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WO2024196937A1 - Cancer therapy with il-2 peg conjugates - Google Patents

Cancer therapy with il-2 peg conjugates Download PDF

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Publication number
WO2024196937A1
WO2024196937A1 PCT/US2024/020545 US2024020545W WO2024196937A1 WO 2024196937 A1 WO2024196937 A1 WO 2024196937A1 US 2024020545 W US2024020545 W US 2024020545W WO 2024196937 A1 WO2024196937 A1 WO 2024196937A1
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weeks
conjugate
subject
formula
once
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French (fr)
Inventor
Silke Elisabeth GASTINE
Wan-Ju MENG
Sergej RAMUSOVIC
Michael R.W. STREIT
Rui Wang
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Synthorx Inc
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Synthorx Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • T cells modulate the immune system to maintain immune homeostasis and tolerance.
  • regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological self-reactivity while cytotoxic T cells target and destroy infected cells and/or cancerous cells.
  • Cytokines comprise a family of cell signaling proteins such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors playing roles in innate and adaptive immune cell homeostasis. Cytokines are produced by immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, endothelial cells, fibroblasts, and different stromal cells. In some instances, cytokines modulate the balance between humoral and cell-based immune responses.
  • Interleukins are signaling proteins that modulate the development and differentiation of T and B lymphocytes, cells of the monocytic lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4+ T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue residents.
  • interleukin 2 (IL-2) signaling is used to modulate T cell responses and subsequently for treatment of a cancer.
  • an exemplary IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 having an unnatural amino acid residue described herein at position 64.
  • an exemplary IL-2 conjuate comprises the amino acid sequence of SEQ ID NO: 2.
  • methods described herein are amenable to a Attorney Docket No.01183-0277-00PCT-SYN solid tumor. In some embodiments, methods described herein are amenable to a hematologic cancer.
  • methods of treating solid tumors in a subject in need thereof comprising administering to a subject an IL-2 conjugate, wherein the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 having an unnatural amino acid residue described herein at position 64.
  • an exemplary IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 2.
  • dosing regimens e.g., doses, dosing frequency, or a combination of both
  • certain immune cells e.g., CD8+ T cells and/or natural killer (NK) cells
  • NK natural killer
  • cancer is a solid tumor.
  • such cancer is a hematologic cancer.
  • provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to a subject an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks.
  • methods of treating solid tumors in a subject comprising administering an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks.
  • метод ⁇ ии in another aspect, provided herein are methods of treating melanoma in a subject comprising administering an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks.
  • IL-2 conjugate a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks.
  • Embodiment 1 is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an IL-2 conjugate, wherein: the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 Attorney Docket No.01183-0277-00PCT-SYN P64 is replaced by the structure of Formula (I): – about 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure: point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue; wherein the IL-2 conjugate is administered to the subject (a) about
  • Embodiment 1.1 is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an IL-2 conjugate, wherein: the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1 wherein the amino acid at position P64 is replaced by the structure of Formula (I): - 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure: point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue; wherein the IL-2 conjugate is administered to the subject (a) about once every week for a first plurality of weeks, followed by (b) about once every two weeks for a second plurality of weeks.
  • the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1 wherein the amino acid at position P64 is replaced by the structure of Formula (I): - 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure: point
  • Embodiment 2 is the method of embodiment 1 or 1.1, wherein in the IL-2 conjugate the PEG group has an average molecular weight of about 30 kDa.
  • Embodiment 3 is the method of embodiment 1, 1.1, or 2, wherein in the IL-2 conjugate 1, 1.1, or 2, wherein in the IL-2 conjugate 1, 1.1, or 2, wherein in the IL-2 conjugate 1, 1.1, or 2, wherein in the IL-2 conjugate 1, 1.1, or 2, wherein the structure of Formula (I) has the structure of Formula (IV) or Formula (V), or is a mixture of Formula (IV) and Formula (V): wherein: q is 1, 2, or 3; X is an L-amino acid having the structure: Attorney Docket No.01183-0277-00PCT-SYN point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
  • Embodiment 8 is the method of embodiment 1, 1.1, or 2, wherein the structure of Formula (I) has the structure of Formula (XII) or Formula (XIII), or is a mixture of Formula (XII) and Formula (XIII): wherein: n is an integer such that -(OCH 2 CH 2 ) n -OCH 3 has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced.
  • Embodiment 9 is the method of any one of embodiments 1, 1.1, and 2-8, wherein q is 1.
  • Embodiment 10 is the method of any one of embodiments 1, 1.1, and 2-8, wherein q is 2.
  • Embodiment 11 is the method of any one of embodiments 1, 1.1, and 2-8, wherein q is 3.
  • Attorney Docket No.01183-0277-00PCT-SYN [0023]
  • Embodiment 12 is the method of any one of embodiments 1, 1.1, and 2-11, wherein the cancer is a solid tumor.
  • Embodiment 13 is the method of embodiment 12, wherein the solid tumor is an advanced or metastatic solid tumor.
  • Embodiment 14 is the method of embodiment 12, wherein the solid tumor is a relapsed or refractory solid tumor, or the solid tumor has relapsed after one or more prior lines of systemic therapy for the solid tumor.
  • Embodiment 15 is the method of any one of embodiments 12-14, further comprising selecting the subject to whom the IL-2 conjugate is administered at least in part on the basis of the subject having received one or more prior lines of systemic therapy for the solid tumor.
  • Embodiment 16 is the method of any one of embodiments 12-15, wherein the subject has received one or more lines of systemic therapy for the solid tumor.
  • Embodiment 17 is the method of embodiment 16, wherein the the subject has received two or more lines of systemic therapy for the solid tumor.
  • Embodiment 18 is the method of embodiment 17, wherein the subject has received three or more lines of systemic therapy for the solid tumor
  • Embodiment 19 is the method of embodiment 12, wherein the solid tumor is melanoma.
  • Embodiment 20 is the method of embodiment 19, wherein the melanoma is metastatic melanoma.
  • Embodiment 21 is the method of embodiment 19, wherein the melanoma is a relapsed or refractory melanoma, or the melanoma has relapsed after one or more prior lines of systemic therapy for the melanoma
  • Embodiment 22 is the method of any one of embodiments 19-21, further comprising selecting the subject to whom the IL-2 conjugate is administered at least in part on the basis of the subject having received one or more prior lines of systemic therapy for the melanoma.
  • Embodiment 23 is the method of any one of embodiments 19-22, wherein the subject has received one or more lines of systemic therapy for the melanoma.
  • Embodiment 23.1 is the method of any one of embodiments 19-22, wherein the subject has received one or more lines of systemic therapy for the solid tumor.
  • Embodiment 24 is the method of embodiment 23 or 23.1, wherein the the subject has received two or more lines of systemic therapy for the melanoma.
  • Embodiment 24.1 is the method of embodiment 23 or 23.1, wherein the the subject has received two or more lines of systemic therapy for the solid tumor.
  • Attorney Docket No.01183-0277-00PCT-SYN [0038]
  • Embodiment 25 is the method of embodiment 24 or 24.1, wherein the subject has received three or more lines of systemic therapy for the melanoma.
  • Embodiment 25.1 is the method of embodiment 24 or 24.1, wherein the subject has received three or more lines of systemic therapy for the solid tumor.
  • Embodiment 26 is the method of any one of embodiments 16-18 and 23-25, wherein the one or more prior lines of systemic therapy for the solid tumor or melanoma comprises an immune checkpoint inhibitor.
  • Embodiment 26.1 is the method of any one of embodiments 23, 23.1, 24, 24.1, 25, and 25.1, wherein the one or more prior lines of systemic therapy for melanoma comprises an immune checkpoint inhibitor.
  • Embodiment 27 is the method of embodiment 26 or 26.1, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG- 3 inhibitor, or combinations thereof.
  • Embodiment 27.1 is the method of 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.
  • Embodiment 28 is the method of 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 about 8 ⁇ g/kg IL-2 as the IL-2 conjugate is administered to the subject.
  • Embodiment 29 is the method of 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 about 16 ⁇ g/kg IL-2 as the IL-2 conjugate is administered to the subject.
  • Embodiment 30 is the method of 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 about 24 ⁇ g/kg IL-2 as the IL-2 conjugate is administered to the subject.
  • Embodiment 31 is the method of 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 about 32 ⁇ g/kg IL-2 as the IL-2 conjugate is administered to the subject.
  • Embodiment 32 is the method of 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 to the subject about 8 ⁇ g/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks.
  • Embodiment 33 is the method of 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 to the subject about 16 ⁇ g/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks.
  • Embodiment 34 is the method of 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 to the subject about 24 ⁇ g/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks.
  • Embodiment 35 is the method of 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 to the subject about 32 ⁇ g/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks.
  • Embodiment 36 is the method of any one of embodiments 32-35, comprising administering to the subject about 8 ⁇ g/kg IL-2 as the IL-2 conjugate about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks.
  • Embodiment 36.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 8 ⁇ g/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks.
  • Embodiment 37 is the method of any one of embodiments 32-35, comprising administering to the subject about 16 ⁇ g/kg IL-2 as the IL-2 conjugate about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks.
  • Embodiment 37.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 16 ⁇ g/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks.
  • Embodiment 38 is the method of any one of embodiments 32-35, comprising administering to the subject about 24 ⁇ g/kg IL-2 as the IL-2 conjugate about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks.
  • Embodiment 38.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 24 ⁇ g/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks.
  • Embodiment 39 is the method of any one of embodiments 32-35, comprising administering to the subject about 32 ⁇ g/kg IL-2 as the IL-2 conjugate about once every at least Attorney Docket No.01183-0277-00PCT-SYN two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks.
  • Embodiment 39.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 32 ⁇ g/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks.
  • Embodiment 40 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, and 39.1, wherein the first plurality of weeks is about 5 weeks, about 6 weeks, or about 7 weeks.
  • Embodiment 41 is the method of embodiment 40, wherein the first plurality of weeks is about 6 weeks.
  • Embodiment 42 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks is at least about 6 weeks.
  • Embodiment 43 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks is at least about 12 weeks.
  • Embodiment 44 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks ranges from about 6 weeks to about 46 weeks.
  • Embodiment 45 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks ranges from about 6 weeks to about 98 weeks.
  • Embodiment 46 is the method of 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.
  • Embodiment 47 is the method of 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.
  • Embodiment 48 is the method of 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 administration.
  • Embodiment 49 is the method of 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 to the subject.
  • Attorney Docket No.01183-0277-00PCT-SYN [0070]
  • Embodiment 50 is the method of 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 subject.
  • Embodiment 51 is the method of embodiment 49 or 50, wherein the acetaminophen and/or diphenhydramine is administered to the subject before administering the IL-2 conjugate.
  • Embodiment 52 is an IL-2 conjugate for use in the method of 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.
  • Embodiment 53 is use of an IL-2 conjugate for the manufacture of a medicament for the method of 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.
  • BRIEF DESCRIPTION OF THE DRAWINGS [0074] The novel features of the invention are set forth with particularity in the appended claims.
  • FIG.1 shows lymphocyte expansion in cynomolgus monkeys treated with an IL-2 conjugate at 0.1 mg/kg at a frequency of either Q1W for a total of 3 doses, Q2W for a total of 3 doses, Q3W for a total of 3 doses, or Q4 weekly for a total of 3 doses.
  • the days of dosing for each are indicated by the small arrows under the X-axis.
  • FIG.2 shows the change in peripheral CD8+ T eff cell counts in the indicated subjects treated with 8 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.3 shows the change in peripheral NK cell counts in the indicated subjects treated with 8 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.4 shows the change in peripheral CD4+ T reg cell counts in the indicated subjects treated with 8 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.5 shows the change in peripheral lymphocyte cell counts in the indicated subjects treated with 8 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.6 shows the change in peripheral eosinophil cell counts in the indicated subjects treated with 8 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.7A and FIG.7B show mean concentrations of the IL-2 conjugate administered to the indicated subjects at 8 ⁇ g/kg [Q2W] after 1 and 2 cycles, respectively, at specified times following administration.
  • FIG.8 shows the levels of IFN- ⁇ , IL-6, and IL-5 in the indicated subjects treated with 8 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.9 shows the change in peripheral CD8+ Teff cell counts in the indicated subjects treated with 16 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.10 shows the change in peripheral NK cell counts in the indicated subjects treated with 16 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.11 shows the changes in peripheral NK cell counts in the indicated subjects treated with 16 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.12 shows the change in peripheral eosinophil cell counts in the indicated subjects treated with 16 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.13 shows the levels of IFN- ⁇ , IL-6, and IL-5 in the indicated subjects treated with 16 ⁇ g/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
  • FIG.14A and FIG.14B show mean concentrations of the IL-2 conjugate administered to the indicated subjects at 16 ⁇ g/kg [Q2W] after 1 and 2 cycles, respectively, at specified times following administration.
  • FIG.15 shows the study design of Example 4. Abbreviations: C: cycle; D: day; DLT: dose-limiting toxicity; EOT: end of treatment; FU: follow-up; IMP: investigational medicinal product; IV: intravenously; QW: once weekly; Q2W: every 2 weeks; Q6W: every 6 weeks; Q8W: every 8 weeks; Q12W: every 12 weeks.
  • FIG.16 shows the study design of Example 5.
  • FIG.17A shows simulated (shaded areas and lines) and observed (dots) fold change in CD8+ T cell counts.
  • FIG.17B shows simulated (shaded areas and lines) and observed (dots) fold change in NK cell counts.
  • FIG.18A shows fold change in CD8+ T cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 ⁇ g/kgin accordance with the indicated dosing schedules.
  • FIG.18B shows fold change in NK cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 ⁇ g/kg in accordance with the indicated dosing schedules.
  • FIG.19A shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 8, 16, 24, or 32 ⁇ g/kg under the Q2W schedule.
  • FIG.18A shows fold change in CD8+ T cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 ⁇ g/kgin accordance with the indicated dosing schedules.
  • FIG.18B shows fold change in NK cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 ⁇ g/kg in accordance with the indicated dosing schedules.
  • FIG.19A shows fold
  • FIG. 19B shows fold change in Treg cell counts in blood collected from participants receiving the IL- 2 conjugate at a dose of 8, 16, 24, 32, or 40 ⁇ g/kg under the Q3W schedule.
  • FIG.19C shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 or 24 ⁇ g/kg QW, as would be administered during part of the QW/Q2W schedule.
  • the terms “subject(s)” and “patient(s)” mean any mammal.
  • the mammal is a human.
  • the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent) of a health care worker (e.g., a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker).
  • a health care worker e.g., a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker.
  • unnatural amino acid refers to an amino acid other than one of the 20 naturally occurring amino acids. Exemplary unnatural amino acids are described in Young et al., “Beyond the canonical 20 amino acids: expanding the genetic lexicon,” J. of Biological Chemistry 285(15): 11039-11044 (2010), the disclosure of which is incorporated herein by reference.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • 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.
  • nucleotide refers to a compound comprising a nucleoside moiety and a phosphate moiety.
  • Exemplary natural nucleotides include, without limitation, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), and guanosine monophosphate (GMP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine diphosphate (dADP), thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxygua
  • Exemplary natural deoxyribonucleotides which comprise a deoxyribose as the sugar moiety, include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP.
  • Exemplary natural ribonucleotides, which comprise a ribose as the sugar moiety include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.
  • base refers to at least the nucleobase portion of a nucleoside or nucleotide (nucleoside and nucleotide encompass the ribo or deoxyribo variants), which may in some cases contain further modifications to the sugar portion of the nucleoside or nucleotide.
  • base is also used to represent the entire nucleoside or nucleotide (for example, a “base” may be incorporated by a DNA polymerase into DNA, or by an RNA polymerase into RNA).
  • base should not be interpreted as necessarily representing the entire nucleoside or nucleotide unless required by the context.
