates to subjects is not contraindicated in subjects with existing regimens of nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic agents. In some embodiments, administration of IL-2 conjugates to subjects is not contraindicated in subjects with existing regimens of aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase. In some embodiments, administration of IL-2 conjugates to subjects is not contraindicated in subjects receiving combination regimens containing antineoplastic agents. In some embodiments, the antineoplastic agent is selected from dacarbazine, cis-platinum, tamoxifen, and interferon-alpha.

[0254] In some embodiments, administration of an IL-2 conjugate to a subject does not cause one or more grade 4 adverse events in the subject after administration. In some embodiments, Grade 4 adverse events are selected from hypothermia, shock, bradycardia, ventricular premature contractions, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, AV block grade 2, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disorder, 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, restlessness, neuropathy, paranoid reaction, convulsions, grand mal seizures, 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 IL-2 conjugates to the target group does not cause one or more grade 4 adverse events in more than 1% of the subjects after administration. In some embodiments, Grade 4 adverse events are selected from hypothermia, shock, bradycardia, ventricular premature contractions, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, AV block grade 2, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disorder, 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, restlessness, neuropathy, paranoid reaction, convulsions, grand mal seizures, delirium, asthma, pulmonary edema, hyperventilation, hypoxia, hemoptysis, hypoventilation, pneumothorax, mydriasis, pupillary disorders, renal dysfunction, renal failure, and acute tubular necrosis.

[0255] In some embodiments, administration of an IL-2 conjugate to a group of subjects does not cause one or more adverse events in more than 1% of the subjects after administration, and one or more adverse events are selected from duodenal ulcer formation, intestinal necrosis, myocarditis, supraventricular tachycardia, permanent or transient blindness secondary to optic neuritis, transient ischemic attack, meningitis, cerebral edema, pericarditis, allergic interstitial nephritis, and tracheoesophageal fistula.

[0256] In some embodiments, administration of an IL-2 conjugate to a group of subjects does not cause one or more adverse events in more than 1% of subjects after administration, and one or more adverse events are selected from malignant hyperthermia, cardiac arrest, myocardial infarction, pulmonary embolism, stroke, bowel perforation, hepatic or renal failure, severe depression leading to suicide, pulmonary edema, respiratory arrest, and respiratory failure.

[0257] In some embodiments, administration of an IL-2 conjugate to a subject stimulates the subject's CD8+ cells. In some embodiments, administration of an IL-2 conjugate to a subject stimulates the subject's NK cells. Stimulation may include, for example, an increase in the number of CD8+ cells in the subject about 4, 5, 6, or 7 days after administration or about 1, 2, 3, or 4 weeks after administration. In some embodiments, the CD8+ cells include memory CD8+ cells. In some embodiments, the CD8+ cells include effector CD8+ cells. Stimulation may include, for example, an increase in the percentage of Ki67-positive CD8+ cells in the subject about 4, 5, 6, or 7 days after administration or about 1, 2, 3, or 4 weeks after administration. Stimulation may include, for example, an increase in the number of NK cells in the subject about 4, 5, 6, or 7 days after administration or about 1, 2, 3, or 4 weeks after administration.

[0258] In some embodiments, CD8+ cells proliferate at least 1.5 times in the control after administration of the IL-2 conjugate, for example, at least 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, or 2.5 times. In some embodiments, NK cells proliferate at least 3.5 times in the control after administration of the IL-2 conjugate, for example, at least 4.0 times, 4.5 times, 5.5 times, 6 times, 6.5 times, 7.0 times, or 7.5 times. In some embodiments, eosinophils proliferate at about 2 times or less in the control after administration of the IL-2 conjugate, for example, at about 1.5 times, 1.4 times, or 1.3 times. In some embodiments, CD4+ cells proliferate at about 2 times or less in the control after administration of the IL-2 conjugate, for example, at about 1.8 times, 1.7 times, or 1.6 times. In some embodiments, the proliferation of CD8+ cells and / or NK cells in subjects after administration of IL-2 conjugate is greater than the proliferation of CD4+ cells and / or eosinophils. In some embodiments, the proliferation of CD8+ cells is greater than the proliferation of CD4+ cells. In some embodiments, the proliferation of NK cells is greater than the proliferation of CD4+ cells. In some embodiments, the proliferation of CD8+ cells is greater than the proliferation of eosinophils. In some embodiments, the proliferation of NK cells is greater than the proliferation of eosinophils. Polyploidy is determined relative to baseline values ​​measured before administration of IL-2 conjugate. In some embodiments, polyploidy is determined at any point after administration, for example, about 4, 5, 6 or 7 days after administration or about 1, 2, 3 or 4 weeks after administration.

[0259] In some embodiments, administration of an IL-2 conjugate to a subject increases the number of peripheral CD8+ T cells and NK cells in the subject without increasing the number of peripheral CD4+ regulatory T cells in the subject. In some embodiments, administration of an IL-2 conjugate to a subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject. In some embodiments, administration of an IL-2 conjugate to a subject increases the number of peripheral CD8+ T cells and NK cells in the subject without increasing the number of intratumoral CD8+ T cells and NK cells in the subject, and without increasing the number of intratumoral CD4+ regulatory T cells in the subject.

[0260] In some embodiments, the administration of IL-2 conjugates to a subject does not require the availability of an intensive care facility or a specialist skilled in cardiopulmonary or critical care medicine. In some embodiments, the administration of IL-2 conjugates to a subject does not require the availability of an intensive care facility or a specialist skilled in cardiopulmonary or critical care medicine. In some embodiments, the administration of IL-2 conjugates to a subject does not require the availability of an intensive care facility. In some embodiments, the administration of IL-2 conjugates to a subject does not require the availability of a specialist skilled in cardiopulmonary or critical care agents.

[0261] In some embodiments, administration of IL-2 conjugate does not cause dose-limiting toxicity (DLT). In some embodiments, administration of IL-2 conjugate causes dose-limiting toxicity (DLT). In some embodiments, DLT is defined as an adverse event occurring within ±1 day of day 1 to day 42 (6-week cycle) (inclusive) of a treatment cycle, not related only to obviously or obviously unrelated causes, and meeting the criteria for DLT described in Example 4.

[0262] In some embodiments, administration of IL-2 conjugates does not cause severe cytokine release syndrome. In some embodiments, IL-2 conjugates do not induce anti-drug antibodies (ADAs), i.e., antibodies against IL-2 conjugates. In some embodiments, the absence of ADA introduction is determined by direct immunoassay of antibodies against PEG and / or ELISA of antibodies against IL-2 conjugates. IL-2 conjugates are considered not to induce ADAs if the measured ADA levels are not statistically distinguishable from baseline (pre-treatment) levels or untreated control levels.

[0263] Kit / Manufactured product This specification discloses kits and articles for use with one or more methods and compositions described in particular embodiments. Such a kit includes a carrier, package, or container partitioned to accept one or more containers, such as vials and tubes, each of which contains one of the distinct elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from a variety of materials, such as glass or plastic.

[0264] The kit typically includes a label listing the contents and / or instructions for use, as well as accompanying documentation containing instructions for use. A set of instructions is also usually included.

[0265] In one embodiment, the label is on or attached to the container. In one embodiment, letters, numbers, or other characters forming the label are attached, molded, or etched onto the container itself. The label is present on the container and, if present in a receiver or carrier that also holds the container, is associated with the container, for example, as accompanying documentation. In one embodiment, the label is used to indicate that the contents must be used for a specific therapeutic purpose. The label also indicates instructions for the use of the contents, such as those described herein.