  • the wavy line represents connection to a nucleoside or nucleotide, in which the sugar portion of the nucleoside or nucleotide may be further modified. In some embodiments, the wavy line represents attachment of the base or nucleobase to the sugar portion, such as a pentose, of the nucleoside or nucleotide.
  • a nucleobase is generally the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring, may be modified, may bear no similarity to natural bases, and/or may be synthesized, e.g., by organic synthesis. In certain embodiments, a nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide, where the atom or group of atoms is capable of interacting with a base of another nucleic acid with or without the use of hydrogen bonds.
  • an unnatural nucleobase is not derived from a natural nucleobase. It should be noted that unnatural nucleobases do not necessarily possess basic properties, however, they are referred to as nucleobases for simplicity. In some embodiments, when referring to a nucleobase, a “(d)” indicates that the nucleobase can be attached to a deoxyribose or a ribose, while “d” without parentheses indicates that the nucleobase is attached to deoxyribose. [0104] As used herein, a “nucleoside” is a compound comprising a nucleobase moiety and a sugar moiety.
  • Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups. Nucleosides include nucleosides comprising any variety of Attorney Docket No.01183-0277-00PCT-SYN substituents. A nucleoside can be a glycoside compound formed through glycosidic linking between a nucleic acid base and a reducing group of a sugar.
  • an “analog” of a chemical structure refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure.
  • a nucleotide analog is an unnatural nucleotide.
  • a nucleoside analog is an unnatural nucleoside.
  • a related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a “derivative.”
  • “advanced solid tumor” refers to a malignant solid neoplasm that has spread extensively to other anatomic sites or is no longer responding to treatment.
  • Line of treatment or “line of therapy” describes a treatment or therapy given as part of a sequence or in order to a patient as the patient’s disease progresses.
  • Initial treatment first- line therapy
  • second-line therapy a second different treatment
  • Subsequent lines of therapy may be given when a second-line therapy does not work or stops working.
  • “severe cytokine release syndrome” refers to level 4 or 5 cytokine release syndrome as described in Teachey et al., Cancer Discov.2016; 6(6); 664–79, the disclosure of which is incorporated herein by reference.
  • CDRs complementarity-determining regions
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences.
  • the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide Attorney Docket No.01183-0277-00PCT-SYN sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences.
  • a comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences may be accomplished using a mathematical algorithm.
  • the "percent identity” or “percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters.
  • Expressions such as “comprises a sequence with at least X% identity to Y” with respect to a test sequence mean that, when aligned to sequence Y as described above, the test sequence comprises residues identical to at least X% of the residues of Y.
  • Interleukin 2 is a pleiotropic type-1 cytokine whose structure comprises a 15.5 kDa four ⁇ -helix bundle.
  • 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 produced primarily by CD4+ T cells post antigen stimulation and to a lesser extent, by CD8+ cells, Natural Killer (NK) cells, and Natural killer T (NKT) cells, activated dendritic cells (DCs), and mast cells.
  • IL-2 signaling occurs through interaction with specific combinations of IL-2 receptor (IL-2R) subunits, IL-2R ⁇ (also known as CD25), IL-2R ⁇ (also known as CD122), and IL-2R ⁇ (also known as CD132). Interaction of IL-2 with the IL-2R ⁇ forms the “low- affinity” IL-2 receptor complex with a K d of about 10 -8 M.
  • IL-2R IL-2 receptor subunits
  • IL-2R ⁇ also known as CD25
  • IL-2R ⁇ also known as CD122
  • IL-2R ⁇ also known as CD132
  • Interaction of IL-2 with IL-2R ⁇ and IL-2R ⁇ forms the “intermediate-affinity” IL-2 receptor complex with a Kd of about 10 -9 M.
  • Interaction of IL-2 with all three subunits, IL-2R ⁇ , IL-2R ⁇ , and IL-2R ⁇ forms the “high- affinity” IL-2 receptor complex with a K d of about >10 -11 M.
  • IL-2 signaling via the “high-affinity” IL-2R ⁇ complex modulates the activation and proliferation of regulatory T cells.
  • Treg cells mediate maintenance of immune homeostasis by suppression of effector cells such as CD4 + T cells, CD8 + T cells, B cells, NK cells, and NKT cells.
  • Treg cells are generated from the thymus (tTreg cells) or are induced from na ⁇ ve T cells in the periphery (pTreg cells).
  • Treg cells are considered as the mediator of Attorney Docket No.01183-0277-00PCT-SYN peripheral tolerance.
  • IL-2 signaling via the “intermediate-affinity” IL-2R ⁇ complex modulates the activation and proliferation of CD8 + effector T (Teff) cells, NK cells, and NKT cells.
  • CD8 + Teff cells also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, Tcon, or killer T cells
  • NK and NKT cells are types of lymphocytes that, similar to CD8 + Teff cells, target cancerous cells and pathogen-infected cells.
  • IL-2 signaling is utilized to modulate certain immune cell responses (e.g., but not limited to T cell responses and/or NK cell responses) and subsequently for treatment of a cancer.
  • IL-2 is administered in a high-dose form to induce expansion of Teff cell populations for treatment of a cancer.
  • high-dose IL-2 further leads to concomitant stimulation of Treg cells that dampen anti-tumor immune responses.
  • High-dose IL-2 also induces toxic adverse events mediated by the engagement of IL- 2R alpha chain-expressing cells in the vasculature, including type 2 innate immune cells (ILC- 2), eosinophils and endothelial cells.
  • ILC- 2 type 2 innate immune cells
  • a cancer in one aspect, provides methods of stimulating and/or expanding certain immune cells that are useful for treatment of cancer, including, e.g., Teff cells, NK cells, and/or NKT cells, in a subject in need thereof, e.g., a subject having cancer, wherein such methods comprise administering to the subject an IL-2 conjugate as described herein.
  • a cancer is a solid tumor.
  • such a cancer is a hematologic cancer.
  • the IL-2 sequence included in an IL-2 conjugate described herein comprises an amino acid sequence that is at least about 90% (including, e.g., 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 sequence of SEQ ID NO: 1: Attorney Docket No.01183-0277-00PCT-SYN PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFSQ
  • the IL-2 sequence included in an IL-2 conjugate described herein comprises the Attorney Docket No.01183-0277-00PCT-SYN sequence of SEQ ID NO: 1, wherein the amino acid at position P64 as set forth in the SEQ ID NO: 1 is replaced by the structure of Formula (I) as described herein.
  • average molecular weight encompasses both weight average molecular weight and number average molecular weight; in other words, for example, both a 30 kDa number average molecular weight and a 30 kDa weight average molecular weight qualify as a 30 kDa molecular weight.
  • the average molecular weight is weight average molecular weight. In other embodiments, the average molecular weight is number average molecular weight. It is understood that in the methods provided herein, administering an IL-2 conjugate as described herein to a subject comprises administering more than a single molecule of IL-2 conjugate; as such, use of the term “average” to describe the molecular weight of the PEG group refers to the average molecular weight of the PEG groups of the IL-2 conjugate molecules in a dose administered to the subject. [0119] In any of the embodiments or variations of Formula (I) described herein, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.
  • 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. [0120] In some embodiments of Formula (I), Z is CH 2 and Y is . In some embodiments of Formula (I), Y is CH2 and Z is some embodiments of Formula embodiments of Formula [0121] In some (I), q is 2. In some embodiments of Formula (I), q is 3. [0122] In some embodiments of Formula (I), W is a PEG group having an average molecular weight of about 25 kDa.
  • 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.
  • q is 1 and structure of Formula (I) is the structure of Formula (Ia): – about 35 kDa;
  • X is an L-amino acid having the structure: point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
  • the IL-2 conjugate comprises the sequence of SEQ ID NO: 2: PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELK[AzK_L1_PEG30kD]LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 2) wherein [AzK_L1_PEG30kD] is N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to PEG via DBCO-mediated click chemistry to form a compound comprising a structure of Formula (IV) or Formula (V), where
  • DBCO means a chemical moiety comprising a dibenzocyclooctyne group, such as comprising the mPEG-DBCO compound illustrated in Schemes 1 and 2 of Example 1.
  • the ratio of regioisomers generated from the click reaction is about 1:1 or greater than 1:1.
  • PEGs typically comprise a number of (OCH2CH2) monomers (or (CH2CH2O) monomers, depending on how the PEG is defined). In some embodiments, the number of (OCH 2 CH 2 ) monomers (or (CH 2 CH 2 O) monomers) is such that the average molecular weight of the PEG group is about 30 kDa.
  • the PEG is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower C 1-6 alkoxy group, or a hydroxyl group.
  • the PEG group is a methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG wherein one terminus of the polymer is a methoxy (-OCH3) group, and the other terminus is a hydroxyl or other functional group that can be optionally chemically modified.
  • mPEG methoxy-PEG
  • the PEG group is a linear or branched PEG group.
  • the PEG group is a linear PEG group.
  • 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 Attorney Docket No.01183-0277-00PCT-SYN embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da ⁇ 3,000 Da, or 30,000 Da ⁇ 4,500 Da, or 30,000 Da ⁇ 5,000 Da.
  • the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V): wherein: W is a PEG group having an average molecular weight of about 25 kDa - 35kDa; q is 1, 2, or 3; and X has the structure: point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
  • W is a PEG group having an average molecular weight of about 25 kDa - 35kDa
  • q is 1, 2, or 3
  • X has the structure
  • an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID Attorney Docket No. 01183-0277-00PCT-SYN NO: 1, in which the amino acid residue P64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V).
  • q is 1.
  • q is 2.
  • q is 3.
  • 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.
  • the structure of Formula (I) has the structure of Formula (IV) or Formula (V), or is a mixture of Formula (IV) and Formula (V).
  • the structure of Formula (I) has the structure of Formula (IV).
  • the structure of Formula (I) has the structure of Formula (V).
  • the structure of Formula (I) is 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): wherein: W is a PEG group having an average molecular weight of about 25 kDa – about 35kDa; and X has the structure: Attorney Docket No.01183-0277-00PCT-SYN point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
  • the PEG group has an average molecular weight of about 30 kDa.
  • the structure of Formula (I) has the structure of Formula (IVa) or Formula (Va), or is a mixture of Formula (IVa) and Formula (Va).
  • the structure of Formula (I) has the structure of Formula (IVa).
  • the structure of Formula (I) has the structure of Formula (Va).
  • the structure of Formula (I) is a mixture of Formula (IVa) and Formula (Va).
  • the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII): wherein: Attorney Docket No.01183-0277-00PCT-SYN n is is an integer such that -(OCH 2 CH 2 ) n -OCH 3 has a molecular weight of about 25 kDa – about 35 kDa; q is 1, 2, or 3; and the wavy lines indicate convalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced
  • an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue P64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII).
  • q is 1.
  • q is 2.
  • q is 3.
  • n is is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 30 kDa.
  • the structure of Formula (I) has the structure of Formula (XII) or Formula (XIII), or is a mixture of Formula (XII) and Formula (XIII).
  • the structure of Formula (I) has the structure of Formula (XII).
  • the structure of Formula (I) has the structure of Formula (XIII).
  • the structure of Formula (I) is a mixture of Formula (XII) and Formula (XIII).
  • q is 1
  • the structure of Formula (XII) is the structure of Formula (XIIa)
  • the structure of Formula (XIII) is the structure of Formula (XIIIa): Attorney Docket No.01183-0277-00PCT-SYN Formula (XIIIa); wherein: n is is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 25 kDa – about 35 kDa; and the wavy lines indicate convalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced.
  • n is is an integer such that -(OCH 2 CH 2 ) n -OCH 3 has a molecular weight of about 30 kDa.
  • the structure of Formula (I) has the structure of Formula (XIIa) or Formula (XIIIa), or is a mixture of Formula (XIIa) and Formula (XIIIa).
  • the structure of Formula (I) has the structure of Formula (XIIa).
  • the structure of Formula (I) has the structure of Formula (XIIIa).
  • the structure of Formula (I) is a mixture of Formula (XIIa) and Formula (XIIIa).
  • the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV): wherein: Attorney Docket No.01183-0277-00PCT-SYN m is an integer from 0 to 20; p is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa – about 35 k
  • an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue P64 is replaced by the structure 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.
  • 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.
  • 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. [0148] In some embodiments, p is an integer from 0 to 15. In some embodiments, p is an integer from 0 to 10. In some embodiments, p is an integer from 0 to 5. In some embodiments, p is an integer from 1 to 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. [0149] In some embodiments, m and p are each 2.
  • 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).
  • the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII): Attorney Docket No.01183-0277-00PCT-SYN wherein: m is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa - 35 kDa; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced.
  • an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue P64 is replaced by the structure 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.
  • 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.
  • 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. [0154] In any of the embodiments 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).
  • the IL-2 conjugates described herein can be prepared by a conjugation reaction comprising a 1,3-dipolar cycloaddition reaction.
  • the 1,3-dipolar cycloaddition reaction comprises reaction of an azide and an alkyne (“Click” reaction).
  • a conjugation reaction described herein comprises the reaction outlined in Scheme I, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1.
  • Scheme I [0156]
  • the conjugating moiety comprises a PEG group as described herein.
  • a reactive group comprises an alkyne or azide.
  • a conjugation reaction described herein comprises the reaction outlined in Scheme II, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1.
  • Scheme II [0158]
  • a conjugation reaction described herein comprises the reaction outlined in Scheme III, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1.
  • Scheme III [0159]
  • a conjugation reaction described herein comprises the reaction outlined in Scheme IV, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1.
  • Attorney Docket No.01183-0277-00PCT-SYN comprises a cycloaddition reaction between an azide moiety, such as that contained in a protein containing an amino acid residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as that derived from DBCO, which is a chemical moiety comprising a dibenzocyclooctyne group.
  • PEG groups comprising a DBCO moiety are commercially available or may be prepared by methods known to those of ordinary skill in the art. Exemplary reactions are shown in Schemes V and VI.
  • Conjugation reactions such as a click reaction described herein may generate a single regioisomer, or a mixture of regioisomers.
  • the ratio of regioisomers is about 1:1.
  • the ratio of regioisomers is about 2:1.
  • the ratio of regioisomers is about 1.5:1.
  • the ratio of regioisomers is about 1.2:1.
  • the ratio of regioisomers is about 1.1:1.
  • the ratio of regioisomers is greater than 1:1.
  • IL-2 conjugates described herein are generated recombinantly or are synthesized chemically.
  • IL-2 conjugates described herein are generated recombinantly, for example, either by a host cell system, or in a cell-free system.
  • IL-2 conjugates are generated recombinantly through a host cell system.
  • the host cell is a eukaryotic cell (e.g., mammalian cell, insect cells, yeast cells or plant cell) or a prokaryotic cell (e.g., Gram-positive bacterium or a Gram-negative bacterium).
  • a eukaryotic host cell is a mammalian host cell.
  • a mammalian host cell is a stable cell line, or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division.
  • a mammalian host cell is a transient cell line, or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division.
  • Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells , 293 H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO- K1 cells, Expi293FTM cells, Flp-InTM T-RExTM 293 cell line, Flp-InTM-293 cell line, Flp-InTM- 3T3 cell line, Flp-InTM-BHK cell line, Flp-InTM-CHO cell line, Flp-InTM-CV-1 cell line, Flp- InTM-Jurkat cell line, FreeStyleTM 293-F cells, FreeStyleTM CHO-S cells, GripTiteTM 293 MSR cell line, GS
  • a eukaryotic host cell is an insect host cell.
  • Exemplary insect host cells include Drosophila S2 cells, Sf9 cells, Sf21 cells, High FiveTM cells, and expresSF+® cells.
  • a 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 strain such as INVSc1.
  • a eukaryotic host cell is a plant host cell.
  • the plant cells comprise a cell from algae.
  • Exemplary plant cell lines include strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.
  • a host cell is a prokaryotic host cell.
  • Exemplary prokaryotic host cells include BL21, Mach1TM, DH10BTM, TOP10, DH5 ⁇ , DH10BacTM, OmniMaxTM, MegaXTM, DH12STM, INV110, TOP10F’, INV ⁇ F, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2TM, Stbl3TM, or Stbl4TM.
  • suitable polynucleic acid molecules or vectors for the production of an IL-2 polypeptide described herein include any suitable vectors derived from either a eukaryotic or prokaryotic source.
  • Exemplary polynucleic acid molecules or vectors include vectors from bacteria (e.g., E. coli), insects, yeast (e.g., Pichia pastoris, K. phaffii), algae, or mammalian source.
  • Bacterial vectors include, for example, pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.
  • Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.
  • FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2
  • MAT vectors such as pPolh-MAT1, or pPolh-MAT2.
  • Yeast vectors include, for example, Gateway ® pDEST TM 14 vector, Gateway ® pDEST TM 15 vector, Gateway ® pDEST TM 17 vector, Gateway ® pDEST TM 24 vector, Gateway ® pYES- DEST52 vector, pBAD-DEST49 Gateway ® destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris (K. phaffii) vector, pGAPZA, B, & C Pichia pastoris (K.
  • Algae vectors include, for example, pChlamy-4 vector or MCS vector.
  • Mammalian vectors include, for example, transient expression vectors or stable expression vectors.
  • Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG- Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP- CMV 4.
  • Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG- CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.
  • a cell-free system is used for the production of an IL-2 polypeptide described herein.
  • a cell-free system comprises a mixture of cytoplasmic and/or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis.
  • a cell-free system utilizes prokaryotic cell components.
  • a cell-free system utilizes eukaryotic cell components.
  • Nucleic acid synthesis is obtained in a cell-free Attorney Docket No.01183-0277-00PCT-SYN system based on, for example, Drosophila cell, Xenopus egg, Archaea, or HeLa cells.
  • Exemplary cell-free systems include E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+.
  • Cell-free translation systems variously comprise components such as plasmids, mRNA, DNA, tRNAs, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and/or unnatural amino acids, and/or other components used for protein expression. Such components are optionally modified to improve yields, increase synthesis rate, increase protein product fidelity, or incorporate unnatural amino acids.
  • cytokines described herein are synthesized using cell-free translation systems described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or US 8,778,631, the disclosure of each of which is incorporated herein by reference.
  • cell-free translation systems comprise modified release factors, or even removal of one or more release factors from the system.
  • cell-free translation systems comprise a reduced protease concentration.
  • cell-free translation systems comprise modified tRNAs with re-assigned codons used to code for unnatural amino acids.
  • the synthetases described herein for the incorporation of unnatural amino acids are used in cell-free translation systems.
  • tRNAs are pre- loaded with unnatural amino acids using enzymatic or chemical methods before being added to a cell-free translation system.
  • components for a cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organism.
  • an IL-2 polypeptide is generated as a circularly permuted form, either via an expression host system or through a cell-free system.
  • Production of Cytokine Polypeptide Comprising an Unnatural Amino Acid [0177]
  • An orthogonal or expanded genetic code can be used in the present disclosure, in which one or more specific codons present in the nucleic acid sequence of an IL-2 polypeptide are allocated to encode the unnatural amino acid so that it can be genetically incorporated into the IL-2 by using an orthogonal tRNA synthetase/tRNA pair.
  • the orthogonal tRNA synthetase/tRNA pair is capable of charging a tRNA with an unnatural amino acid and is capable of incorporating that unnatural amino acid into the polypeptide chain in response to the codon.
  • the codon is the codon amber, ochre, opal or a quadruplet codon.
  • the codon corresponds to the orthogonal tRNA which will be used to carry the unnatural amino acid.
  • the codon is amber.
  • the codon is an orthogonal codon.
  • the codon is a quadruplet codon, which can be decoded by an orthogonal ribosome ribo-Q1.
  • the quadruplet codon is as illustrated in Neumann, et al., “Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome,” Nature, 464(7287): 441-444 (2010), the disclosure of which is incorporated herein by reference.
  • a codon used in the present disclosure is a recoded codon, e.g., a synonymous codon or a rare codon that is replaced with alternative codon.
  • the recoded codon is as described in Napolitano, et al., “Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli,” PNAS, 113(38): E5588-5597 (2016), the disclosure of which is incorporated herein by reference.
  • the recoded codon is as described in Ostrov et al., “Design, synthesis, and testing toward a 57-codon genome,” Science 353(6301): 819-822 (2016), the disclosure of which is incorporated herein by reference.
  • unnatural nucleic acids are utilized leading to incorporation of one or more unnatural amino acids into the IL-2.
  • Exemplary unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted a
  • Certain unnatural 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 duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5- methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of aden
  • the heterocyclic base includes, in some cases, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4- aminopyrrolo [2.3-d] pyrimidin-5-yl, 2-amino-4-oxopyrolo [2, 3-d] pyrimidin-5-yl, 2- amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the nucleic acid via Attorney Docket No.01183-0277-00PCT-SYN the 9-position, the pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1-position.
  • nucleotide analogs are also modified at the phosphate moiety.
  • Modified phosphate moieties include, but are not limited to, those with modification at the linkage between two nucleotides and contains, for example, a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkage between two nucleotides are through a 3’-5’ linkage or a 2’-5’ linkage, and the linkage contains inverted 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.
  • nucleotides containing modified phosphates include but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 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 disclosure of each of which is incorporated herein by reference.
  • unnatural nucleic acids include 2’,3’-dideoxy-2’,3’-didehydro- nucleosides (PCT/US2002/006460), 5’-substituted DNA and RNA derivatives (PCT/US2011/033961; Saha et al., J.
  • unnatural nucleic acids include modifications at the 5’-position and the 2’-position of the sugar ring (PCT/US94/02993), such as 5’-CH2-substituted 2’-O- protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem.1999, 10, 921-924).
  • unnatural nucleic acids include amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3’ linked nucleoside in the dimer (5’ to 3’) comprises a 2’-OCH 3 and a 5’-(S)-CH 3 (Mesmaeker et al., Synlett, 1997, 1287-1290).
  • Unnatural nucleic acids can include 2’-substituted 5’-CH 2 (or Attorney Docket No.01183-0277-00PCT-SYN O) modified nucleosides (PCT/US92/01020).
  • Unnatural nucleic acids can 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).
  • Unnatural nucleic acids can include 5’-phosphonate monomers having a 2’-substitution (US2006/0074035) and other modified 5’-phosphonate monomers (WO1997/35869).
  • Unnatural nucleic acids can include 5’-modified methylenephosphonate monomers (EP614907 and EP629633).
  • Unnatural nucleic acids can include analogs of 5’ or 6’-phosphonate ribonucleosides comprising a hydroxyl group at the 5’ and/or 6’-position (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033).
  • Unnatural nucleic acids can include 5’-phosphonate deoxyribonucleoside monomers and dimers having a 5’-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82).
  • Unnatural nucleic acids can include nucleosides having a 6’-phosphonate group wherein the 5’ or/and 6’-position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH 3 ) 3 ) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med.
  • unnatural nucleic acids also include modifications of the sugar moiety.
  • nucleic acids contain one or more nucleosides wherein the sugar group has been modified.
  • nucleic acids comprise a chemically modified ribofuranose ring moiety.
  • a modified nucleic acid comprises modified sugars or sugar analogs.
  • the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group.
  • the sugar can be in a pyranosyl or furanosyl form.
  • the sugar moiety may be the furanoside of ribose, deoxyribose, arabinose or 2’-O-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric configuration.
  • Sugar modifications include, but are not limited to, 2’-alkoxy-RNA analogs, 2’-amino-RNA analogs, 2’-fluoro-DNA, and 2’-alkoxy- or amino-RNA/DNA chimeras.
  • a sugar modification may include 2’-O-methyl-uridine or 2’-O-methyl-cytidine.
  • Sugar modifications include 2’-O-alkyl-substituted deoxyribonucleosides and 2’-O-ethyleneglycol like ribonucleosides. The preparation of these sugars or sugar analogs and the respective “nucleosides” wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known. Sugar modifications may also be made and combined with other modifications. [0188] Modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications.
  • Sugar modifications include, but are not limited to, the following modifications at the 2’ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 , alkyl or C 2 to C 10 alkenyl and alkynyl.2’ sugar modifications also include but are not limited to -O[(CH2)nO]m CH3, -O(CH2)nOCH3, - O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)nONH2, and -O(CH2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10.
  • Modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH 2 and S.
  • Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5’-vinyl, 5’-methyl (R or S), 4’-S, 2’-F, 2’-OCH3, and 2’- O(CH 2 ) 2 OCH 3 substituent groups.
  • nucleic acids described herein include one or more bicyclic nucleic acids.
  • the bicyclic nucleic acid comprises a bridge between the 4’ and the 2’ ribosyl ring atoms.
  • nucleic acids provided herein include one or more bicyclic nucleic acids wherein the bridge comprises a 4’ to 2’ bicyclic nucleic acid.
  • 4’ to 2’ bicyclic nucleic acids include, but are not limited to, one of the formulae: 4’-(CH2)-O-2’ (LNA); 4’-(CH2)-S-2’; 4’-(CH2)2-O-2’ (ENA); 4’-CH(CH3)-O- 2’ and 4’-CH(CH 2 OCH 3 )-O-2’, and analogs thereof (see, U.S.
  • nucleic acids comprise linked nucleic acids.
  • Nucleic acids can be linked together using any inter nucleic acid linkage.
  • the two main classes of inter nucleic acid linking groups are defined by the presence or absence of a phosphorus atom.
  • non-phosphorus containing inter nucleic acid linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N*-dimethylhydrazine (-CH 2 -N(CH 3 )-N(CH 3 )).
  • inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates.
  • Unnatural nucleic acids can contain a single modification. Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.
  • Backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in any combination. Other non- phosphate linkages may also be used.
  • backbone modifications e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages
  • backbone modifications can confer immunomodulatory activity on the modified nucleic acid and/or enhance their stability in vivo.
  • a phosphorous derivative or modified phosphate group is attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like.
  • Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found in Peyrottes et al., 1996, Nucleic Acids Res.24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res.24:2318-2323; Schultz et al., (1996) Nucleic Acids Res.24:2966- 2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview” in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, “Oligonucleoside Phosphorothioates” in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp.165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Bio
  • backbone modification comprises replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group.
  • modifications include: anionic internucleoside linkage; N3’ to P5’ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and Attorney Docket No.01183-0277-00PCT-SYN thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179, the disclosure of which is incorporated herein by reference).
  • anionic internucleoside linkage N3’ to P5’ phosphoramidate modification
  • boranophosphate DNA prooligonucleotides
  • neutral internucleoside linkages such as methylphosphonates
  • a modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g. a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages.
  • phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages.
  • Substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts.
  • nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA).
  • PNA aminoethylglycine
  • United States Patent Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake.
  • Conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et
  • a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol 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), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O- hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651- Attorney Docket No.01183
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • the unnatural nucleic acids further form unnatural base pairs.
  • exemplary unnatural nucleotides capable of forming an unnatural DNA or RNA base pair (UBP) under conditions in vivo includes, but is not limited to, TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof.
  • unnatural nucleotides include: .
  • Exemplary unnatural base pairs include: (d)TPT3-(d)NaM; (d)5SICS-(d)NaM; (d)CNMO- (d)TAT1; (d)NaM-(d)TAT1; (d)CNMO-(d)TPT3; and (d)5FM-(d)TAT1.
  • unnatural nucleotides include: .
  • R 2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azido; and the wavy line indicates a bond to a ribosyl or 2’-deoxyribosyl, wherein the 5’-hydroxy group of the ribosyl or 2’-deoxyribosyl moiety is in free form, is connected to a monophosphate, diphosphate, triphosphate, ⁇ -thiotriphosphate, ⁇ -thiotriphosphate, or ⁇ -thiotriphosphate group, or is included in an RNA
  • the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein may be derived from a compound of the Formula Attorney Docket No.01183-0277-00PCT-SYN wherein: each X is independently carbon or nitrogen; R2 is absent when X is nitrogen, and 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 secondary amine; E is oxygen, sulfur, or selenium; and the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety or an analog thereof, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof is in free form, is connected to
  • 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. [0203] 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. [0204] In some embodiments, E is sulfur. In some embodiments, E is oxygen.
  • E is selenium.
  • R2 is halogen, such as chloro, bromo, or fluoro.
  • R 2 is cyano.
  • R 2 is azide.
  • E is sulfur, Y is sulfur, and each X is independently carbon or nitrogen.
  • E is sulfur, Y is sulfur, and each X is carbon.
  • Attorney Docket No. 01183-0277-00PCT-SYN [0207]
  • the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein may be derived from ,
  • an unnatural base pair generate an unnatural amino acid described in Dumas et al., “Designing logical codon reassignment – Expanding the chemistry in biology,” Chemical Science, 6: 50-69 (2015), the disclosure of which is incorporated herein by reference.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a synthetic codon comprising an unnatural nucleic acid.
  • the unnatural amino acid is incorporated into the cytokine by an orthogonal, modified Attorney Docket No.01183-0277-00PCT-SYN synthetase/tRNA pair.
  • Such orthogonal pairs comprise an unnatural synthetase that is capable of charging the unnatural tRNA with the unnatural amino acid, while minimizing charging of a) other endogenous amino acids onto the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs.
  • Such orthogonal pairs comprise tRNAs that are capable of being charged by the unnatural synthetase, while avoiding being charged with a) other endogenous amino acids by endogenous synthetases.
  • an orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from two different organisms. In some embodiments, an orthogonal synthetase/tRNA pair comprising components that prior to modification, promote translation of two different amino acids. In some embodiments, an orthogonal synthetase is a modified alanine synthetase. In some embodiments, an orthogonal synthetase is a modified arginine synthetase.
  • an orthogonal synthetase is a modified asparagine synthetase. In some embodiments, an orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, an orthogonal synthetase is a modified cysteine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamic acid synthetase. In some embodiments, an orthogonal synthetase is a modified alanine glycine.
  • an orthogonal synthetase is a modified histidine synthetase. In some embodiments, an orthogonal synthetase is a modified leucine synthetase. In some embodiments, an orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, an orthogonal synthetase is a modified lysine synthetase. In some embodiments, an orthogonal synthetase is a modified methionine synthetase. In some embodiments, an orthogonal synthetase is a modified phenylalanine synthetase.
  • an orthogonal synthetase is a modified proline synthetase. In some embodiments, an orthogonal synthetase is a modified serine synthetase. In some embodiments, an orthogonal synthetase is a modified threonine synthetase. In some embodiments, an orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, an orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, an orthogonal synthetase is a modified valine synthetase.
  • an orthogonal synthetase is a modified phosphoserine synthetase.
  • an orthogonal tRNA is a modified alanine tRNA.
  • an orthogonal tRNA is a modified arginine tRNA.
  • an orthogonal tRNA is a modified asparagine tRNA.
  • an orthogonal tRNA is a modified aspartic acid tRNA.
  • an orthogonal tRNA is a modified cysteine tRNA.
  • an orthogonal tRNA is a modified glutamine tRNA.
  • an orthogonal tRNA is Attorney Docket No.01183-0277-00PCT-SYN a modified glutamic acid tRNA.
  • an orthogonal tRNA is a modified alanine glycine.
  • an orthogonal tRNA is a modified histidine tRNA.
  • an orthogonal tRNA is a modified leucine tRNA.
  • an orthogonal tRNA is a modified isoleucine tRNA.
  • an orthogonal tRNA is a modified lysine tRNA.
  • an orthogonal tRNA is a modified methionine tRNA.
  • an orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, an orthogonal tRNA is a modified proline tRNA. In some embodiments, an orthogonal tRNA is a modified serine tRNA. In some embodiments, an orthogonal tRNA is a modified threonine tRNA. In some embodiments, an orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, an orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, an orthogonal tRNA is a modified valine tRNA. In some embodiments, an orthogonal tRNA is a modified phosphoserine tRNA.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair.
  • aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus tRNACUA pairs, E. coli LeuRS (Ec-Leu)/B. stearothermophilus tRNACUA pairs, and pyrrolysyl-tRNA pairs.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Mj- TyrRS/tRNA pair.
  • exemplary UAAs that can be incorporated by a Mj-TyrRS/tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p- aminophenylalanine and p-methoyphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronophenylalanine; and o-nitrobenzyltyrosine.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Ec-Tyr/tRNA CUA or a Ec-Leu/tRNA CUA pair.
  • exemplary UAAs that can be incorporated by a Ec-Tyr/tRNACUA or a Ec-Leu/tRNACUA pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O- propargyltyrosine; ⁇ -aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3- (naphthalene-2-ylamino)-2-amino-propanoic acid.
  • the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a pyrrolysyl-tRNA pair.
  • the PylRS is obtained from an archaebacterial, e.g., from a methanogenic archaebacterial.
  • the PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.
  • Exemplary UAAs that can be incorporated by a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2- Attorney Docket No.01183-0277-00PCT-SYN carboxamido)hexanoic acid, N- ⁇ - D -prolyl- L -lysine, and N- ⁇ -cyclopentyloxycarbonyl- L -lysine; N- ⁇ -Acryloyl- L -lysine; N- ⁇ -[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]- L -lysine; and N- ⁇ -(1-methylcyclopro-2-enecarboxamido)lysine.
  • amide and carbamate substituted lysines such as 2-amino-6-
  • the IL-2 conjugates disclosed herein may be prepared by use of M. mazei tRNA which is selectively charged with a non-natural amino acid such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) by the M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS).
  • M. mazei tRNA which is selectively charged with a non-natural amino acid such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) by the M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS).
  • Mb PylRS M. barkeri pyrrolysyl-tRNA synthetase
  • an unnatural amino acid is incorporated into a cytokine described herein (e.g., the IL polypeptide) by a synthetase disclosed in US 9,988,619 and US 9,938,516, the disclosure of each of which is incorporated herein by reference.
  • the host cell into which the constructs or vectors disclosed herein are introduced is cultured or maintained in a suitable medium such that the tRNA, the tRNA synthetase and the protein of interest are produced.
  • the medium also comprises the unnatural amino acid(s) such that the protein of interest incorporates the unnatural amino acid(s).
  • a nucleoside triphosphate transporter from bacteria, plant, or algae is also present in the host cell.
  • the IL-2 conjugates disclosed herein are prepared by use of a host cell that expresses a NTT.
  • the nucleotide nucleoside triphosphate transporter used in the host cell may be selected from TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P.
  • the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, the NTT is PtNTT1.
  • the NTT is PtNTT2. In some embodiments, the NTT is PtNTT3. In some embodiments, the NTT is PtNTT4. In some embodiments, the NTT is PtNTT5. In some embodiments, the NTT is PtNTT6.
  • 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.
  • the orthogonal tRNA synthetase/tRNA pair charges a tRNA with an unnatural amino acid and incorporates the unnatural amino acid into the polypeptide chain in response to the codon.
  • exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus tRNA CUA pairs, E. coli LeuRS (Ec-Leu)/B. stearothermophilus tRNA CUA pairs, and pyrrolysyl-tRNA pairs.
  • the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, and M. mazei.
  • the NTT is PtNTT1 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei.
  • the NTT is PtNTT2 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei.
  • the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei.
  • the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, E.
  • the NTT is PtNTT4 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei.
  • the NTT is PtNTT5 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei.
  • the NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei.
  • the IL-2 conjugates disclosed herein may be prepared in a cell, such as E.
  • nucleotide triphosphate transporter PtNTT2 including a truncated variant in which the first 65 amino acid residues of the full-length protein are deleted
  • a plasmid comprising a double-stranded oligonucleotide that encodes an IL-2 variant having a desired amino acid sequence and that contains a unnatural base pair comprising a first unnatural nucleotide and a second unnatural nucleotide to provide a codon at the desired position at which an unnatural amino acid, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), will be incorporated, (c) a plasmid encoding a tRNA derived from M.
  • PtNTT2 including a truncated variant in which the first 65 amino acid residues of the full-length protein are deleted
  • a plasmid comprising a double-stranded oligonucleotide that encodes an IL
  • the cell is further supplemented with Attorney Docket No. 01183-0277-00PCT-SYN deoxyribo triphosphates comprising one or more unnatural bases.
  • the cell is further supplemented with ribo triphosphates comprising one or more unnatural bases.
  • the cell is further supplemented with one or more unnatural amino acids, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK).
  • the double- stranded oligonucleotide that encodes the amino acid sequence of the desired IL-2 variant contains a codon AXC at position 64 of the sequence that encodes the protein having SEQ ID NO: 1, wherein X is an unnatural nucleotide.
  • the cell further comprises a plasmid, which may be the protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene from M.
  • Y is an unnatural nucleotide that is complementary and may be the same or different as the unnatural nucleotide in the codon.
  • the unnatural nucleotide in the codon is different than and complimentary to the unnatural nucleotide in the anti-codon.
  • the unnatural nucleotide in the codon is the same as the unnatural nucleotide in the anti-codon.
  • the first and second unnatural nucleotides comprising the unnatural base pair in the double-stranded oligonucleotide the stranded oligonucleotide may be derived from some embodiments, the triphosphates of the Attorney Docket No. 01183-0277-00PCT-SYN CH 3 salts thereof.
  • the mRNA a first unnatural nucleotide and a second unnatural nucleotide may comprise a codon comprising an unnatural nucleotide derived from some embodiments, the that recognizes the codon comprising the unnatural nucleotide of the mRNA.
  • 01183-0277-00PCT-SYN tRNA may comprise an unnatural nucleotide derived from , nucleotide derived from some embodiments, the mRNA comprises an unnatural nucleotide derived from some embodiments, the mRNA Attorney Docket No. 01183-0277-00PCT-SYN comprises an unnatural nucleotide derived from the mRNA comprises an unnatural nucleotide derived from embodiments, the mRNA comprises an unnatural nucleotide derived from . In some embodiments, the tRNA comprises an unnatural nucleotide nucleotide derived from some embodiments, the tRNA comprises an Attorney Docket No.
  • the tRNA comprises an unnatural nucleotide derived from the tRNA comprises an unnatural nucleotide derived from embodiments
  • the mRNA comprises an unnatural derived from the tRNA comprises an unnatural nucleotide derived from Attorney Docket No.01183-0277-00PCT-SYN .
  • the mRNA comprises an unnatural nucleotide derived from the tRNA comprises an unnatural nucleotide derived from some embodiments, the mRNA comprises an unnatural nucleotide derived from the tRNA comprises an unnatural nucleotide derived from .
  • the host cell is cultured in a medium containing appropriate nutrients, (a) the triphosphates of the deoxyribo nucleosides comprising one or more unnatural bases that are necessary for replication of the plasmid(s) encoding the cytokine gene harboring the codon, (b) the triphosphates of the ribo nucleosides comprising one or more unnatural bases necessary for transcription of (i) the mRNA corresponding to the coding sequence of the cytokine and containing the codon comprising one or more unnatural bases, and (ii) the tRNA containing the anticodon comprising one or more unnatural bases, and (c) the unnatural amino acid(s) to be incorporated in to the polypeptide sequence of the cytokine of Attorney Docket No.01183-0277-00PCT-SYN interest.
  • the host cells are then maintained under conditions which permit expression of the protein of interest.
  • the resulting AzK-containing protein that is expressed may be purified by methods known to those of ordinary skill in the art and may then be allowed to react with an alkyne, such as DBCO comprising a PEG chain having a desired average molecular weight as disclosed herein, under conditions known to those of ordinary skill in the art, to afford the IL-2 conjugates disclosed herein.
  • an alkyne such as DBCO comprising a PEG chain having a desired average molecular weight as disclosed herein
  • the resulting protein comprising the one or more unnatural amino acids, Azk for example, that is expressed may be purified by methods known to those of ordinary skill in the art and may then be allowed to react with an alkyne, such as DBCO comprising a PEG chain having a desired average molecular weight as disclosed herein, under conditions known to those of ordinary skill in the art, to afford the IL-2 conjugates disclosed herein.
  • an alkyne such as DBCO comprising a PEG chain having a desired average molecular weight as disclosed herein
  • an IL-2 polypeptide comprising an unnatural amino acid(s) is prepared by introducing the nucleic acid constructs described herein comprising the tRNA and aminoacyl tRNA synthetase and comprising a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons into a host cell.
  • the host cell is cultured in a medium containing appropriate nutrients, is supplemented with (a) the triphosphates of the deoxyribo nucleosides comprising one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene harboring the new codon and anticodon, (b) the triphosphates of the ribo nucleosides required for transcription of the mRNA corresponding to (i) the cytokine sequence containing the codon, and (ii) the orthogonal tRNA containing the anticodon, and (c) the unnatural amino acid(s).
  • the host cells are then maintained under conditions which permit expression of the protein of interest.
  • the unnatural amino acid(s) is incorporated into the polypeptide chain in response to the unnatural codon.
  • one or more unnatural amino acids are incorporated into the IL-2 polypeptide.
  • two or more unnatural amino acids may be incorporated into the IL-2 polypeptide at two or more sites in the protein.
  • Attorney Docket No.01183-0277-00PCT-SYN [0221]
  • Suitable host cells may include bacterial cells (e.g., E. coli, BL21(DE3)), but most suitably host cells are eukaryotic cells, for example insect cells (e.g., Drosophila such as Drosophila melanogaster), yeast cells, nematodes (e.g., C.
  • mice e.g., Mus musculus
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells
  • suitable host cells are known to those skilled in the art.
  • the host cell is a mammalian cell - such as a human cell or an insect cell.
  • the suitable host cells comprise E. coli.
  • Other suitable host cells which may be used generally in the embodiments of the invention are those mentioned in the examples section.
  • Vector DNA can be introduced into host cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of well-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art. [0224] When creating cell lines, it is generally preferred that stable cell lines are prepared. For stable transfection of mammalian cells for example, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome.
  • a gene that encodes a selectable marker is generally introduced into the host cells along with the gene of interest.
  • Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, or methotrexate.
  • Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (for example, cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • the constructs described herein are integrated into the genome of the host cell.
  • an advantage of stable integration is that the uniformity between individual cells Attorney Docket No.01183-0277-00PCT-SYN or clones is achieved. Another advantage is that selection of the best producers may be carried out. Accordingly, it is desirable to create stable cell lines.
  • the constructs described herein are transfected into a host cell. An advantage of transfecting the constructs into the host cell is that protein yields may be maximized.
  • a cell comprising the nucleic acid construct or the vector described herein.
  • a subject in need thereof e.g., a subject having cancer
  • methods of stimulating and/or expanding certain immune cells comprise administering to the subject an IL-2 conjugate as described herein at a first dose (a) about once every week for a first plurality of weeks, followed by a second dose (b) about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a second plurality of weeks as described herein.
  • such a cancer is a solid tumor. In some embodiments, such a cancer is a hematologic cancer.
  • a method of treating a cancer in a subject in need thereof comprising administering to the subject an IL-2 conjugate as described herein at a first dose (a) about once every week for a first plurality of weeks, followed by a second dose (b) about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a second plurality of weeks as described herein.
  • the first dose and/or the second dose are the same. In some embodiments of various aspects described herein, the first dose and/or the second dose are different. In some embodiments of various aspects described herein, the second dose is lower than the first dose. In some embodiments of various aspects described herein, the second dose is higher than the first dose. [0229] In some embodiments of various aspects described herein, the first dose and/or the second dose is about 8 ⁇ g/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments of various aspects described herein, the first dose and/or the second dose is about 16 ⁇ g/kg IL-2 as an IL-2 conjugate as described herein.
  • the first dose and/or the second dose is about 24 ⁇ g/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments of various aspects described herein, the first dose and/or the second dose is about 32 ⁇ g/kg IL-2 as an IL-2 conjugate as described herein.
  • Cancer Types [0230] In some embodiments, the cancer is a solid tumor as described herein. In some embodiments, the cancer is a hematologic cancer, including, e.g., but not limited to leukemia, lymphoma, and multiple myeloma.
  • the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (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, esophageal squamous cell carcinoma (ESCC), glioblastoma, mesothelioma, breast cancer, triple- negative breast cancer, prostate cancer, castrate-resistant prostate cancer, metastatic castrate- resistant prostate cancer, or metastatic castrate-resistant prostate cancer having DNA damage response (DDR) defects, bladder cancer, ovarian cancer, tumors of moderate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), low- to non-expressing PD
  • RCC
  • the cancer is a solid tumor. In one aspect, the cancer is melanoma. In one aspect, the cancer is renal cell carcinoma (RCC). In one aspect, the cancer is a carcinoma such as an ovarian, colorectal, pancreatic, or hepatocellular carcinoma.
  • RCC renal cell carcinoma
  • the cancer is a carcinoma such as an ovarian, colorectal, pancreatic, or hepatocellular carcinoma.
  • provided herein is a method of treating a cancer (e.g., solid tumor or a hematologic cancer) in a subject in need thereof, comprising administering to the subject an IL-2 conjugate as described herein.
  • the method of treating the cancer (e.g., solid tumor or hematologic cancer) in a subject in need thereof comprises administering to the subject about 8 ⁇ g/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating the cancer (e.g., solid tumor or hematologic cancer) in a subject in need thereof comprises administering to the subject about 16 ⁇ g/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating the cancer (e.g., solid tumor or hematologic cancer) in a subject in need thereof comprises administering to the subject about 24 ⁇ g/kg IL-2 as an IL-2 conjugate as described herein.
  • an IL-2 conjugate for the manufacture of a medicament for a method disclosed herein of treating cancer, including a solid tumor, for example, melanoma or renal cell carcinoma (RCC), in a subject in need thereof.
  • the IL-2 conjugate is administered as at least a second or subsequent line of therapy. In some embodiments, the IL-2 conjugate is administered as at least a third or subsequent line of therapy. In some embodiments, the IL-2 conjugate is administered as at least a fourth or subsequent line of therapy. In some embodiments, the IL-2 conjugate is administered as at least a fifth or subsequent line of therapy.
  • the prior line of therapy comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a LAG-3 inhibitor.
  • the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration.
  • the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, or intramuscular administration.
  • the IL-2 conjugate is administered to the subject by intravenous administration.
  • the IL-2 conjugate is administered to the subject by subcutaneous administration.
  • the IL-2 conjugate is administered to the subject by intramuscular administration.
  • the IL-2 conjugate is administered to the subject by intravenous administration.
  • the IL-2 conjugate may be administered more than once, e.g., twice, three times, four times, five times, or more.
  • the duration of the treatment is up to 24 months, such as 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months or 24 months. In some embodiments, the duration of treatment is further extended by up to another 24 months.
  • the IL-2 conjugate is administered to a subject in need thereof about once every week, about once every two weeks, about once every three weeks, or about once every 4 weeks.
  • the IL-2 conjugate is administered to a subject in need thereof once every week. In some embodiments, the IL-2 conjugate is administered to a Attorney Docket No.01183-0277-00PCT-SYN subject in need thereof once every two weeks. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once every three weeks. In some embodiments, the IL- 2 conjugate is administered to a subject in need thereof once every 4 weeks. In some embodiments, the IL-2 conjugate is administered about once every 14, 15, 16, 17, 18, 19, 20, or 21 days. [0240] Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the IL-2 conjugate is administered to a subject in need thereof about once every week for a first plurality of weeks (induction period) followed by about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a second plurality of weeks (maintenance period).
  • the plurality of weeks of the induction period is about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks.
  • the plurality of weeks of the maintenance period is about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 16 weeks, about 18 weeks, about 20 weeks, about 30 weeks, about 40 weeks, about 50 weeks, about 60 weeks, about 70 weeks, about 80 weeks, about 90 weeks, or about 100 weeks.
  • the plurality of weeks of the maintenance period ranges from about 4 weeks to about 100 weeks, or from about 6 weeks to about 100 weeks, or from about 6 weeks to about 98 weeks, or from about 6 weeks to about 46 weeks.