[0266] In certain embodiments, the pharmaceutical composition is supplied in a pack or dispenser device containing one or more unit dosage forms containing the compounds provided herein. For example, the pack contains metal foil or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser device is also accompanied by a notice relating to the container in the form prescribed by the government agency that regulates the manufacture, use or sale of pharmaceuticals. This notice reflects the agency's approval of the drug form for administration to humans or livestock. Such notice is, for example, a label approved by the U.S. Food and Drug Administration or a package insert for an approved product. In one embodiment, the composition containing the compounds provided herein, formulated in a suitable pharmaceutical carrier, is also prepared, placed in a suitable container, and labeled for the treatment of the indicated condition. [Examples]

[0267] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0268] Example 1. Preparation of PEGylated IL-2 Conjugate This embodiment provides an exemplary method having details for preparing the IL-2 conjugate described herein.

[0269] IL-2 used for bioconjugation was expressed as an inclusion body in Escherichia coli (E. coli) using the method disclosed herein: (a) an expression plasmid encoding tRNA from M. mazei Pyl, comprising (i) a protein having a desired amino acid sequence, including a first non-natural base pair to provide a codon at a desired position in which the non-natural amino acid N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) is incorporated, and (ii) a second non-natural nucleotide to provide an anticodon that fits in place of the natural sequence; and (b) pyrrolidine-tRNA synthetase (Mb) from M. barkeri. Plasmids encoding PylRS, (c) N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and (d) a cleaved variant of the nucleotide triphosphate transporter PtNTT2 lacking the first 65 amino acid residues of the full-length protein were expressed as inclusion bodies in Escherichia coli (E. coli). Double-stranded oligonucleotides encoding the amino acid sequence of the desired IL-2 variant contained codon AXC as codon 64 of the sequence encoding the protein of SEQ ID NO: 1, with P64 substituted with a non-natural amino acid described herein. Plasmids encoding orthogonal tRNA genes from M. mazei contained the AXC-matched anticodon GYT instead of its natural sequence, where Y is a non-natural nucleotide disclosed herein. X and Y were selected from the non-natural nucleotides dTPT3 and dNaM disclosed herein. The expressed protein was extracted from the inclusion body and refolded using a standard procedure. The AzK-containing IL-2 product was then site-specifically pegylated using DBCO-mediated copper-free click chemistry to attach a stable covalent mPEG moiety to AzK. Exemplary reactions are shown in Schemes 1 and 2 (where n is the number of repeating PEG units). The reaction between the AzK moiety and the DBCO alkynyl moiety yields either a single positional isomer product or a mixture of positional isomer products. [ka] [ka]

[0270] Example 2. Preclinical safety, PK, and PDy studies of IL-2 conjugates. To evaluate the safety, PK, and PDy of the IL-2 conjugate, studies were conducted in cynomolgus monkeys. The animals received a total of three intravenous doses of 0.1 mg / kg of the IL-2 conjugate at QW, Q2W, Q3W, or Q4W. The IL-2 conjugate contains Sequence ID No. 2, where position 64 is AzK_L1_PEG30kD, defined as the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30 kDa linear mPEG chain. This IL-2 conjugate can also be described as the IL-2 conjugate containing Sequence ID No. 1, where position 64 is substituted with the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30 kDa linear mPEG chain. This IL-2 conjugate may also be described as the IL-2 conjugate containing Sequence ID No. 1, in which position 64 is substituted with the structure of formula (XII) or formula (XIII), or a mixture of formulas (XII) and (XIII), and a 30 kDa linear mPEG chain. This IL-2 conjugate has the proposed international common name (pINN) of Pegenjirokin.

[0271] Peripheral CD8+ T cell proliferation peaked on day 6. Peripheral CD8+ T cell proliferation was comparable across all dose schedules at day 6. CD8+ T cells remained elevated in the QW administration schedule. In the lower-frequency administration schedules, CD8+ T cells gradually returned to pre-treatment levels by day 11 and remained at this level until subsequent administrations (see Figure 1). There were no significant changes in clinical signs, body weight, blood pressure, or hematological parameters. Troponin I levels in the IL-2 conjugate treatment group were comparable to those in the vehicle control group. These results support the conclusion that the QW regimen increases the potential for more sustained CD8+ T cell growth compared to the Q2W and Q3W regimens.

[0272] Example 3. Clinical trial of biomarker effects after administration of IL-2 conjugate (8, 16, 24 and 32 μg / kg [Q2W], 8, 16, 24, 32 and 40 μg / kg [Q3W]). Studies were conducted to characterize the immunological effects of in vivo administration of IL-2 conjugates. The IL-2 conjugate contains Sequence ID No. 2, where position 64 is AzK_L1_PEG30kD, defined as the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30kDa linear mPEG chain. This IL-2 conjugate can also be described as the IL-2 conjugate containing Sequence ID No. 1, where position 64 is substituted with the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30kDa linear mPEG chain. This IL-2 conjugate can also be described as the IL-2 conjugate containing Sequence ID No. 1, where position 64 is substituted with the structure of formula (XII) or formula (XIII) or a mixture of formulas (XII) and (XIII) and a 30kDa linear mPEG chain. This IL-2 conjugate has the proposed international generic name (pINN) of peggenjirokin. The IL-2 conjugate was administered by IV infusion at doses of 8, 16, 24, or 32 μg / kg (e.g., over approximately 30 minutes) [Q2W] every two weeks, or by IV infusion at doses of 8, 16, 24, 32, or 40 μg / kg (e.g., over 30 minutes) [Q3W] every three weeks.

[0273] The following biomarkers were analyzed as surrogate predictors of safety and / or efficacy: Eosinophilia (increased peripheral eosinophil count): A cell surrogate marker for IL-2-induced proliferation of cells (eosinophils) associated with vascular leak syndrome (VLS). Interleukin 5 (IL-5): A cytokine surrogate marker for the release of this chemoattractant that leads to IL-2-induced activation and eosinophilia and potentially VLS in type 2 innate lymphoid cells. 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 induction activation of CD8+ cytotoxic T lymphocytes.

[0274] The following biomarkers were analyzed as surrogate predictors of antitumor immune activity: Peripheral CD8+ effector cells: Markers of IL-2-induced proliferation of these target cells in the periphery, serving as surrogate markers for inducing potential therapeutic responses during invasion. Peripheral CD8+ memory cells: When infiltrated, they serve as surrogate markers for inducing the treatment and maintenance of potentially persistent latent memory populations, and are markers for IL-2-induced proliferation of these target cells in the periphery. Peripheral NK cells: Markers of IL-2-induced proliferation of these target cells in the periphery, which can serve as surrogate markers to potentially induce a rapid therapeutic response during infiltration, and Peripheral CD4+ regulatory cells: These peripheral target cells induce immunosuppressive TMEs upon infiltration and serve as surrogate markers for counteracting effector-based therapeutic effects, acting as markers for IL-2-induced proliferation.

[0275] The subjects were human males or females aged 18 years or older at the time of screening. All subjects had previously been treated with anticancer therapy and met at least one of the following criteria: treatment-related toxicity (excluding alopecia) improved to grade 0 or 1 according to NCI CTCAE v5.0, or treatment-related toxicity improved to at least grade 2 according to NCI CTCAE v5.0 with prior approval from Medical Monitor. The most common tumors were colorectal or melanoma.

[0276] The subjects also met the following criteria: provided informed consent; had an ECOG (East Coast Cancer Group) performance status of 0 or 1; had a mean life expectancy of 12 weeks or more as determined by the principal investigator; had a histologically or cytologically confirmed diagnosis of advanced and / or metastatic solid tumor; had an advanced or metastatic solid tumor that had refused standard treatment, or for which there was no reasonable standard of care that would provide a clinical benefit, or for which standard treatment was unacceptable, ineffective, or unavailable; had a disease measured according to RECIST v1.1; and met appropriate laboratory parameters, e.g., absolute lymphocyte count greater than 0.5 times the lower limit of normal, platelet count ≥ 100 × 10⁶ 9 / L, hemoglobin ≥ 9.0 g / dL (no growth factor or transfection within the past two weeks, a one-week washout for ESA and CSF administration is sufficient), absolute neutrophil count ≥ 1.5 × 10 9 / L (no growth factors within 2 weeks), prothrombin time (PT) and partial thromboplastin time (PTT) ≤ 1.5 times the upper limit of normal (ULN), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 times ULN and possibly ≤ 5 times ULN, except in the presence of liver metastases. Total bilirubin ≤ 1.5 × ULN. Premenopausal women and women less than 12 months postmenopausal had a negative serum pregnancy test within 7 days prior to the start of the study treatment.

[0277] The following data pertain to treatment-related adverse events (TEAEs) and efficacy biomarkers for the 8, 16, and 24 μg / kg [Q2W] cohorts.

[0278] The first cohort ([Q2W]) used an 8 μg / kg dose. Four individuals (4 males [100%], 0 females [0%], Caucasian, median age: 64 years, range: 49–70 years) with progressive or metastatic solid tumors were administered IL-2 conjugate at a dose of 8 μg / kg Q2W (1 dose per cycle). Tumor types included colorectal, pancreatic, and sarcoma. Here, throughout the cohort of Example 3, the drug mass per kg of subject (e.g., 8 μg / kg) refers to the mass of IL-2 excluding the mass of PEG and linker. The treatment duration ranged from 1.4 to 9.0 months (median 2.0 months), and subjects received a total dose of 4 to 20 (median 5.0 doses).

[0279] Three subjects (75%) experienced at least one TEAE, all of which were grade 1 or 2. There were no drug interruptions due to TEAEs, and no dose-limiting toxicities. One subject died as a result of disease progression (grade 5 AE). No cumulative toxicity, end-organ toxicity, QTc prolongation, or other cardiotoxicities were observed. Furthermore, no significant elevation of IL-5 was observed. TEAEs are detailed in Table 1.

[0280] [Table 1]

[0281] Efficacy biomarker: Peripheral CD8+T eff The number of cells was measured (Figure 2), and the number of peripheral NK cells is shown in Figure 3. Peripheral CD4+T reg The number of cells is shown in Figure 4. The number of peripheral lymphocytes is shown in Figure 5, and the number of peripheral eosinophils is shown in Figure 6.

[0282] The average concentrations of IL-2 conjugate after one and two cycles are shown in Figures 7A and 7B, respectively.

[0283] Cytokine levels (IFN-γ, IL-6, and IL-5) are shown in Figure 8.

[0284] Overall, the IL-2 conjugate appeared to be well-tolerated. Overall, the results support the non-alpha preferred activity of the IL-2 conjugate, accompanied by an acceptable safety profile and preliminary evidence of activity in patients with immunosensitive tumors.

[0285] The second cohort ([Q2W]) used a dose of 16 μg / kg. Four individuals with advanced or metastatic solid tumors were administered IL-2 conjugate at a dose of 16 μg / kg Q2W (1 dose per cycle). Tumor types included melanoma, prostate cancer, and colon cancer.

[0286] All four subjects (100%) experienced at least one TEAE, and three of the four subjects (75%) experienced at least one grade 3-4 related TEAE (one grade 3 and two grade 4). One subject experienced grade 3 lymphopenia, and two subjects experienced grade 4 lymphopenia (one with grade 3 hypophosphatemia), with the lymphopenia lasting for two days. There were no SAEs associated with these subjects (one unrelated bowel obstruction SAE). There were no drug discontinuations due to TEAEs. No DLTs were observed. One patient could not be evaluated for DLTs because disease progression prevented administration of C2D1. One subject showed elevated IL-6 (1000 pg / mL) without symptoms suggestive of CRS. TEAEs are detailed in Table 2.

[0287] [Table 2]

[0288] Efficacy biomarker: Peripheral CD8+T eff Cell counts were measured (Figure 9). CD8+ enlargement was approximately 2x, similar to the enlargement observed in the initial [Q2W] cohort (8 μg / kg dose). Peripheral NK cell counts are shown in Figure 10. NK cell proliferation was approximately 1 to 20 times higher than in the initial [Q2W] cohort (8 μg / kg dose). Peripheral CD4+T regThe number of cells is shown in Figure 11. The number of peripheral eosinophil cells is shown in Figure 12. CD4+T reg Eosinophil proliferation was similar to that of the first [Q2W] cohort (8 μg / kg dose).

[0289] Cytokine levels (IFN-γ, IL-6, and IL-5) are shown in Figure 13.

[0290] The average concentrations of IL-2 conjugate after one and two cycles are shown in Figures 14A and 14B, respectively.

[0291] Therefore, IL-2 conjugates showed promising PD data and were generally well-tolerated. Overall, the results support the non-alpha preferred activity of IL-2 conjugates, suggest a tolerable safety profile, and encourage preliminary evidence of activity in patients with PD and immunosensitive tumors.

[0292] Third cohort ([Q2W]) using a 24 μg / kg dose. Three individuals with progressive or metastatic solid tumors were administered IL-2 conjugate at a dose of 16 μg / kg Q2W (once per cycle). Tumor types included melanoma and lung cancer.

[0293] All three subjects (100%) experienced at least one TEAE, and two of the three subjects (33.3%) experienced at least one grade 3-4 related TEAE (2 grades 4). There were two cases of grade 4 lymphopenia (one subject had grade 1 transaminasis and the other had grade 1 decreased TSH). No DLTs were observed. No related SAEs were observed. One subject required dose retention to receive treatment for a particular adverse event (COVID-19 infection) and subsequently discontinued IL-2 conjugate therapy as a result of disease progression (PD). There were no drug discontinuations due to TEAEs. One subject had C2D1 administration withheld due to GI bleeding (gastric ulcer) unrelated to IL-2 conjugate therapy. TEAEs are detailed in Table 3.

[0294] [Table 3]

[0295] Therefore, IL-2 conjugates showed promising PD data and were generally well-tolerated. Overall, the results support the non-alpha preferred activity of IL-2 conjugates, suggest a tolerable safety profile, and encourage preliminary evidence of activity in patients with PD and immunosensitive tumors.

[0296] Example 4. Clinical trial of solid tumor treatment using IL-2 conjugates We will conduct an open-label, multicenter, phase 1 / 2 dose-escalation study to evaluate the clinical benefit of IL-2 conjugate as a monotherapy in adult patients with advanced or metastatic solid tumors.

[0297] The IL-2 conjugate includes Sequence ID No. 2, where position 64 is AzK_L1_PEG30kD, defined as the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30kDa linear mPEG chain. This IL-2 conjugate can also be described as the IL-2 conjugate including Sequence ID No. 1, where position 64 is substituted with the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30kDa linear mPEG chain. This IL-2 conjugate can also be described as the IL-2 conjugate including Sequence ID No. 1, where position 64 is substituted with the structure of formula (XII) or formula (XIII) or a mixture of formulas (XII) and (XIII) and a 30kDa linear mPEG chain. This IL-2 conjugate has the proposed international common name (pINN) of Pegenjirokin.

[0298] Participants received IL-2 conjugate monotherapy once weekly (QW) for 6 weeks (induction period), followed by every 2 weeks (Q2W) (maintenance period), also known as QW / Q2W. The administration regimen consisted of IV infusions at QW for 30 minutes on day 1 of each week for 6 weeks (i.e., on D1, D8, D15, D22, D29, and D36) for dose escalation (induction), and Q2W at the start of each cycle (from cycle 2 onwards, each cycle = 6 weeks) for dose expansion (maintenance). See Figure 15.

[0299] During Cycle 1 (six weekly doses), each subject was continuously monitored in the clinic for at least 24 hours after administration of the study drug. The first subject enrolled at each cohort's dose level was observed for at least 72 hours before the second subject was enrolled at the same dose level.