  • the plurality of weeks of the maintenance period 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.
  • the IL-2 conjugate is administered to a subject in need thereof about once every week for about six weeks followed by about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a plurality of weeks (maintenance period).
  • the plurality of weeks for the maintenance period is about 6 weeks, about 12 weeks, 18 weeks, 24 Attorney Docket No.01183-0277-00PCT-SYN weeks, about 30 weeks, about 36 weeks, about 42 weeks, about 48 weeks, about 54 weeks, about 60 weeks or longer.
  • the IL-2 conjugate is administered to a subject in need thereof at a dose of about 8 ⁇ g/kg IL-2 as the IL-2 conjugate, about 16 ⁇ g/kg IL-2 as the IL-2 conjugate, about 24 ⁇ g/kg IL-2 as the IL-2 conjugate, about 32 ⁇ g/kg IL-2 as the IL-2 conjugate at a frequency of about once every week for the first plurality of weeks.
  • the IL-2 conjugate is administered to a subject in need thereof at a dose of about 8 ⁇ g/kg IL-2 as the IL-2 conjugate, about 16 ⁇ g/kg IL-2 as the IL-2 conjugate, about 24 ⁇ g/kg IL-2 as the IL-2 conjugate, about 32 ⁇ g/kg IL-2 as the IL-2 conjugate at a frequency of about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks.
  • the IL-2 conjugate is administered to a subject in need thereof on days 1, 8, 15, 22, 29, and 36 ⁇ 1 day of a first 6-week cycle, followed by administration on days 1, 15, and 29 ⁇ 1 day of each subsequent 6-week cycle.
  • 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.
  • the desired doses are conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.
  • a method described herein further comprises administering one or more additional therapeutic agents.
  • the additional therapeutic agent comprises one or more chemotherapeutic agent.
  • the chemotherapeutic agent comprises pemetrexed.
  • the chemotherapeutic agent comprises a platinum agent, such as carboplatin.
  • the chemotherapeutic agent comprises cisplatin.
  • the chemotherapeutic agent comprises nab- paclitaxel.
  • the chemotherapeutic agent comprises pemetrexed and a platinum agent, such as carboplatin.
  • the chemotherapeutic agent comprises pemetrexed and cisplatin.
  • the additional therapeutic agent comprises one or more immunotherapeutics.
  • the additional therapeutic agent comprises an antihistamine, such as diphenhydramine. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent and an antihistamine, such as diphenhydramine. In some embodiments, the additional therapeutic agent comprises any one of the foregoing chemotherapeutic agents and an antihistamine, such as diphenhydramine. Attorney Docket No.01183-0277-00PCT-SYN [0247] In some embodiments, the additional therapeutic agent comprises an analgesic, such as acetaminophen. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent and an analgesic, such as acetaminophen.
  • the additional therapeutic agent comprises any one of the foregoing chemotherapeutic agents and an analgesic, such as acetaminophen.
  • the additional therapeutic agent comprises one or more vitamins, such as folic acid and/or vitamin B12.
  • the additional therapeutic agent comprises a chemotherapeutic agent and one or more vitamins, such as folic acid and/or vitamin B12.
  • the additional therapeutic agent comprises any one of the foregoing chemotherapeutic agents and one or more vitamins, such as folic acid and/or vitamin B12.
  • the additional therapeutic agent comprises an antihistamine and an analgesic, such as diphenhydramine and acetaminophen.
  • the additional therapeutic agent comprises an antihistamine and one or more vitamins, such as 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, such as 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, such as diphenhydramine, acetaminophen, and one or both of folic acid and vitamin B12. In any of the foregoing embodiments, the additional therapeutic agent can further comprise a chemotherapeutic agent, such as any one of the foregoing chemotherapeutic agents.
  • administration of the IL-2 conjugate is to an adult subject.
  • the adult subject is a male.
  • the adult subject is a female.
  • the adult subject is at least age 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age.
  • the adult subject has relapsed or refractory cancer, such as a relapsed or refractory solid tumor or a relapsed or refractory hematologic cancer.
  • the subject has relapsed or refractory melanoma or relapsed or refractory renal cell carcinoma (RCC).
  • RRCC renal cell carcinoma
  • the subject has refractory melanoma. In some embodiments, the subject has relapsed melanoma. In some embodiments, the subject has an advanced solid tumor. In some embodiments, the subject has a metastatic solid tumor. In some embodiments, the metastatic solid tumor is metastatic melanoma or metastatic renal cell carcinoma (RCC). Attorney Docket No.01183-0277-00PCT-SYN [0251] In some embodiments, the subject is ⁇ 18 years of age. In some embodiments, the subject has received prior anti-cancer therapy (eg, radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy treatment.
  • prior anti-cancer therapy eg, radiotherapy, chemotherapy, surgery, targeted therapy
  • the subject has received at least one prior line of therapy for metastatic melanoma and/or does not have any standard of care (SoC) treatment option or participant declines or is intolerant to be treated with SoC treatment.
  • SoC standard of care
  • the subject has advanced or metastatic solid tumors and has either refused standard of care; or for whom no reasonable standard of care exists that would confer clinical benefit; or for whom standard therapy is intolerable, not effective, or not accessible.
  • the subject has measurable disease per RECIST v1.1 and at least 1 measurable lesion.
  • the subject has adequate laboratory parameters including: (1) Absolute lymphocyte count ⁇ 0.5 times lower limit of normal, (2) Platelet count ⁇ 100 ⁇ 10 9 /L (3) Hemoglobin ⁇ 9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for erythropoiesis-stimulating agent (ESA) and colony-stimulating factor (CSF) administration is sufficient), (4) Absolute neutrophil count ⁇ 1.5 ⁇ 10 9 /L (absence of growth factors within 2 weeks), (5) Prothrombin time (PT) and partial thromboplastin time (PTT) ⁇ 1.5 times ULN, (6) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ⁇ 2.5 times ULN except if liver metastases are present may be ⁇ 5 times ULN, and (7) Total bilirubin ⁇ 1.5 ⁇ ULN.
  • ESA erythropoiesis-stimulating agent
  • CSF colony-sti
  • the subject meets each of the foregoing criteria.
  • the subject did not receive radiotherapy ⁇ 14 days prior to first dose of IL-2 conjugate, or palliative radiation or stereotactic radiosurgery within 7 days prior to first dose of IL-2 conjugate.
  • the subject did not receive systemic anti- cancer therapy or an investigational agent within 2 weeks prior to first dose of IL-2 conjugate, or within 4 weeks for immunotherapy and tyrosine kinase inhibitor therapy.
  • the subject did not experience a Grade 3 or higher immune-related toxicity from prior immuno- oncology therapy.
  • the subject did not have major surgery ⁇ 30 days prior to first dose of IL-2 conjugate, or did not recover to at least Grade 1 from adverse effects from Attorney Docket No.01183-0277-00PCT-SYN such procedure, or anticipates the need for major surgery during treatment with the IL-2 conjugate.
  • the subject does not have active autoimmune disease requiring systemic treatment within the past 3 months or does not have a documented history of clinically severe autoimmune disease that requires systemic steroids or immunosuppressive agents; or did not receive corticosteroids at a dose >10 mg daily of prednisone or equivalent (except inhaled, intranasal, intraocular, and topical steroids, or steroids as prophylaxis against contrast reactions, for disease assessments).
  • the subject does not have primary central nervous system (CNS) disease or leptomeningeal disease, known CNS metastases unless the subject has been treated, is asymptomatic, is without evidence of radiological progression for at least 8 weeks and has had no requirement for steroids or enzyme inducing anticonvulsants in the last 14 days prior to screening; abnormal pulmonary function within the previous 6 months, including pneumonitis, active pneumonitis, interstitial lung disease requiring the use of steroids, idiopathic pulmonary fibrosis, confirmed pleural effusion, severe dyspnea at rest or requiring supplementary oxygen therapy.
  • the subject does not have a history of allogenic or solid organ transplant.
  • the subject does not have uncontrolled diabetes mellitus or other uncontrolled immune-related endocrinopathies in the opinion of a physician; In some embodiments, the subject did not receive parenteral antibiotics ⁇ 14 days prior to first dose of IL-2 conjugate, and does not have any serious systemic fungal, bacterial, viral, or other infection that is not controlled or requires IV antibiotics.
  • the subject does not have known human immunodeficiency virus (HIV) infection or active infection with hepatitis C, or a known uncontrolled hepatitis B virus (HBV) infection (unless the subject has received anti-HBV therapy before initiation of first dose of IL-2 conjugate and the subject has an HBV viral load ⁇ 2000 IU/mL (104 copies/mL), or the subject has positive anti-HBc, positive anti HBs, negative HBsAg, and HBV virus load without HBV therapy).
  • the subject did not receive a live or live attenuated virus vaccination (except seasonal flu vaccines or SARS-CoV-2 vaccines that do not contain live virus) ⁇ 14 days of treatment.
  • the subject did not experience clinically significant bleeding within 2 weeks prior to the initial IL-2 conjugate dose (eg, gastrointestinal bleeding, intracranial hemorrhage). In some embodiments, the subject did not have a prior diagnosis of deep vein thrombosis or pulmonary embolism within 3 months of screening.
  • the subject did not have a severe or unstable cardiac condition within 6 months prior to first dose of IL-2 conjugate, such as congestive heart failure (New York Heart Association Class III or IV), cardiac bypass surgery or coronary artery stent placement, angioplasty, cardiac ejection fraction below the lower limit of normal, unstable angina, medically uncontrolled hypertension (eg, ⁇ 160 mm Hg systolic or ⁇ 100 mm Hg diastolic), Attorney Docket No.01183-0277-00PCT-SYN uncontrolled cardiac arrhythmia requiring medication ( ⁇ Grade 2, according to NCI CTCAE v5.0), or myocardial infarction.
  • congestive heart failure New York Heart Association Class III or IV
  • cardiac bypass surgery or coronary artery stent placement angioplasty
  • cardiac ejection fraction below the lower limit of normal, unstable angina
  • medically uncontrolled hypertension eg, ⁇ 160 mm Hg systolic or ⁇ 100 mm Hg diasto
  • the subject did not have a history of non- pharmacologically induced prolonged corrected QT interval determined using Fridericia's formula (QTcF) (>450 milliseconds (msec) for a male subject or >470 msec for a female subject.
  • QTcF Fridericia's formula
  • the subject did not have known hypersensitivity or contraindications to any components of the IL-2 conjugate, PEG, pegylated drugs and E. coli derived-protein.
  • the subject did not have an active second malignancy, or history of previous malignancy that would impact the assessment of any study endpoints (except subjects with non-melanomatous skin cancer or cervical cancer that has been curatively surgically resected).
  • the subject did not have a serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concurrent medical illness that in the opinion of a physician would preclude protocol therapy or would make the subject inappropriate for the study, for example subjects with active SARS-CoV-2 (COVID-19) infections with clinically significant symptoms.
  • the subject is not pregnant or breast- feeding, or expecting to conceive or father children within the projected duration of the trial, starting with the screening visit through for at least 7 days for female subjects, and for at least 3 days for male subjects after the last dose of IL-2 conjugate.
  • the subject did not receive concurrent therapy with any other investigational agent, vaccine, or device (except concomitant participation in observational studies). In some embodiments, the subject did not have baseline oxygen saturation of ⁇ 92%. In some embodiments, the subject does not have uveal or ocular or desmoplastic metastatic melanoma. In some embodiments, the subject does not have any of the characteristics listed in this paragraph. In some embodiments, the subject does not have any of the characteristics listed in this paragraph and meets each of the criteria in the preceding paragraph.
  • the subject is a female of childbearing potential and has (1) agreed to use a medically accepted method of birth control during the treatment and for at least 7 days after the last dose of the IL-2 conjugate, and (2) has a negative serum pregnancy test within 7 days prior to first dose of IL-2 conjugate.
  • the subject is a male who is not surgically sterile, and (1) has agreed to use medically accepted method of birth control during the treatment and for at least 3 days after the last dose of the IL-2 conjugate, and (2) has agreed to refrain from donating or banking sperm during the treatment period and for at least 3 days after last dose of the IL-2 conjugate.
  • the subject has no known hypersensitivity or contraindications to the IL-2 conjugate being administered or to PEG. In some embodiments, the subject has no Attorney Docket No.01183-0277-00PCT-SYN known hypersensitivity or contraindications to the IL-2 conjugate being administered, PEG, or pegylated drugs. [0255] In some embodiments, the subject does not have any serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concurrent medical illness that would preclude treatment or would make treatment inappropriate. [0256] In some embodiments, the subject is not pregnant or breastfeeding.
  • the subject is not expecting to conceive or father children during the course of the treatment and following up to 1, 2, 3, 4, 5, 6, or 7 months after administration of the final treatment dose.
  • the subject is not receiving a concurrent therapy with any investigational agent, vaccine, or device during the course of treatment.
  • the subject is receiving concurrent therapy during the course of treatment with an IL-2 conjugate described herein.
  • the subject is receiving concurrent therapy with an investigational agent, vaccine, or device during the course of treatment with an IL-2 conjugate described herein after physician approval.
  • administration of the IL-2 conjugate provides a complete response, a partial response, or stable disease.
  • the subject experiences a complete response (CR) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1.
  • the subject experiences a partial response (PR) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1
  • the subject experiences stable disease (SD) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1.
  • administration of the IL-2 conjugate to the subject does not cause vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2, Grade 3, or Grade 4 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 3 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade Attorney Docket No.01183-0277-00PCT-SYN 4 vascular leak syndrome in the subject.
  • administration of the IL-2 conjugate to the subject does not cause loss of vascular tone in the subject. [0261] In some embodiments, administration of the IL-2 conjugate to the subject does not cause extravasation of plasma proteins and fluid into the extravascular space in the subject. [0262] In some embodiments, administration of the IL-2 conjugate to the subject does not cause hypotension and reduced organ perfusion in the subject. [0263] In some embodiments, administration of the IL-2 conjugate to the subject does not cause impaired neutrophil function in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause reduced chemotaxis in the subject.
  • administration of the IL-2 conjugate to the subject is not associated with an increased risk of disseminated infection in the subject.
  • the disseminated infection is sepsis or bacterial endocarditis.
  • the disseminated infection is sepsis.
  • the disseminated infection is bacterial endocarditis.
  • the subject is treated for any preexisting bacterial infections prior to administration of the IL-2 conjugate.
  • the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate.
  • administration of the IL-2 conjugate to the subject does not exacerbate a pre-existing or initial presentation of an autoimmune disease or an inflammatory disorder in the subject. In some embodiments, the administration of the IL-2 conjugate to the subject does not exacerbate a pre-existing or initial presentation of an autoimmune disease in the subject. In some embodiments, the administration of the IL-2 conjugate to the subject does not exacerbate a pre-existing or initial presentation of an inflammatory disorder in the subject.
  • the autoimmune disease or inflammatory disorder in the subject is selected from Crohn’s disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, oculo- bulbar myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome and bullous pemphigoid.
  • the autoimmune disease or inflammatory disorder in the subject is Crohn’s disease.
  • the autoimmune disease or inflammatory disorder in the subject is scleroderma.
  • the autoimmune disease or inflammatory disorder in the subject is thyroiditis.
  • 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 oculo-bulbar myasthenia gravis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is crescentic IgA Attorney Docket No.01183-0277-00PCT-SYN glomerulonephritis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cholecystitis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cerebral vasculitis.
  • 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. [0266] In some embodiments, administration of the IL-2 conjugate to the subject does not cause changes in mental status, speech difficulties, cortical blindness, limb or gait ataxia, hallucinations, agitation, obtundation, or coma in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause seizures in the subject. In some embodiments, administration of the IL-2 conjugate to the subject is not contraindicated in subjects having a known seizure disorder.
  • administration of the IL-2 conjugate to the subject does not cause capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2, Grade 3, or Grade 4 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 3 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 4 capillary leak syndrome in the subject.