[0300] Dose-limiting toxicity was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE version 5.0). Neurological events associated with cytokine release syndrome were assessed using CTCAE version 5.0, supplemented with the American Society for Transplantation and Cell Therapy (ASTCT) criteria.

[0301] The DLT evaluation period is from day 1 to day 42 (6-week cycle) (including both ends) ± 1 day. The dose administered on day 1 of cycle 2 was excluded from the DLT observation period. If the patient receives the drug early, the DLT observation period will end before the C2D1 dose.

[0302] DLT is defined as any of the following events occurring during the first treatment cycle in which a causal relationship with the IL-2 conjugate is determined to be at least likely associated:

[0303] blood toxicity • Grade 3 neutropenia lasting for more than 7 days (absolute neutrophil count [ANC] < 1000 / mm³) 3 >500 / mm 3 ) or Grade 4 neutropenia for any period of time • Grade 3+ Febrile Neutropenia • Grade 4+ thrombocytopenia (platelet count <25,000 / mm³) 3 ) Grade 3+ thrombocytopenia (platelet count <50,000 to 25,000 / mm³) that lasts for more than 5 days, involves clinically significant bleeding, or requires platelet transfusion. 3 ) • Within 10 days ANC1000 cells / mm 3 Over, platelet count 75000 cells / mm 3 Failure to meet the above recovery criteria • Any other Grade 4+ hematological toxicity lasting more than 5 days

[0304] non-hematological toxicity Grade 3+ ALT or AST in combination with bilirubin levels exceeding twice the upper limit of normal (ULN) without evidence of other causes such as cholestasis, viral infection, or other medications (i.e., Hy's Law). • Grade 3 infusion-related reactions occurring with premedication: Grade 4 infusion-related reactions • Grade 3 vascular leak syndrome (VLS) is defined as hypotension associated with fluid retention and pulmonary edema. • Grade 3+ anaphylaxis • Grade 3+ Hypotension Grade 3+ Cytokine Release Syndrome • Grade 3+ Acceptable Care: AEs that do not improve to Grade 2 or below within 7 days of initiating medical management. The following Grade 3 non-hematological AEs are exceptions: • Grade 3: Fatigue, nausea, vomiting, or diarrhea that improves to Grade 2 or lower within 3 days with optimal medical management. • Grade 3 fever (defined as a temperature below 40°C for 24 hours or less) • Grade 3 infusion-related reactions occurring without premedication should be treated with premedication for subsequent administrations, and if the reaction recurs, it becomes a DLT (Delayed Liver Therapy). • Grade 3 joint pain or rash improves to Grade 2 or lower within 7 days of initiating acceptable standard treatment (e.g., systemic corticosteroid therapy). In patients with grade 1 or 2 elevated ALT or AST levels at baseline, which are thought to be secondary to liver metastases, grade 3 elevations must also be at least three times the baseline level and persist for more than 7 days.

[0305] Participants received IL-2 conjugate monotherapy QW / Q2W. The starting dose of IL-2 conjugate was 16 μg / kg (DL1). Provided that no DLT occurred and escalation was permitted considering the overall safety assessment, the dose was increased to 24 μg / kg (DL2), and then to 32 μg / kg (DL3). If DL1 was determined to be unsafe, a dose of 8 μg / kg (dose level 1) could be used. Herein, throughout the examples and specification described herein, the drug mass per kg of subject (e.g., 16 μg / kg) refers to the IL-2 mass excluding the PEG and linker mass. In other words, any indication of the drug mass per kg of subject (e.g., "16 μg / kg" or "16 μg / kg of IL-2 as an IL-2 conjugate" or "16 μg / kg as an IL-2 conjugate") throughout the specification, including the examples described herein, refers to the dose based on the IL-2 protein portion of the IL-2 conjugate described herein.

[0306] The following inclusion criteria apply. Participants must be able to voluntarily provide informed consent and comply with protocol requirements throughout the study period. Participants must be 18 years of age or older. Prior anticancer therapy (e.g., radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy, is possible. If previously treated with any anticancer therapy, at least one of the following must be met: (1) treatment-related toxicity improved to Grade 0 or 1 (excluding alopecia) according to NCI CTCAE v5.0, and (2) treatment-related toxicity improved to at least Grade 2 according to NCI CTCAE v5.0 with prior approval from the medical monitor. Participants must have a Performance Status of 0 or 1 with the East Coast Cancer Group (ECOG). Participants must have a mean life expectancy of at least 12 weeks as determined by the principal investigator. Participants must have a histologically or cytologically confirmed diagnosis of advanced and / or metastatic solid tumor with at least one tumor lesion in a location accessible for safe biopsy as determined by the principal investigator's clinical judgment. Patients with advanced or metastatic solid tumors who have refused standard treatment, or for whom reasonable standards of care providing clinical benefit are unavailable, or for whom standard treatment is unacceptable, ineffective, or unavailable. Measurable disease according to RECIST v1.1. Participants must have at least one measurable lesion. Biopsies during treatment may be omitted upon written request from the principal investigator and with written approval from the sponsor, taking into consideration the clinical feasibility of the biopsy.

[0307] Participants must have appropriate laboratory parameters, including: (1) an absolute lymphocyte count of 0.5 times or more below the lower limit of normal, and (2) a platelet count of 100 × 10⁶ 9 (3) Hemoglobin of 9.0 g / dL or higher (no growth factors or transfection within the past two weeks, and a one-week washout for erythropoiesis-stimulating agents (ESAs) and colony-stimulating factors (CSFs) is sufficient), (4) Absolute neutrophil count of 1.5 × 10⁻¹⁴ 9Appropriate experimental parameters are met, including (5) prothrombin time (PT) and partial thromboplastin time (PTT) being ≤1.5 times ULN, (6) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) being ≤2.5 times ULN, except in the presence of liver metastases, and (7) total bilirubin being ≤1.5 × ULN. Women of childbearing potential and men who are not surgically infertile must agree to use medically acceptable methods of contraception during the study and for at least 7 days in the case of women, and for at least 3 days in the case of men after the last dose of the study intervention. Female participants must have a negative serum pregnancy test within 7 days prior to the start of the study treatment, both for premenopausal women less than 12 months postmenopausal and for women. Male participants must agree to refrain from sperm donation or storage during the treatment period and for at least 3 days after the last dose of the study treatment.

[0308] Participants will be excluded from the exam if any of the following criteria apply: - Radiotherapy within 14 days prior to the first dose of the investigational drug (palliative radiation or stereotactic radiosurgery within 7 days prior to the start of the investigational treatment). - The patient had received systemic anticancer therapy or the investigational drug within two weeks prior to the start of investigational drug treatment (within four weeks for immunotherapy and tyrosine kinase inhibitor therapy). - Participants who have experienced grade 3 or higher immuno-related toxicity from previous immuno-oncology therapy. - Whether the patient underwent major surgery within 30 days prior to the first dose of the study drug, or has not recovered to at least Grade 1 from adverse effects of such procedure, or whether there is a predicted need for major surgery during the study treatment. - Active autoimmune disease with a recorded history of clinically severe autoimmune disease requiring systemic treatment or systemic steroids or immunosuppressants within the past three months; subjects receiving corticosteroids are excluded from doses of prednisone or equivalent greater than 10 mg per day. Inhaled steroids, intranasal steroids, intraocular steroids, and topical steroids are acceptable. Steroids as prophylaxis (for disease evaluation) are also acceptable. - Primary central nervous system (CNS) disease or pia mater disease; known CNS metastases were asymptomatic unless untreated, showed no evidence of radiological progression for at least 8 weeks, and did not require steroids or enzyme-induced anticonvulsants in the last 14 days prior to screening. - Abnormal lung function within the past six months, including pneumonia, active pneumonia, interstitial lung disease requiring steroid use, idiopathic pulmonary fibrosis, confirmed pleural effusion, severe dyspnea at rest, or interstitial lung disease requiring supplemental oxygen therapy. - A history of allogeneic organ transplantation or solid organ transplantation. - Poorly controlled diabetes or other poorly controlled immune-related endocrine disorders, as determined by the principal investigator. - If parenteral antibiotics have been used within 14 days prior to the first dose of the study drug, or if there is poor control of a severe systemic fungal, bacterial, viral or other infection, or if intravenous antibiotics are required. - Active infection due to known human immunodeficiency virus (HIV) infection or hepatitis C: (a) Known uncontrolled hepatitis B virus (HBV) infection: (i) Anti-HBV therapy initiated before the start of IMP and an HBV viral load of less than 2000 IU / mL (10⁴ copies / mL) are eligible. Anti-HBV therapy should be continued throughout the treatment period. (ii) Anti-HBc positive, anti-HBs positive, HbsAg negative and HBV viral load without HBV treatment are eligible. - If a live virus vaccine was received within 14 days prior to the first dose of the investigational drug. Seasonal influenza and other inactivated vaccines that do not contain live viruses are permitted. - Clinically significant bleeding (e.g., gastrointestinal bleeding, intracranial hemorrhage) within two weeks prior to the first dose of IL-2 conjugate. - Prior diagnosis of deep vein thrombosis or pulmonary embolism within the past three months. - Severe or unstable cardiac conditions within 6 months prior to the start of the study treatment, such as congestive heart failure (New York Heart Association class III or IV), cardiac bypass surgery or coronary stenting, angioplasty, ejection fraction below the lower limit of normal, unstable angina, medically uncontrolled hypertension (e.g., systolic ≥ 160 mmHg or diastolic ≥ 100 mmHg), uncontrolled arrhythmia requiring drug therapy (grade 2 or higher, according to NCI CTCAE v5.0), or myocardial infarction. - A history of non-pharmacologically induced, extended, and corrected QT intervals determined using Fridericia's formula (QTcF) with intervals greater than 450 milliseconds (msec) in males and greater than 470 msec in females. - Known hypersensitivity or contraindications to any component of IL-2 conjugate, PEG, PEGylated drugs, and Escherichia coli (E. coli)-derived proteins. - A history of active secondary malignancy or prior malignancy that would affect the evaluation of any study endpoint. Subjects with curatively surgically resected non-melanoma skin cancer or cervical cancer are eligible. - Any serious medical condition (including pre-existing autoimmune or inflammatory disorders), laboratory abnormality, psychiatric condition, or any other serious or unstable concurrent medical condition that the principal investigator believes would interfere with protocol therapy or make the subject unsuitable for the study: (a) patients with clinically significant active SARS-CoV-2 (COVID-19) infection. - Beginning with a screening visit of at least 7 days for women, or at least 3 days after the last dose of the trial intervention for men, participants must be pregnant or breastfeeding, or planning to become pregnant or give birth, during the expected duration of the trial. - Concurrent treatment with any other investigational drug, vaccine, or device. Concurrent participation in observational studies is permitted only after approval by the sponsor. - Subjects with a baseline oxygen saturation of less than 92% are not eligible for registration.

[0309] Safety Assessment: Throughout the entire test, safety was assessed as follows: • AES (Type, Incidence, Severity, Timing, Criticality, and Associated) • Measurement of vital signs (blood pressure, heart rate, respiratory rate, body temperature, pulse oximetry) • Physical examination • Clinical laboratory ·electro-cardiogram • All adjunctive medications and procedures, including all supportive therapies provided.

[0310] To monitor delayed immunotoxicity associated with immunotherapy products, adverse events were evaluated up to approximately 90 days after the last dose of the study drug or until subsequent anticancer treatment was administered (whichever came first).

[0311] Efficacy Evaluation: Antitumor activity was determined by repeated radiographic imaging and response assessments using RECIST 1.1 and iRECIST. All subjects were required to undergo scans using a modality appropriate for the target tumor (e.g., PET-CT scan, CT scan, MRI). All anatomical sites imaged during screening were required to be evaluated at each time point during the study using the same imaging modality.

[0312] Test endpoints Key evaluation criteria DLTs were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. • The MTD of IL-2 conjugate as monotherapy was the highest study dose level with a probability of causing DLT less than 33% (i.e., a dose level where 0 / 6 or 1 / 6 subjects experienced DLT in the first cycle, and at least 2 / 3 or 2 / 6 subjects experienced DLT in the next higher dose level). Based on available safety, tolerability, PK, and Pdy (including, but not limited to, Ki67 expression in lymphocytes) data from different dose levels and schedules tested, RP2D as a monotherapy was below the MTD.

[0313] Secondary endpoints: Clinical antitumor activity was evaluated according to RECIST v1.1, as determined by the principal investigator. • ORR (defined as the percentage of subjects with confirmed complete response (CR) or partial response (PR). A confirmed response is defined as a sustained response observed in a follow-up scan at least four weeks after the initial confirmation of the response.) • DOR (Defined as the period from the date of the first objective response (either complete response (CR) or partial response (PR)) until the first radiographic progression of the disease or death due to any cause is recorded.) • PFS (defined as the earlier of the time from the first dose of IL-2 conjugate to the first confirmed progression of a radiation-induced illness or death due to any cause). • OS (defined as the time from the first dose of IL-2 conjugate to death from any cause.) • TTR is defined as the time from the initial dose of IL-2 conjugate to the first recorded objective response (either complete response (CR) or partial response (PR)). • DCR (Defined as the percentage of subjects who achieved CR, PR, or disease stabilization (a period of disease stabilization of 3 months or more)). • The percentage of subjects who did not experience disease progression 6 months after treatment.

[0314] Example 5. Clinical trial of melanoma treatment using IL-2 conjugate We will conduct an open-label, multicenter, phase 1 / 2 dose-expansion trial to evaluate the clinical benefit of IL-2 conjugate as a monotherapy in adult patients with late-stage metastatic melanoma.

[0315] The IL-2 conjugate includes Sequence ID No. 2, where position 64 is AzK_L1_PEG30kD, defined as the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30kDa linear mPEG chain. This IL-2 conjugate can also be described as the IL-2 conjugate including Sequence ID No. 1, where position 64 is substituted with the structure of formula (IV) or formula (V) or a mixture of formulas (IV) and (V) and a 30kDa linear mPEG chain. This IL-2 conjugate can also be described as the IL-2 conjugate including Sequence ID No. 1, where position 64 is substituted with the structure of formula (XII) or formula (XIII) or a mixture of formulas (XII) and (XIII) and a 30kDa linear mPEG chain. This IL-2 conjugate has the proposed international common name (pINN) of Pegenjirokin.

[0316] Based on the determination of the recommended dose from Example 4 (e.g., 8 μg / kg, 16 μg / kg, 24 μg / kg, or 32 μg / kg), Example 5 will be initiated during the dose escalation phase. The goal is to evaluate the antitumor activity, safety, and PK / Pdy of IL-2 conjugate monotherapy once a week (QW) for 6 weeks (induction period), and then every two weeks (Q2W) thereafter (maintenance period). See Figure 16. Example 5 will include approximately 40 participants with late metastatic melanoma. DLT will not be applied.