  • administration of the IL-2 conjugate to the subject does not cause a drop in mean arterial blood pressure in the subject following administration. In some embodiments, administration of the IL-2 conjugate to the subject does cause hypotension in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause the subject to experience a systolic blood pressure below 90 mm Hg or a 20 mm Hg drop from baseline systolic pressure. [0269] In some embodiments, administration of the IL-2 conjugate to the subject does not cause edema or impairment of kidney or liver function in the subject. [0270] In some embodiments, administration of the IL-2 conjugate to the subject does not cause eosinophilia in the subject.
  • administration of the IL-2 conjugate to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 500 per ⁇ L. In some embodiments, administration of the IL-2 conjugate to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 500 ⁇ L to 1,500 per ⁇ L. In some embodiments, administration of the IL-2 conjugate to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 1,500 per ⁇ L to 5,000 per ⁇ L.
  • administration of the IL-2 conjugate to the subject does not cause the Attorney Docket No.01183-0277-00PCT-SYN eosinophil count in the peripheral blood of the subject to exceed 5,000 per ⁇ L.
  • administration of the IL-2 conjugate to the subject is not contraindicated in subjects on an existing regimen of psychotropic drugs. [0271] In some embodiments, administration of the IL-2 conjugate to the subject is not contraindicated in subjects on an existing regimen of nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs.
  • administration of the IL-2 conjugate to the subject is not contraindicated in subjects on an existing regimen of aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase. In some embodiments, administration of the IL-2 conjugate to the subject 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. [0272] In some embodiments, administration of the IL-2 conjugate to the subject does not cause one or more Grade 4 adverse events in the subject following administration.
  • Grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystoles; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; AV block second degree; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function tests abnormal; gastrointestinal hemorrhage; hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; alkaline phosphatase increase; blood urea nitrogen (BUN) increase; hyperuricemia; non-protein nitrogen (NPN) increase; respiratory acidosis; somnolence; agitation; neuropathy; paranoid reaction; convulsion; grand mal convulsion; delirium; asthma, lung edema; hypervent
  • Grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystoles; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; AV block second degree; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function tests abnormal; gastrointestinal hemorrhage; hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; alkaline phosphatase increase; blood urea nitrogen (BUN) increase; hyperuricemia; non-protein nitrogen (NPN) increase; respiratory acidosis; somn
  • administration of the IL-2 conjugate to a group of subjects does not cause one or more adverse events in greater than 1% of the subjects following administration, wherein the one or more adverse events is selected from duodenal ulceration; bowel necrosis; myocarditis; supraventricular tachycardia; permanent or transient blindness secondary to optic neuritis; transient ischemic attacks; meningitis; cerebral edema; pericarditis; allergic interstitial nephritis; and tracheo-esophageal fistula.
  • administration of the IL-2 conjugate to a group of subjects does not cause one or more adverse events in greater than 1% of the subjects following administration, wherein the one or more adverse events is selected from malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary emboli; stroke; intestinal perforation; liver or renal failure; severe depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure.
  • administration of the IL-2 conjugate to the subject stimulates CD8+ cells in a subject.
  • administration of the IL-2 conjugate to the subject stimulates NK cells in a subject.
  • Stimulation may comprise an increase in the number of CD8+ cells in the subject, e.g., about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration.
  • the CD8+ cells comprise memory CD8+ cells.
  • the CD8+ cells comprise effector CD8+ cells.
  • Stimulation may comprise an increase in the proportion of CD8+ cells that are Ki67 positive in the subject, e.g., about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration.
  • Stimulation may comprise an increase in the number of NK cells in the subject, e.g., about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration.
  • CD8+ cells are expanded in the subject following administration of the IL-2 conjugate by at least 1.5-fold, such as by at least 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, or 2.5-fold.
  • NK cells are expanded in the subject following administration of the IL-2 conjugate by at least 3.5-fold, such as by at least 4.0-fold, 4.5-fold, 5.5-fold, 6-fold, 6.5-fold, 7.0-fold, or 7.5-fold.
  • eosinophils are expanded in the subject following administration of the IL-2 conjugate by no more than about 2-fold, such as no more than about 1.5-fold, 1.4-fold, or 1.3-fold.
  • CD4+ cells are expanded in the subject following administration of the IL-2 conjugate by no more than about 2-fold, such as no more than about 1.8-fold, 1.7-fold, or 1.6-fold.
  • the expansion of CD8+ cells and/or NK cells in the subject following administration of the IL-2 conjugate is greater than the Attorney Docket No.01183-0277-00PCT-SYN expansion of CD4+ cells and/or eosinophils.
  • the expansion of CD8+ cells is greater than the expansion of CD4+ cells. In some embodiments, the expansion of NK cells is greater than the expansion of CD4+ cells. In some embodiments, the expansion of CD8+ cells is greater than the expansion of eosinophils. In some embodiments, the expansion of NK cells is greater than the expansion of eosinophils. Fold expansion is determined relative to a baseline value measured before administration of the IL-2 conjugate. In some embodiments, fold expansion is determined at any of the times after administration, such as about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration.
  • administration of the IL-2 conjugate to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral CD4+ regulatory T cells in the subject. In some embodiments, administration of the IL-2 conjugate to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject. In some embodiments, administration of the IL-2 conjugate to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of intratumoral CD8+ T and NK cells in the subject and without increasing the number of intratumoral CD4+ regulatory T cells in the subject.
  • administration of the IL-2 conjugate to the subject does not require the availability of an intensive care facility or skilled specialists in cardiopulmonary or intensive care medicine. In some embodiments, administration of the IL-2 conjugate to the subject does not require the availability of an intensive care facility or skilled specialists in cardiopulmonary or intensive care medicine. In some embodiments, administration of the IL-2 conjugate to the subject does not require the availability of an intensive care facility. In some embodiments, administration of the IL-2 conjugate to the subject does not require the availability of skilled specialists in cardiopulmonary or intensive care medicine. [0279] In some embodiments, administration of the IL-2 conjugate does not cause dose- limiting toxicity (DLT). In some embodiments, administration of the IL-2 conjugate causes dose-limiting toxicity (DLT).
  • DLT dose-limiting toxicity
  • a DLT is defined as an adverse event occurring within Day 1 through Day 42 (6 week-cycle) (inclusive) ⁇ 1 day of a treatment cycle that was not clearly or incontrovertibly solely related to an extraneous cause and that meets the criteria set forth in Example 4 for DLT.
  • administration of the IL-2 conjugate does not cause severe cytokine release syndrome.
  • the IL-2 conjugate does not induce anti-drug antibodies (ADAs), i.e., antibodies against the IL-2 conjugate.
  • ADAs anti-drug antibodies
  • kits and articles of manufacture for use with one or more methods and compositions described herein.
  • kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers are formed from a variety of materials such as glass or plastic.
  • a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions isalso typically be included.
  • a label is on or associated with the container.
  • a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • a label is used to indicate that the contents are to be used for a specific therapeutic application.
  • the label also indicates directions for use of the contents, such as in the methods described herein.
  • the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein.
  • the pack for example, contains metal or plastic foil, such as a blister pack.
  • the pack or dispenser device is accompanied by instructions for administration.
  • the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration.
  • a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration.
  • Such notice for example, is the labeling approved by the U.S. Food and Drug Administration for drugs, or the approved product insert.
  • compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Example 1 Preparation of pegylated IL-2 conjugates.
  • An exemplary method with details for preparing IL-2 conjugates described herein is provided in this Example.
  • IL-2 employed for bioconjugation was expressed as inclusion bodies in E.
  • an expression plasmid encoding (i) the protein with the desired amino acid sequence, which gene contains a first unnatural base pair to provide a codon at the desired position at which an unnatural amino acid N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) was incorporated and (ii) a tRNA derived from M. mazei Pyl, which gene comprises a second unnatural nucleotide to provide a matching anticodon in place of its native sequence; (b) a plasmid encoding a M.
  • barkeri derived pyrrolysyl-tRNA synthetase (Mb PylRS), (c) N6-((2- azidoethoxy)-carbonyl)-L-lysine (AzK); and (d) a truncated variant of nucleotide triphosphate transporter PtNTT2 in which the first 65 amino acid residues of the full-length protein were deleted.
  • the double-stranded oligonucleotide that encodes the amino acid sequence of the desired IL-2 variant contained a codon AXC as codon 64 of the sequence that encodes the protein having SEQ ID NO: 1 in which P64 is replaced with an unnatural amino acid described herein.
  • the plasmid encoding an orthogonal tRNA gene from M. mazei comprised an AXC- matching anticodon GYT in place of its native sequence, wherein Y is an unnatural nucleotide as disclosed herein.
  • Y is an unnatural nucleotide as disclosed herein.
  • X and Y were selected from unnatural nucleotides dTPT3 and dNaM as disclosed herein.
  • the expressed protein was extracted from inclusion bodies and re-folded using standard procedures before site-specifically pegylating the AzK-containing IL-2 product using DBCO-mediated copper-free click chemistry to attach stable, covalent mPEG moieties to the AzK.
  • Examplary reactions are shown in Schemes 1 and 2 (wherein n indicates the number of repeating PEG units).
  • the IL-2 conjugate comprised SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate can also be described as an IL-2 conjugate comprising Attorney Docket No.01183-0277-00PCT-SYN SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • the IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin.
  • pINN International Nonproprietary Name
  • the IL-2 conjugate comprised SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • the IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin.
  • the IL-2 conjugate was administered via IV infusion at a dose of 8, 16, 24, or 32 ⁇ g/kg (e.g., for about 30 minutes) every 2 weeks [Q2W], or was administered via IV infusion at a dose of 8, 16, 24, 32, or 40 ⁇ g/kg (e.g., for 30 minutes) every 3 weeks [Q3W].
  • Eosinophilia (elevated peripheral eosinophil count): Cell surrogate marker for IL-2-induced proliferation of cells (eosinophils) linked to vascular leak syndrome (VLS); Interleukin 5 (IL-5): Cytokine surrogate marker for IL-2 induced activation of type 2 innate lymphoid cells and release of this chemoattractant that leads to eosinophilia and potentially VLS; Interleukin 6 (IL-6): Cytokine surrogate marker for IL-2 induced cytokine release syndrome (CRS); and Interferon ⁇ (IFN- ⁇ ): Cytokine surrogate marker for IL-2 induced activation of CD8+ cytotoxic T lymphocytes.
  • Peripheral CD8+ Effector Cells Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing a potentially latent therapeutic response
  • Peripheral CD8+ Memory Cells Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing a potentially durable latent therapeutic and maintenance of the memory population
  • Peripheral NK Cells Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing a potentially rapid therapeutic response
  • Peripheral CD4+ Regulatory Cells Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing an immunosuppressive TME and offsetting of an effector-based
  • Subjects were human males or females aged ⁇ 18 years at screening. All subjects had been previously treated with an anti-cancer therapy and met at least one of the following: Treatment related toxicity resolved to grade 0 or 1 (alopecia excepted) according to NCI CTCAE v5.0; or Treatment related toxicity resolved to at least grade 2 according to NCI CTCAE v5.0 with prior approval of the Medical Monitor. The most common tumors were colorectal or melanoma. [0294] Subjects also met the following criteria: Provided informed consent. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Life expectancy greater than or equal to 12 weeks as determined by the Investigator. Histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors.
  • ECOG Eastern Cooperative Oncology Group
  • Adequate laboratory parameters including: Absolute lymphocyte count > 0.5 times lower limit of normal; Platelet count ⁇ 100 ⁇ 10 9 /L; Hemoglobin ⁇ 9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for ESA and CSF administration is sufficient); Absolute neutrophil count ⁇ 1.5 ⁇ 10 9 /L (absence of growth factors within 2 weeks); Prothrombin time (PT) and partial thromboplastin time (PTT) ⁇ 1.5 times upper limit of normal (ULN); Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ⁇ 2.5 times ULN except if liver metastases are present may be ⁇ 5 times ULN; Total bilirubin ⁇ 1.5 ⁇ ULN.
  • Tumor types included colorectal, pancreactic, and sarcoma.
  • drug mass per kg subject e.g., 8 ⁇ g/kg refers to IL-2 mass exclusive of PEG and linker mass.
  • Treatment duration ranged from 1.4-9.0 months (2.0 months, median), and subjects received from 4-20 total doses (5.0 doses, median).
  • Three of the subjects experienced at least one TEAE, all of which were Grade 1 or 2. No drug discontinuations resulted from TEAE, and there were no dose-limiting toxicities.
  • TEAEs are detailed in Table 1.
  • Peripheral lymphocyte cell counts are shown in FIG.5, and peripheral eosinophil cell counts are shown in FIG.6.
  • Mean concentrations of the IL-2 conjugate after 1 and 2 cycles are shown in FIG.7A and FIG.7B, respectively.
  • Cytokine levels are shown in FIG.8.
  • the IL-2 conjugate was generally well-tolerated. Overall, the results are considered to support non-alpha preferential activity of the IL-2 conjugate, with a tolerable safety profile, and preliminary evidence of activity in patients with immune-sensitive tumors.
  • e CD8+ expansion was about 2-fold, similar to the observed expansion of the first [Q2W] cohort (8 ⁇ g/kg dose).
  • Peripheral NK cell counts are shown in FIG.10. The NK cell expansion was about 1- to 20-fold higher than the first [Q2W] cohort (8 ⁇ g/kg dose).
  • Peripheral CD4+ Treg cell counts are shown in FIG.11.
  • Peripheral eosinophil cell counts are shown in FIG.12.
  • the CD4+ Attorney Docket No.01183-0277-00PCT-SYN T reg and eosinophil cell expansions were similar to the expansion of the first [Q2W] cohort (8 ⁇ g/kg dose).
  • Cytokine levels (IFN- ⁇ , IL-6, and IL-5) are shown in FIG.13.
  • Mean concentrations of the IL-2 conjugate after 1 and 2 cycles are shown in FIG.14A and FIG.14B, respectively.
  • the IL-2 conjugate demonstrated encouraging PD data and was generally well-tolerated. Overall, the results are considered to support non-alpha preferential activity of the IL-2 conjugate, with a tolerable safety profile, encouraging PD and preliminary evidence of activity in patients with immune-sensitive tumors.
  • the IL-2 conjugate comprises SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • the IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin.
  • the first subject enrolled into a dose level for each cohort was observed for a minimum of 72 hours before the second subject is enrolled at the same dose level.
  • Dose-limiting toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) Version 5.0. Neurologic events occurring in the context of cytokine release syndrome were evaluated using CTCAE Attorney Docket No.01183-0277-00PCT-SYN Version 5.0 supplemented with the American Society for Transplantation and Cellular Therapy (ASTCT) criteria.
  • ASTCT American Society for Transplantation and Cellular Therapy
  • a DLT is defined as any of the following events occurring during the first treatment cycle for which causal relationship to the IL-2 conjugate is determined to be at least possibly related: [0318] Hematologic toxicities • Grade 3 neutropenia (absolute neutrophil count [ANC] ⁇ 1000/mm 3 >500/mm 3 ) lasting ⁇ 7 days, or Grade 4 neutropenia of any duration • Grade 3+ febrile neutropenia • Grade 4+ thrombocytopenia (platelet count ⁇ 25000/mm 3 ) • Grade 3+ thrombocytopenia (platelet count ⁇ 50000-25000/mm 3 ) lasting ⁇ 5 days, or associated with clinically significant bleeding or requiring platelet transfusion • Failure to meet recovery criteria of an ANC of at least 1000 cells/mm 3 and a platelet count of at least 75000 cells/mm 3 within 10 days • Any other Grade 4+ hematologic toxicity lasting ⁇ 5 days [0319] Non-
  • IL-2 conjugate monotherapy QW/Q2W received the IL-2 conjugate monotherapy QW/Q2W.
  • the starting dose of the IL-2 conjugate was 16 ⁇ g/kg (DL1).