[0317] The following inclusion criteria apply. Participants must be able to voluntarily provide informed consent and comply with protocol requirements throughout the study period. Participants must be 18 years of age or older. Prior anticancer therapy (e.g., radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy, is possible. If previously treated with any anticancer therapy, at least one of the following must be met: (1) treatment-related toxicity improved to Grade 0 or 1 (excluding alopecia) according to NCI CTCAE v5.0, and (2) treatment-related toxicity improved to at least Grade 2 according to NCI CTCAE v5.0 with prior approval from the medical monitor. Participants must have a Performance Status of 0 or 1 with the East Coast Cancer Group (ECOG). Participants must have a mean life expectancy of at least 12 weeks as determined by the principal investigator. Participants must have a histologically or cytologically confirmed diagnosis of advanced and / or metastatic solid tumor with at least one tumor lesion in a location accessible for safe biopsy as determined by the principal investigator's clinical judgment. Participants must not have received at least one prior therapy line for metastatic melanoma and / or have no standard of care (SoC) options, or participants must refuse or be intolerant of SoC treatment. Participants must have advanced or metastatic solid tumors that have refused standard treatment, or for which reasonable standards of care providing clinical benefit are unavailable, or for which standard treatment is unacceptable, ineffective, or unavailable. Measurable disease according to RECIST v1.1. Participants must have at least two measurable lesions to safely perform mandatory pre-treatment and intra-treatment biopsies. Intra-treatment biopsies may be omitted upon written request from the principal investigator and written approval from the sponsor, taking into consideration the clinical feasibility of the biopsy.

[0318] Participants must have appropriate laboratory parameters, including: (1) an absolute lymphocyte count of 0.5 times or more below the lower limit of normal, and (2) a platelet count of 100 × 10⁶ 9(3) Hemoglobin of 9.0 g / dL or higher (no growth factors or transfection within the past two weeks, and a one-week washout for erythropoiesis-stimulating agents (ESAs) and colony-stimulating factors (CSFs) is sufficient), (4) Absolute neutrophil count of 1.5 × 10⁻¹⁴ 9 Appropriate experimental parameters are met, including (5) prothrombin time (PT) and partial thromboplastin time (PTT) being ≤1.5 times ULN, (6) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) being ≤2.5 times ULN, except in the presence of liver metastases, and (7) total bilirubin being ≤1.5 × ULN. Women of childbearing potential and men who are not surgically infertile must agree to use medically acceptable methods of contraception during the study and for at least 7 days in the case of women, and for at least 3 days in the case of men after the last dose of the study intervention. Female participants must have a negative serum pregnancy test within 7 days prior to the start of the study treatment, both for premenopausal women less than 12 months postmenopausal and for women. Male participants must agree to refrain from sperm donation or storage during the treatment period and for at least 3 days after the last dose of the study treatment.

[0319] Participants will be excluded from the exam if any of the following criteria apply: - Radiotherapy within 14 days prior to the first dose of the investigational drug (palliative radiation or stereotactic radiosurgery within 7 days prior to the start of the investigational treatment). - The patient had received systemic anticancer therapy or the investigational drug within two weeks prior to the start of investigational drug treatment (within four weeks for immunotherapy and tyrosine kinase inhibitor therapy). - Participants who have experienced grade 3 or higher immuno-related toxicity from previous immuno-oncology therapy. - Whether the patient underwent major surgery within 30 days prior to the first dose of the study drug, or has not recovered to at least Grade 1 from adverse effects of such procedure, or whether there is a predicted need for major surgery during the study treatment. - Active autoimmune disease with a recorded history of clinically severe autoimmune disease requiring systemic treatment or systemic steroids or immunosuppressants within the past three months; subjects receiving corticosteroids are excluded from doses of prednisone or equivalent greater than 10 mg per day. Inhaled steroids, intranasal steroids, intraocular steroids, and topical steroids are acceptable. Steroids as prophylaxis (for disease evaluation) are also acceptable. - Primary central nervous system (CNS) disease or pia mater disease; known CNS metastases were asymptomatic unless untreated, showed no evidence of radiological progression for at least 8 weeks, and did not require steroids or enzyme-induced anticonvulsants in the last 14 days prior to screening. - Abnormal lung function within the past six months, including pneumonia, active pneumonia, interstitial lung disease requiring steroid use, idiopathic pulmonary fibrosis, confirmed pleural effusion, severe dyspnea at rest, or interstitial lung disease requiring supplemental oxygen therapy. - A history of allogeneic organ transplantation or solid organ transplantation. - Poorly controlled diabetes or other poorly controlled immune-related endocrine disorders, as determined by the principal investigator. - If parenteral antibiotics have been used within 14 days prior to the first dose of the study drug, or if there is poor control of a severe systemic fungal, bacterial, viral or other infection, or if intravenous antibiotics are required. - Active infection due to known human immunodeficiency virus (HIV) infection or hepatitis C: (a) Known uncontrolled hepatitis B virus (HBV) infection: (i) Anti-HBV therapy initiated before the start of IMP and an HBV viral load of less than 2000 IU / mL (10⁴ copies / mL) are eligible. Anti-HBV therapy should be continued throughout the treatment period. (ii) Anti-HBc positive, anti-HBs positive, HBsAg negative and HBV viral load without HBV treatment are eligible. - If a live virus vaccine was received within 14 days prior to the first dose of the investigational drug. Seasonal influenza and other inactivated vaccines that do not contain live viruses are permitted. - Clinically significant bleeding (e.g., gastrointestinal bleeding, intracranial hemorrhage) within two weeks prior to the first dose of IL-2 conjugate. - Prior diagnosis of deep vein thrombosis or pulmonary embolism within the past three months. - Severe or unstable cardiac conditions within 6 months prior to the start of the study treatment, such as congestive heart failure (New York Heart Association class III or IV), cardiac bypass surgery or coronary stenting, angioplasty, ejection fraction below the lower limit of normal, unstable angina, medically uncontrolled hypertension (e.g., systolic ≥ 160 mmHg or diastolic ≥ 100 mmHg), uncontrolled arrhythmia requiring drug therapy (grade 2 or higher, according to NCI CTCAE v5.0), or myocardial infarction. - A history of non-pharmacologically induced, extended, and corrected QT intervals determined using Fridericia's formula (QTcF) with intervals greater than 450 milliseconds (msec) in males and greater than 470 msec in females. - Known hypersensitivity or contraindications to any component of IL-2 conjugate, PEG, PEGylated drugs, and Escherichia coli (E. coli)-derived proteins. - A history of active secondary malignancy or prior malignancy that would affect the evaluation of any study endpoint. Subjects with curatively surgically resected non-melanoma skin cancer or cervical cancer are eligible. - Any serious medical condition (including pre-existing autoimmune or inflammatory disorders), laboratory abnormality, psychiatric condition, or any other serious or unstable concurrent medical condition that the principal investigator believes would interfere with protocol therapy or make the subject unsuitable for the study: (a) patients with clinically significant active SARS-CoV-2 (COVID-19) infection. - Beginning with a screening visit of at least 7 days for women, or at least 3 days after the last dose of the trial intervention for men, participants must be pregnant or breastfeeding, or planning to become pregnant or give birth, during the expected duration of the trial. - Concurrent treatment with any other investigational drug, vaccine, or device. Concurrent participation in observational studies is permitted only after approval by the sponsor. - Subjects with a baseline oxygen saturation of less than 92% are not eligible for registration. - Participants with uveal, ocular, or fibrous metastatic melanoma.

[0320] Participants will receive IL-2 conjugate monotherapy QW / Q2W. The starting dose of IL-2 conjugate will be RP2D from Example 4.

[0321] Safety Assessment: Safety is assessed throughout the testing process by the following: • AE (type, incidence, severity, timing, severity, and association) • Measurement of vital signs (blood pressure, heart rate, respiratory rate, body temperature, pulse oximetry) • Physical examination • Clinical laboratory ·electro-cardiogram • All adjunctive medications and procedures, including all supportive therapies provided.

[0322] To monitor delayed immunotoxicity associated with immunotherapy products, adverse events will be evaluated for approximately 90 days after the last dose of the investigational drug or until subsequent anticancer treatment is administered (whichever comes first).

[0323] Efficacy Evaluation: Antitumor activity is determined by repeated radiographic imaging and response assessed using RECIST 1.1 and iRECIST. All subjects must undergo scans performed using a modality appropriate for the target tumor (e.g., PET-CT scan, CT scan, MRI). All anatomical sites imaged during screening must be evaluated at each point in time during the study using the same imaging modality.