  • dosage was escalated to 24 ⁇ g/kg (DL2) and subsequently to 32 ⁇ g/kg (DL3).
  • a dose of 8 ⁇ g/kg (dose level -1) may be utilized if DL1 is determined to be unsafe.
  • drug mass per kg subject e.g., 16 ⁇ g/kg refers to IL-2 mass exclusive of PEG and linker mass.
  • the presentation of drug mass per kg subject refers to the dose based on the IL-2 protein part of an IL-2 conjugate described herein.
  • the following inclusion criteria apply. The participant must be willing and able to provide informed consent and comply with protocol requirements for the duration of the study. The participant must be ⁇ 18 years of age. Prior anti-cancer therapy is allowed (eg, radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy treatment.
  • treatment related toxicity resolved to Grade 0 or 1 (alopecia excepted) according to NCI CTCAE v5.0
  • treatment related toxicity resolved to at least Grade 2 according to NCI CTCAE v5.0 with prior approval of the Medical Monitor.
  • Participants must have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
  • Participants must have a life expectancy greater than or equal to 12 weeks as determined by the Investigator.
  • Participants must have histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors with at least one tumor lesion with location accessible to safely biopsy per clinical judgment of the Investigator.
  • On-treatment biopsy may be Attorney Docket No.01183-0277-00PCT-SYN omitted upon written request from the Investigators considering biopsy clinical feasibility and upon written approval by the Sponsor.
  • Participants must have adequate laboratory parameters including: (1) Absolute lymphocyte count ⁇ 0.5 times lower limit of normal, (2) Platelet count ⁇ 100 ⁇ 10 9 /L (3) Hemoglobin ⁇ 9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for erythropoiesis-stimulating agent (ESA) and colony-stimulating factor (CSF) administration is sufficient), (4) Absolute neutrophil count ⁇ 1.5 ⁇ 10 9 /L (absence of growth factors within 2 weeks), (5) Prothrombin time (PT) and partial thromboplastin time (PTT) ⁇ 1.5 times ULN, (6) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ⁇ 2.5 times ULN except if liver metastases are present may be ⁇ 5 times ULN, and (7) Total bilirubin ⁇ 1.5 ⁇ ULN.
  • ESA erythropoiesis-stimulating agent
  • CSF colony-sti
  • Females of childbearing potential and men who are not surgically sterile must agree to use medically accepted method of birth control during the study and for at least 7 days for females, and for at least 3 days for males after the last dose of study intervention.
  • Female participants must have a negative serum pregnancy test within 7 days prior to initiating study treatment in premenopausal women and women less than 12 months after menopause.
  • Male participants must agree to refrain from donating or banking sperm during the treatment period and for at least 3 days after last dose of study treatment.
  • Participants are excluded from the study if any of the following criteria applies: – Radiotherapy ⁇ 14 days prior to first dose of study drug (palliative radiation or stereotactic radiosurgery within 7 days prior to start of study treatment).
  • ⁇ Treated with systemic anti-cancer therapy or an investigational agent within 2 weeks prior to start of study drug treatment (within 4 weeks for immunotherapy and tyrosine kinase inhibitor therapy).
  • Participants who experienced Grade 3 or higher immune-related toxicity from prior immuno- oncology therapy.
  • Major surgery ⁇ 30 days prior to first dose of study drug, or has not recovered to at least Grade 1 from adverse effects from such procedure, or anticipation of the need for major surgery during study treatment.
  • Active autoimmune disease requiring systemic treatment within the past 3 months or have a documented history of clinically severe autoimmune disease that requires systemic steroids or immunosuppressive agents; subjects on corticosteroids are excluded for a dose >10 mg daily of prednisone or equivalent.
  • hepatitis C Known human immunodeficiency virus (HIV) infection or active infection with hepatitis C: (a) known uncontrolled hepatitis B virus (HBV) infection: (i) anti-HBV therapy started before initiation of IMP and HBV viral load ⁇ 2000 IU/mL (104 copies/mL) are eligible. The anti-HBV therapy should continue throughout the treatment period; (ii) positive anti-HBc, positive anti HBs, negative HbsAg, and HBV virus load without HBV therapy are eligible. – Received a live-virus vaccination ⁇ 14 days prior to first dose of study drug. Seasonal flu and other inactivated vaccines that do not contain live virus are permitted.
  • Any serious medical condition including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concurrent medical illness that in the opinion of the Investigator would preclude protocol therapy or would make the subject inappropriate for the study: (a) patients with active SARS-CoV-2 (COVID-19) infections with clinically significant symptoms. – Is pregnant or breast-feeding, or expecting to conceive or father children within the projected duration of the trial, starting with the screening visit through for at least 7 days for females, and for at least 3 days for males after the last dose of study intervention. — Concurrent therapy with any other investigational agent, vaccine, or device. Concomitant participation in observational studies is acceptable after Sponsor approval.
  • Safety Assessments Safety was assessed throughout the study by the following: • Aes (type, incidence, severity, timing, seriousness, and relatedness) • Vital sign measurements (blood pressure, heart rate, respiratory rate, body temperature, pulse oximetry) • Physical examinations • Clinical laboratory tests • Electrocardiogram(s) • Concomitant medications and procedures, including all supportive care provided. [0325] Adverse events were assessed until approximately 90 days after the last dose of study drug(s) to monitor for delayed immune toxicities that have been associated with immunotherapy products, or until a subsequent anti-cancer treatment is administered (whichever comes first).
  • Efficacy Assessments Anti-tumor activity was determined by repeat radiological imaging and response assessed using RECIST 1.1 and iRECIST. All subjects must have scans performed using a modality appropriate for the subject’s tumor (eg, PET-CT scan, CT scans, MRI). All anatomical areas that are imaged during Screening should be assessed at every on- study time point using the same imaging modality.
  • Study Endpoints [0328] Primary Endpoints • DLTs were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0.
  • the MTD of the IL-2 conjugate as a single agent was the highest tested dose level that has ⁇ 33% probability of causing a DLT (ie, a dose level at which 0/6 or 1/6 subjects experience first-cycle DLT and at least 2/3 or 2/6 subjects experience first-cycle DLT at the next higher dose level).
  • the RP2D of the IL-2 conjugate as a single agent was based on available safety, tolerability, PK, and Pdy (which may include, but is not limited to, Ki67 expression in lymphocytes) data from different dose levels and schedules tested, but was no higher than the MTD.
  • Clinical anti-tumor activity was assessed according to RECIST v1.1, as determined by the Investigator.
  • ORR defined as the proportion of subjects with confirmed complete response (CR) or partial response (PR); a confirmed response is a response that persists on repeat-imaging ⁇ 4 weeks after initial documentation of response.
  • DOR defined as time from date of first objective response (either CR or PR) to first documentation of radiographic disease progression or death due to any cause, whichever occurs first.
  • PFS defined as the time from first dose of the IL-2 conjugate to first documentation of radiographic disease progression or death due to any cause, whichever occurs first.
  • OS defined as the time from first dose of the IL-2 conjugate to the date of death due to any cause.
  • TTR defined as the time from first dose of the IL-2 conjugate to first documentation of objective response (either CR or PR).
  • DCR defined as the proportion of subjects who have achieved CR, PR, or stable disease (duration of stable disease should be ⁇ 3 months).
  • the IL-2 conjugate comprises SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain.
  • the IL-2 conjugate can also Attorney Docket No.01183-0277-00PCT-SYN be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain.
  • This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin.
  • pINN International Nonproprietary Name
  • the goal will be to evaluate the anti-tumor activity, safety and PK/Pdy of the IL-2 conjugate monotherapy once weekly (QW) for 6 weeks (induction period), and then every 2 weeks (Q2W) thereafter (maintenance period). See FIG.16.
  • Example 5 will include approximately 40 participants with late-line metastatic melanoma. DLTs will not apply. [0333] The following inclusion criteria apply. The participant must be willing and able to provide informed consent and comply with protocol requirements for the duration of the study. The participant must be ⁇ 18 years of age. Prior anti-cancer therapy is allowed (eg, radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy treatment.
  • Prior anti-cancer therapy is allowed (eg, radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy treatment.
  • treatment related toxicity resolved to Grade 0 or 1 (alopecia excepted) according to NCI CTCAE v5.0
  • treatment related toxicity resolved to at least Grade 2 according to NCI CTCAE v5.0 with prior approval of the Medical Monitor.
  • Participants must have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
  • Participants must have a life expectancy greater than or equal to 12 weeks as determined by the Investigator.
  • Participants must have histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors with at least one tumor lesion with location accessible to safely biopsy per clinical judgment of the Investigator.
  • the participant must have received at least one prior line of therapy for metastatic melanoma and/or does not have any standard of care (SoC) treatment option or participant declines or is intolerant to be treated with SoC treatment.
  • SoC standard of care
  • Subjects with advanced or metastatic solid tumors who have refused standard of care; or for whom no reasonable standard of care exists that would confer clinical benefit; or for whom standard therapy is intolerable, not effective, or not accessible.
  • Measurable disease per RECIST v1.1 Participants must have at least 2 measurable lesions to safely perform mandatory pre & on-treatment biopsy. On-treatment biopsy may be omitted upon written request from the Investigators considering biopsy clinical feasibility and upon written approval by the Sponsor.
  • Participants must have adequate laboratory parameters including: (1) Absolute lymphocyte count ⁇ 0.5 times lower limit of normal, (2) Platelet count ⁇ 100 ⁇ 10 9 /L (3) Hemoglobin ⁇ 9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for erythropoiesis-stimulating agent (ESA) and colony-stimulating factor (CSF) Attorney Docket No.01183-0277-00PCT-SYN administration is sufficient), (4) Absolute neutrophil count ⁇ 1.5 ⁇ 10 9 /L (absence of growth factors within 2 weeks), (5) Prothrombin time (PT) and partial thromboplastin time (PTT) ⁇ 1.5 times ULN, (6) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ⁇ 2.5 times ULN except if liver metastases are present may be ⁇ 5 times ULN, and (7) Total bilirubin ⁇ 1.5 ⁇ ULN.
  • ESA
  • Females of childbearing potential and men who are not surgically sterile must agree to use medically accepted method of birth control during the study and for at least 7 days for females, and for at least 3 days for males after the last dose of study intervention.
  • Female participants must have a negative serum pregnancy test within 7 days prior to initiating study treatment in premenopausal women and women less than 12 months after menopause.
  • Male participants must agree to refrain from donating or banking sperm during the treatment period and for at least 3 days after last dose of study treatment.
  • Participants are excluded from the study if any of the following criteria applies: – Radiotherapy ⁇ 14 days prior to first dose of study drug (palliative radiation or stereotactic radiosurgery within 7 days prior to start of study treatment).
  • ⁇ Treated with systemic anti-cancer therapy or an investigational agent within 2 weeks prior to start of study drug treatment (within 4 weeks for immunotherapy and tyrosine kinase inhibitor therapy).
  • Participants who experienced Grade 3 or higher immune-related toxicity from prior immuno- oncology therapy.
  • Major surgery ⁇ 30 days prior to first dose of study drug, or has not recovered to at least Grade 1 from adverse effects from such procedure, or anticipation of the need for major surgery during study treatment.
  • Active autoimmune disease requiring systemic treatment within the past 3 months or have a documented history of clinically severe autoimmune disease that requires systemic steroids or immunosuppressive agents; subjects on corticosteroids are excluded for a dose >10 mg daily of prednisone or equivalent.
  • CNS disease or leptomeningeal disease are asymptomatic, are without evidence of radiological progression for at least 8 weeks and have had no requirement for steroids or enzyme inducing anticonvulsants in the last 14 days prior to Screening.
  • hepatitis C Known human immunodeficiency virus (HIV) infection or active infection with hepatitis C: (a) known uncontrolled hepatitis B virus (HBV) infection: (i) anti-HBV therapy started before initiation of IMP and HBV viral load ⁇ 2000 IU/mL (104 copies/mL) are eligible. The anti-HBV therapy should continue throughout the treatment period; (ii) positive anti-HBc, positive anti HBs, negative HBsAg, and HBV virus load without HBV therapy are eligible. – Received a live-virus vaccination ⁇ 14 days prior to first dose of study drug. Seasonal flu and other inactivated vaccines that do not contain live virus are permitted.
  • Adverse events will be assessed until approximately 90 days after the last dose of study drug(s) to monitor for delayed immune toxicities that have been associated with immunotherapy products, or until a subsequent anti-cancer treatment is administered (whichever comes first).
  • Efficacy Assessments Anti-tumor activity will be determined by repeat radiological imaging and response assessed using RECIST 1.1 and iRECIST. All subjects must have scans performed using a modality appropriate for the subject’s tumor (eg, PET-CT scan, CT scans, MRI). All anatomical areas that are imaged during Screening should be assessed at every on- study time point using the same imaging modality.
  • Study Endpoints [0341] Primary Endpoints Attorney Docket No.01183-0277-00PCT-SYN [0342] Objective Response Rate (ORR), defined as the proportion of subjects with confirmed complete response (CR) or partial response (PR); a confirmed response is a response that persists on repeat-imaging ⁇ 4 weeks after initial documentation of response. [0343] Secondary Endpoints • DOR, defined as time from date of first objective response (either CR or PR) to first documentation of radiographic disease progression or death due to any cause, whichever occurs first. • PFS, defined as the time from first dose of the IL-2 conjugate to first documentation of radiographic disease progression or death due to any cause, whichever occurs first.
  • • OS defined as the time from first dose of the IL-2 conjugate to the date of death due to any cause.
  • TTR defined as the time from first dose of the IL-2 conjugate to first documentation of objective response (either CR or PR).
  • DCR defined as the proportion of subjects who have achieved CR, PR, or stable disease (duration of stable disease should be ⁇ 3 months).
  • TEAE(s) by Preferred Term and Adverse Events of Specific Interest For participants receiving the IL-2 conjugate at a dose of 16, 24, or 32 ⁇ g/kg under the Q2W, Q3W, or QW/Q2W schedules, as described in Examples 3 and 4, data regarding TEAE(s) by preferred term (PT) (worst grade by participant) are summarized in Tables 4-6, respectively. [0345] In Tables 4-6, the adverse events of specific interest (AESI) and their data are underlined. If an adverse event is shown in a line in one table, but there is no corresponding line in another table, then that adverse event was not observed at any of the dose levels described in the other table.
  • AESI adverse events of specific interest
  • Table 6 has a line for capillary leak syndrome (CLS), but Tables 4 and 5 do not; this means that no CLS of any grade was observed at any of the dose levels described in the Tables 4 and 5.
  • CLS capillary leak syndrome
  • Tables 4-6 incidences of Grade 3 or higher AESI are not high among the different schedules, and there was no observable increase in the TEAE profile among the different schedules.
  • cytokine release syndrome increased aspartate aminotransferase, increased alanine aminotransferase, increased blood bilirubin, infusion related reaction, hyperbilirubinaemia, and capillary leak syndrome, in the Attorney Docket No.01183-0277-00PCT-SYN QW/Q2W dosing schedule (at all tested dose levels), relative to the Q2W dosing schedule (at all tested dose levels), or the Q3W dosing schedule (at all tested dose levels).
  • the QW/Q2W dosing schedule is more aggressive than the Q2W dosing schedule or the Q3W dosing schedule, surprisingly the QW/Q2W dosing schedule is relatively safe (e.g., with no observable increase in incidences of any of the aforementioned adverse events of Grade 3 or higher).
  • N Y S - T 3 C > ) 8 ) 9 ) 3 ) ) P 6 3 ) 6 ) 6 ) 6 0 0 - 7 7 2 0 - 3 8 1 1 0.
  • o N t e k c o D y e n r ot t A p f c e d ) ) 5 ) 0 ) ) ) ) o m ) 6 a r 8 . 2 . 5 6 5 . 0 2 2 1 0 . 5 5 . 5 2 0 2 .