[0324] Test endpoints Key evaluation criteria Objective response rate (ORR) is defined as the percentage of subjects with a confirmed complete response (CR) or partial response (PR). A confirmed response is defined as a sustained response observed in a follow-up scan at least four weeks after the initial confirmation of the response.

[0325] Secondary outcome items • DOR (Defined as the period from the date of the first objective response (either complete response (CR) or partial response (PR)) until the first radiographic progression of the disease or death due to any cause is recorded.) • PFS (defined as the earlier of the time from the first dose of IL-2 conjugate to the first confirmed progression of a radiation-induced illness or death due to any cause). • OS (defined as the time from the first dose of IL-2 conjugate to death from any cause.) • TTR is defined as the time from the initial dose of IL-2 conjugate to the first recorded objective response (either complete response (CR) or partial response (PR)). • DCR (Defined as the percentage of subjects who achieved CR, PR, or disease stabilization (a period of disease stabilization of 3 months or more)). • The percentage of subjects who did not experience disease progression 6 months after treatment. • Incidence rates of TEAE, SAE, and laboratory abnormalities according to NCI CTCAE v5.0 and ASTCT consensus grades.

[0326] Example 6. TEAEs due to priority period and specific target adverse events For participants who received IL-2 conjugate at doses of 16 μg / kg, 24 μg / kg, or 32 μg / kg under a Q2W, Q3W, or QW / Q2W schedule, as described in Examples 3 and 4, data on TEAEs in preferred terminology (PT) (worst grade by participant) are summarized in Tables 4-6, respectively.

[0327] In Tables 4-6, adverse events for specific purposes (AESIs) and their data are underlined. If an adverse event is shown in a row in one table but not in another table, it means that the adverse event was not observed at any of the dose levels listed in the other table. For example, Table 6 has a line for capillary leak syndrome (CLS), but Tables 4 and 5 do not. This means that no grade of CLS was observed at any of the dose levels listed in Tables 4 and 5.

[0328] As shown in Tables 4-6, the incidence of Grade 3 or higher AESIs was not high between the different schedules, and there was no observable increase in the TEAE profile between the different schedules. For example, as shown in Tables 4-6, there was no observable increase in the incidence of the following Grade 3 or higher adverse events: cytokine release syndrome, increased aspartate aminotransferase, increased alanine aminotransferase, increased serum bilirubin, infusion-related reactions, hyperbilirubinemia, and capillary leak syndrome were observed in the QW / Q2W administration schedule (at all study dose levels), the Q2W administration schedule (at all study dose levels), or the Q3W administration schedule (at all study dose levels). In other words, the QW / Q2W administration schedule is more aggressive than the Q2W or Q3W administration schedules, but surprisingly, the QW / Q2W administration schedule is relatively safe (e.g., there is no observable increase in the incidence of any of the aforementioned Grade 3 or higher adverse events).

[0329] [Table 4]

[0330] [Table 5]

[0331] [Table 6]

[0332] [Table 7]

[0333] [Table 8]

[0334] [Table 9]

[0335] [Table 10]

[0336] [Table 11]

[0337] [Table 12]

[0338] Example 7. Modeling of lymphocyte dynamics As described in Examples 3 and 4, for participants administered IL-2 conjugates at doses of 16 or 24 μg / kg under a Q2W, Q3W, or QW schedule (where the QW schedule corresponds to the introductory period of the QW / Q2W schedule), a semi-mechanistic population pharmacokinetic / pharmacodynamic (PK / PD) model was developed to explain the complex dynamics of IL-2 conjugate-induced lymphocyte transport from the blood to proliferation sites immediately after administration, as well as the subsequent reappearance of proliferating lymphocytes in the blood.

[0339] The model results are shown in the figures. Figures 17A and 17B also include observed data. Figure 17A shows the magnification changes of the simulated values ​​(shaded area and line) and observed values ​​(dots) of the number of CD8+ T cells. Figure 17B shows the magnification changes of the simulated (shaded area and line) and observed (dots) number of NK cells.

[0340] This model was developed to fully capture the inherent complexities of the pharmacokinetics and pharmacodynamics of IL-2 conjugates. Notably, body weight appeared as a statistically significant covariate that influenced both the clearance and volume terms.

[0341] The model's components of lymphocyte recirculation and expansion explained their dynamics by utilizing the plutonization changes of each immune cell. By using plasma concentrations to drive cell marginalization and migration, and individual area under the curve (AUC) values ​​to determine the exposure-response relationship governing cell expansion, the model cleverly captured the interaction between drug exposure and immune cell dynamics.

[0342] Simulation activity demonstrated a dose-dependent effect of IL-2 conjugates on CD8+ T cell and NK cell proliferation dynamics. Notably, IL-2 conjugates showed the ability to stimulate proliferation in these immune cell populations. The QW dosing schedule was also observed to induce increased CD8+ T cell and NK cell proliferation over multiple cycles compared to the Q2W and Q3W schedules. Such data suggest that the QW dosing schedule as an induction period (followed by maintenance periods, e.g., every two weeks) has a higher chance of eliciting therapeutic effects compared to the Q2W and Q3W schedules as induction periods.

[0343] Example 8. Observation of lymphocyte dynamics As described in Examples 3 and 4, blood was collected from participants receiving the IL-2 conjugate described herein at doses of (i) 8, 16, 24, or 32 μg / kg under a Q2W schedule, (ii) 8, 16, 24, 32, or 40 μg / kg under a Q3W schedule, or (iii) 16 or 24 μg / kg under a QW schedule (the QW schedule corresponds to the introductory period of the QW / Q2W schedule). These cells were identified in the blood collected by flow cytometry using specific antibodies against CD8 (for CD8+ T cells), CD56 (for NK cells), and FoxP3 (for regulatory T (Treg) cells).

[0344] Figure 18A shows the multiplicative change in the number of CD8+ T cells in blood samples taken from participants who received IL-2 conjugate at a dose of 16 μg / kg according to the prescribed administration schedule. Figure 18B shows the multiplicative change in the number of NK cells in blood samples taken from participants who received IL-2 conjugate at a dose of 16 μg / kg according to the prescribed administration schedule.

[0345] Figures 19A–19C show the flattening of peripheral CD4+ Treg cell counts across different test dosing schedules. Figure 19A shows the multiplier changes in Treg cell counts in blood samples taken from participants administered 8, 16, 24, and 32 μg / kg of IL-2 conjugate under the Q2W schedule. Figure 19B shows the multiplier changes in Treg cell counts in blood samples taken from participants administered 8, 16, 24, 32, or 40 μg / kg of IL-2 conjugate under the Q3W schedule. Figure 19C shows the multiplier changes in Treg cell counts in blood samples taken from participants administered 16 or 24 μg / kg of IL-2 conjugate under the QW schedule, which will be administered in part of the QW / Q2W schedule.

[0346] As shown in these figures, administration of IL-2 conjugate stimulated the proliferation of peripheral CD8+ T and NK cells without significantly increasing CD4+ Treg cell proliferation. More frequent administration during the induction period (e.g., using a QW / Q2W schedule) can induce increased CD8+ T cell proliferation and NK cell proliferation compared to administration schedules without a QW induction period (e.g., a Q2W or Q3W administration schedule as described in Example 3). Furthermore, no increase in Treg regulation was observed with more frequent administration during the induction period (e.g., using a QW / Q2W schedule) compared to administration schedules without a QW induction period (e.g., a Q2W or Q3W administration schedule as described in Example 3).

[0347] Sequence List The following sequence listings provide the sequences of the claims and referenced herein.

[0348] [Table 13]

Claims

1. A method for treating cancer in a subject in need of such treatment, comprising administering an IL-2 conjugate to the subject, The IL-2 conjugate contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and the amino acid at position P64 is given by formula (I): 【Chemistry 1】 (In the formula, Z is CH ２ And Y is, 【Chemistry 2】 Is it, Y is CH ２ And Z is, 【Transformation 3】 Is it, Z is CH ２ And Y is, 【Chemistry 4】 is or Y is CH ２ And Z is, 【Transformation 5】 And, W is a PEG group having an average molecular weight of approximately 25 kDa to approximately 35 kDa. q is 1, 2, or 3. X is structure: 【Transformation 6】 It is an L-amino acid having the following properties: X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to a subsequent amino acid residue. A method wherein the IL-2 conjugate is replaced by the structure of the IL-2 conjugate, and the IL-2 conjugate is administered to the subject (a) about once a week over a first set of weeks, and then (b) about once every two weeks over a second set of weeks.

2. The method according to claim 1, wherein the PEG group in the IL-2 conjugate has an average molecular weight of about 30 kDa.

3. In the IL-2 conjugate, Z is CH ２ And Y is, 【Transformation 7】 The method according to claim 1 or 2.

4. In the IL-2 conjugate, Y is CH ２ And Z is, 【Transformation 8】 The method according to claim 1 or 2.

5. In the IL-2 conjugate, Z is CH ２ And Y is, 【Chemistry 9】 The method according to claim 1 or 2.

6. In the IL-2 conjugate, Y is CH ２ And Z is, 【Chemistry 10】 The method according to claim 1 or 2.

7. The structure of formula (I) is formula (IV) or formula (V): 【Chemistry 11】 (In the formula, q is 1, 2, or 3. X is structure: 【Chemistry 12】 It is an L-amino acid having the following properties: X-1 indicates a binding site to the preceding amino acid residue, and X+1 indicates a binding site to a subsequent amino acid residue. The method according to claim 1 or 2, wherein the structure is or is a mixture of formula (IV) and formula (V).

8. The structure of formula (I) is formula (XII) or formula (XIII): 【Chemistry 13】 (In the formula, 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 a covalent bond to an unsubstituted amino acid residue in SEQ ID NO: 1.) The method according to claim 1 or 2, wherein the structure is or is a mixture of formula (XII) and formula (XIII).

9. The method according to any one of claims 1 to 8, wherein q is 1.

10. The method according to any one of claims 1 to 8, wherein q is 2.

11. The method according to any one of claims 1 to 8, wherein q is 3.

12. The method according to any one of claims 1 to 11, wherein the cancer is a solid tumor.

13. The method according to claim 12, wherein the solid tumor is a progressive or metastatic solid tumor.

14. The method according to claim 12, wherein the solid tumor is a recurrent or refractory solid tumor, or the solid tumor has recurred after one or more prior lines of systemic therapy for the solid tumor.

15. The method according to any one of claims 12 to 14, further comprising selecting the subject to be administered the IL-2 conjugate on at least partially the basis that the subject has received one or more prior lines of systemic therapy for the solid tumor.

16. The method according to any one of claims 12 to 15, wherein the subject is receiving one or more lines of systemic therapy for the solid tumor.

17. The method according to claim 16, wherein the subject is receiving two or more lines of systemic therapy for the solid tumor.

18. The method according to claim 17, wherein the subject is receiving three or more lines of systemic therapy for the solid tumor.

19. The method according to claim 12, wherein the solid tumor is a melanoma.

20. The method according to claim 19, wherein the melanoma is metastatic melanoma.

21. The method according to claim 19, wherein the melanoma is recurrent or refractory melanoma, or the melanoma has recurred after one or more prior lines of systemic therapy for the melanoma.

22. The method according to any one of claims 19 to 21, further comprising selecting the subject to be administered the IL-2 conjugate on at least partially the basis that the subject has received one or more prior lines of systemic therapy for the melanoma.

23. The method according to any one of claims 19 to 22, wherein the subject is receiving one or more lines of systemic therapy for the melanoma.

24. The method according to claim 23, wherein the subject is receiving two or more lines of systemic therapy for melanoma.

25. The method according to claim 24, wherein the subject is receiving three or more lines of systemic therapy for the melanoma.

26. The method according to any one of claims 16 to 18 or 23 to 25, wherein the one or more preceding lines of systemic therapy for the solid tumor or melanoma comprises an immune checkpoint inhibitor.

27. The method according to claim 26, wherein the immune checkpoint inhibitor includes a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a LAG-3 inhibitor.

28. The method according to any one of claims 1 to 27, wherein a dose of IL-2 of approximately 8 μg / kg is administered to the subject as the IL-2 conjugate.

29. The method according to any one of claims 1 to 27, wherein a dose of IL-2 of approximately 16 μg / kg is administered to the subject as the IL-2 conjugate.

30. The method according to any one of claims 1 to 27, wherein a dose of IL-2 of approximately 24 μg / kg is administered to the subject as the IL-2 conjugate.

31. The method according to any one of claims 1 to 27, wherein a dose of approximately 32 μg / kg of IL-2 is administered to the subject as the IL-2 conjugate.

32. The method according to any one of claims 1 to 27, comprising administering approximately 8 μg / kg of IL-2 as the IL-2 conjugate to the subject approximately once a week over a first number of weeks.

33. The method according to any one of claims 1 to 27, comprising administering approximately 16 μg / kg of IL-2 as the IL-2 conjugate to the subject approximately once a week over a first number of weeks.

34. The method according to any one of claims 1 to 27, comprising administering approximately 24 μg / kg of IL-2 as the IL-2 conjugate to the subject approximately once a week over a first number of weeks.

35. The method according to any one of claims 1 to 27, comprising administering approximately 32 μg / kg of IL-2 as the IL-2 conjugate to the subject approximately once a week over a first number of weeks.

36. The method according to any one of claims 32 to 35, comprising administering approximately 8 μg / kg of IL-2 as the IL-2 conjugate to the subject at least once every two weeks over a second number of weeks.

37. The method according to any one of claims 32 to 35, comprising administering approximately 16 μg / kg of IL-2 as the IL-2 conjugate to the subject at least once every two weeks over a second number of weeks.

38. The method according to any one of claims 32 to 35, comprising administering approximately 24 μg / kg of IL-2 as the IL-2 conjugate to the subject at least once every two weeks over a second number of weeks.

39. The method according to any one of claims 32 to 35, comprising administering approximately 32 μg / kg of IL-2 as the IL-2 conjugate to the subject at least once every two weeks over a second number of weeks.

40. The method according to any one of claims 32 to 39, wherein the first number of weeks is approximately five weeks, approximately six weeks, or approximately seven weeks.

41. The method according to claim 40, wherein the first number of weeks is approximately six weeks.

42. The method according to any one of claims 32 to 40, wherein the second number of weeks is at least about six weeks.

43. The method according to any one of claims 32 to 40, wherein the second number of weeks is at least about 12 weeks.

44. The method according to any one of claims 32 to 40, wherein the second number of weeks is in the range of about 6 weeks to about 46 weeks.

45. The method according to any one of claims 32 to 40, wherein the second number of weeks is in the range of about 6 weeks to about 98 weeks.

46. The method according to any one of claims 1 to 45, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

47. The method according to any one of claims 1 to 46, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

48. The method according to any one of claims 1 to 46, wherein the IL-2 conjugate is administered to the subject by subcutaneous administration.

49. The method according to any one of claims 1 to 48, further comprising administering acetaminophen to the subject.

50. The method according to any one of claims 1 to 49, further comprising administering diphenhydramine to the subject.

51. The method according to claim 49 or 50, wherein the acetaminophen and / or diphenhydramine is administered to the subject before administering the IL-2 conjugate.

52. An IL-2 conjugate for use in the method according to any one of claims 1 to 51.

53. Use of IL-2 conjugate for manufacturing a pharmaceutical product according to any one of claims 1 to 51.

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