  • a semi-mechanistic population pharmacokinetic/pharmacodynamic (PK/PD) model was developed to illustrate the intricate dynamics of the IL-2 conjugate-induced lymphocyte trafficking from blood to expansion sites immediately following administration, as well as the subsequent reappearance of expanded lymphocytes in the blood.
  • PK/PD semi-mechanistic population pharmacokinetic/pharmacodynamic
  • Fig.17A shows simulated (shaded areas and lines) and observed (dots) fold change in CD8+ T cell counts.
  • Fig.17B shows simulated (shaded areas and lines) and observed (dots) fold change in NK cell counts.
  • the model was developed to sufficiently capture the complexities inherent in the IL- 2 conjugate pharmacokinetics and pharmacodynamics. Notably, body weight emerged as a statistically significant covariate exerting influence on both clearance and volume terms.
  • the lymphocyte re-circulation and expansion component of the model utilized the fold-change of the respective immune cells to explain the dynamics at play.
  • Blood was collected from participants receiving an IL-2 conjugate described herein (i) at a dose of 8, 16, 24, 32 ⁇ g/kg under the Q2W schedule, (ii) at a dose of 8, 16, 24, 32, or 40 ⁇ g/kg under the Q3W schedule, or (iii) at a dose of 16 or 24 ⁇ g/kg under the QW schedule (where the QW schedule corresponds to the induction period of a QW/Q2W schedule), as described in Examples 3 and 4.
  • Fig.18A shows fold change in CD8+ T cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 ⁇ g/kg in accordance with the indicated dosing schedule.
  • Fig.18B shows fold change in NK cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 ⁇ g/kg in accordance with the indicated dosing schedule.
  • Figures 19A-19C show flat modulation of peripheral CD4+ Treg cells across different tested dosing schedules.
  • Fig.19A shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 8, 16, 24, or 32 ⁇ g/kg conjugate under the Q2W schedule.
  • Fig.19B shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 8, 16, 24, 32, or 40 ⁇ g/kg under the Q3W schedule.
  • Fig.19C shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 or 24 ⁇ g/kg QW, as would be administered during part of the QW/Q2W schedule.
  • administering stimulated the expansion of peripheral CD8+ T and NK cell counts without significantly expanding CD4+ Treg cells.
  • More frequent dosing during the induction period e.g., using a QW/Q2W schedule
  • can induce increased CD8+ T cell expansion and NK cell expansion, as compared to a dosing schedule without a QW induction period e.g., a Q2W or Q3W dosing schedule as described in Example 3).

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Abstract

Disclosed herein are methods for treating a cancer in a subject in need thereof, comprising administering an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks. Also disclosed herein are methods for treating solid tumors in a subject in need thereof, comprising administering an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks. Also disclosed herein are methods for treating melanoma in a subject in need thereof, comprising administering an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks.

Description

Attorney Docket No.01183-0277-00PCT-SYN CANCER THERAPY WITH IL-2 CONJUGATES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority of United States Provisional Application No.63/453,370, filed March 20, 2023, the content of which is incorporated by reference herein for all purposes. BACKGROUND OF THE DISCLOSURE [0002] Distinct populations of T cells modulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological self-reactivity while cytotoxic T cells target and destroy infected cells and/or cancerous cells. In some instances, modulation of the different populations of T cells provides an option for treatment of a disease or indication. [0003] Cytokines comprise a family of cell signaling proteins such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors playing roles in innate and adaptive immune cell homeostasis. Cytokines are produced by immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, endothelial cells, fibroblasts, and different stromal cells. In some instances, cytokines modulate the balance between humoral and cell-based immune responses. [0004] Interleukins are signaling proteins that modulate the development and differentiation of T and B lymphocytes, cells of the monocytic lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4+ T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue residents. [0005] In some instances, interleukin 2 (IL-2) signaling is used to modulate T cell responses and subsequently for treatment of a cancer. SUMMARY OF THE DISCLOSURE [0006] Described herein are methods of treating a cancer in a subject in need thereof, comprising administering to a subject an IL-2 conjugate, wherein the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 having an unnatural amino acid residue described herein at position 64. In some embodiments, an exemplary IL-2 conjuate comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, methods described herein are amenable to a Attorney Docket No.01183-0277-00PCT-SYN solid tumor. In some embodiments, methods described herein are amenable to a hematologic cancer. [0007] Also described herein are methods of treating solid tumors in a subject in need thereof, comprising administering to a subject an IL-2 conjugate, wherein the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 having an unnatural amino acid residue described herein at position 64. In some embodiments, an exemplary IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 2. [0008] The present disclosure, among other things, provides dosing regimens (e.g., doses, dosing frequency, or a combination of both) for improved stimulation and/or expansion of certain immune cells (e.g., CD8+ T cells and/or natural killer (NK) cells) that are useful for treatment of cancer, and/or improvement in treatment of cancer. In some embodiments, such cancer is a solid tumor. In some embodiments, such cancer is a hematologic cancer. [0009] In one aspect, provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to a subject an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks. In another aspect, provided herein are methods of treating solid tumors in a subject comprising administering an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks. In another aspect, provided herein are methods of treating melanoma in a subject comprising administering an IL-2 conjugate (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks. [0010] Exemplary embodiments include the following. [0011] Embodiment 1 is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an IL-2 conjugate, wherein: the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 Attorney Docket No.01183-0277-00PCT-SYN P64 is replaced by the structure of Formula (I):
Figure imgf000005_0001
– about 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure:
Figure imgf000005_0002
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue; wherein the IL-2 conjugate is administered to the subject (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a second plurality of weeks. Attorney Docket No.01183-0277-00PCT-SYN [0012] Embodiment 1.1 is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an IL-2 conjugate, wherein: the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1 wherein the amino acid at position P64 is replaced by the structure of Formula (I):
Figure imgf000006_0001
- 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure:
Figure imgf000006_0002
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue; wherein the IL-2 conjugate is administered to the subject (a) about once every week for a first plurality of weeks, followed by (b) about once every two weeks for a second plurality of weeks. Attorney Docket No.01183-0277-00PCT-SYN [0013] Embodiment 2 is the method of embodiment 1 or 1.1, wherein in the IL-2 conjugate the PEG group has an average molecular weight of about 30 kDa. [0014] Embodiment 3 is the method of embodiment 1, 1.1, or 2, wherein in the IL-2 conjugate
Figure imgf000007_0001
1, 1.1, or 2, wherein in the IL-2 conjugate
Figure imgf000007_0002
1, 1.1, or 2, wherein in the IL-2 conjugate
Figure imgf000007_0003
1, 1.1, or 2, wherein in the IL-2 conjugate
Figure imgf000007_0004
1, 1.1, or 2, wherein the structure of Formula (I) has the structure of Formula (IV) or Formula (V), or is a mixture of Formula (IV) and Formula (V):
Figure imgf000007_0005
Figure imgf000007_0006
wherein: q is 1, 2, or 3; X is an L-amino acid having the structure: Attorney Docket No.01183-0277-00PCT-SYN
Figure imgf000008_0001
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. [0019] Embodiment 8 is the method of embodiment 1, 1.1, or 2, wherein the structure of Formula (I) has the structure of Formula (XII) or Formula (XIII), or is a mixture of Formula (XII) and Formula (XIII):
Figure imgf000008_0002
wherein: n is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. [0020] Embodiment 9 is the method of any one of embodiments 1, 1.1, and 2-8, wherein q is 1. [0021] Embodiment 10 is the method of any one of embodiments 1, 1.1, and 2-8, wherein q is 2. [0022] Embodiment 11 is the method of any one of embodiments 1, 1.1, and 2-8, wherein q is 3. Attorney Docket No.01183-0277-00PCT-SYN [0023] Embodiment 12 is the method of any one of embodiments 1, 1.1, and 2-11, 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 relapsed or refractory solid tumor, or the solid tumor has relapsed after one or more prior lines of systemic therapy for the solid tumor. [0026] Embodiment 15 is the method of any one of embodiments 12-14, further comprising selecting the subject to whom the IL-2 conjugate is administered at least in part on the basis of the subject having received one or more prior lines of systemic therapy for the solid tumor. [0027] Embodiment 16 is the method of any one of embodiments 12-15, wherein the subject has received one or more lines of systemic therapy for the solid tumor. [0028] Embodiment 17 is the method of embodiment 16, wherein the the subject has received two or more lines of systemic therapy for the solid tumor. [0029] Embodiment 18 is the method of embodiment 17, wherein the subject has received three or more lines of systemic therapy for the solid tumor [0030] Embodiment 19 is the method of embodiment 12, wherein the solid tumor is melanoma. [0031] Embodiment 20 is the method of embodiment 19, wherein the melanoma is metastatic melanoma. [0032] Embodiment 21 is the method of embodiment 19, wherein the melanoma is a relapsed or refractory melanoma, or the melanoma has relapsed after one or more prior lines of systemic therapy for the melanoma [0033] Embodiment 22 is the method of any one of embodiments 19-21, further comprising selecting the subject to whom the IL-2 conjugate is administered at least in part on the basis of the subject having received one or more prior lines of systemic therapy for the melanoma. [0034] Embodiment 23 is the method of any one of embodiments 19-22, wherein the subject has received one or more lines of systemic therapy for the melanoma. [0035] Embodiment 23.1 is the method of any one of embodiments 19-22, wherein the subject has received one or more lines of systemic therapy for the solid tumor. [0036] Embodiment 24 is the method of embodiment 23 or 23.1, wherein the the subject has received two or more lines of systemic therapy for the melanoma. [0037] Embodiment 24.1 is the method of embodiment 23 or 23.1, wherein the the subject has received two or more lines of systemic therapy for the solid tumor. Attorney Docket No.01183-0277-00PCT-SYN [0038] Embodiment 25 is the method of embodiment 24 or 24.1, wherein the subject has received three or more lines of systemic therapy for the melanoma. [0039] Embodiment 25.1 is the method of embodiment 24 or 24.1, wherein the subject has received three or more lines of systemic therapy for the solid tumor. [0040] Embodiment 26 is the method of any one of embodiments 16-18 and 23-25, wherein the one or more prior lines of systemic therapy for the solid tumor or melanoma comprises an immune checkpoint inhibitor. [0041] Embodiment 26.1 is the method of any one of embodiments 23, 23.1, 24, 24.1, 25, and 25.1, wherein the one or more prior lines of systemic therapy for melanoma comprises an immune checkpoint inhibitor. [0042] Embodiment 27 is the method of embodiment 26 or 26.1, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG- 3 inhibitor, or combinations thereof. [0043] Embodiment 27.1 is the method of 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 of 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 about 8 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. [0045] Embodiment 29 is the method of 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 about 16 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. [0046] Embodiment 30 is the method of 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 about 24 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. [0047] Embodiment 31 is the method of 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 about 32 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. [0048] Embodiment 32 is the method of 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 to the subject about 8 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. [0049] Embodiment 33 is the method of 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 to the subject about 16 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. Attorney Docket No.01183-0277-00PCT-SYN [0050] Embodiment 34 is the method of 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 to the subject about 24 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. [0051] Embodiment 35 is the method of 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 to the subject about 32 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. [0052] Embodiment 36 is the method of any one of embodiments 32-35, comprising administering to the subject about 8 μg/kg IL-2 as the IL-2 conjugate about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks. [0053] Embodiment 36.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 8 μg/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks. [0054] Embodiment 37 is the method of any one of embodiments 32-35, comprising administering to the subject about 16 μg/kg IL-2 as the IL-2 conjugate about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks. [0055] Embodiment 37.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 16 μg/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks. [0056] Embodiment 38 is the method of any one of embodiments 32-35, comprising administering to the subject about 24 μg/kg IL-2 as the IL-2 conjugate about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks. [0057] Embodiment 38.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 24 μg/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks. [0058] Embodiment 39 is the method of any one of embodiments 32-35, comprising administering to the subject about 32 μg/kg IL-2 as the IL-2 conjugate about once every at least Attorney Docket No.01183-0277-00PCT-SYN two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks. [0059] Embodiment 39.1 is the method of any one of embodiments 32-35, comprising administering to the subject about 32 μg/kg IL-2 as the IL-2 conjugate about once every two weeks for the second plurality of weeks. [0060] Embodiment 40 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, and 39.1, wherein the first plurality of weeks is about 5 weeks, about 6 weeks, or about 7 weeks. [0061] Embodiment 41 is the method of embodiment 40, wherein the first plurality of weeks is about 6 weeks. [0062] Embodiment 42 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks is at least about 6 weeks. [0063] Embodiment 43 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks is at least about 12 weeks. [0064] Embodiment 44 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks ranges from about 6 weeks to about 46 weeks. [0065] Embodiment 45 is the method of any one of embodiments 32-36, 36.1, 37, 37.1, 38, 38.1, 39, 39.1, and 40, wherein the second plurality of weeks ranges from about 6 weeks to about 98 weeks. [0066] Embodiment 46 is the method of 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 of 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 of 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 administration. [0069] Embodiment 49 is the method of 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 to the subject. Attorney Docket No.01183-0277-00PCT-SYN [0070] Embodiment 50 is the method of 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 subject. [0071] Embodiment 51 is the method of embodiment 49 or 50, wherein the acetaminophen and/or diphenhydramine is administered to the subject before administering the IL-2 conjugate. [0072] Embodiment 52 is an IL-2 conjugate for use in the method of 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 use of an IL-2 conjugate for the manufacture of a medicament for the method of 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. BRIEF DESCRIPTION OF THE DRAWINGS [0074] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [0075] FIG.1 shows lymphocyte expansion in cynomolgus monkeys treated with an IL-2 conjugate at 0.1 mg/kg at a frequency of either Q1W for a total of 3 doses, Q2W for a total of 3 doses, Q3W for a total of 3 doses, or Q4 weekly for a total of 3 doses. The days of dosing for each are indicated by the small arrows under the X-axis. [0076] FIG.2 shows the change in peripheral CD8+ Teff cell counts in the indicated subjects treated with 8 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0077] FIG.3 shows the change in peripheral NK cell counts in the indicated subjects treated with 8 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0078] FIG.4 shows the change in peripheral CD4+ Treg cell counts in the indicated subjects treated with 8 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0079] FIG.5 shows the change in peripheral lymphocyte cell counts in the indicated subjects treated with 8 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. Attorney Docket No.01183-0277-00PCT-SYN [0080] FIG.6 shows the change in peripheral eosinophil cell counts in the indicated subjects treated with 8 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0081] FIG.7A and FIG.7B show mean concentrations of the IL-2 conjugate administered to the indicated subjects at 8 µg/kg [Q2W] after 1 and 2 cycles, respectively, at specified times following administration. [0082] FIG.8 shows the levels of IFN-γ, IL-6, and IL-5 in the indicated subjects treated with 8 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0083] FIG.9 shows the change in peripheral CD8+ Teff cell counts in the indicated subjects treated with 16 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0084] FIG.10 shows the change in peripheral NK cell counts in the indicated subjects treated with 16 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0085] FIG.11. shows the change in peripheral CD4+ Treg cell counts in the indicated subjects treated with 16 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0086] FIG.12 shows the change in peripheral eosinophil cell counts in the indicated subjects treated with 16 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0087] FIG.13 shows the levels of IFN-γ, IL-6, and IL-5 in the indicated subjects treated with 16 µg/kg [Q2W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0088] FIG.14A and FIG.14B show mean concentrations of the IL-2 conjugate administered to the indicated subjects at 16 µg/kg [Q2W] after 1 and 2 cycles, respectively, at specified times following administration. [0089] FIG.15 shows the study design of Example 4. Abbreviations: C: cycle; D: day; DLT: dose-limiting toxicity; EOT: end of treatment; FU: follow-up; IMP: investigational medicinal product; IV: intravenously; QW: once weekly; Q2W: every 2 weeks; Q6W: every 6 weeks; Q8W: every 8 weeks; Q12W: every 12 weeks. [0090] FIG.16 shows the study design of Example 5. Abbreviations: C: cycle; D: day; DLT: EOT: end of treatment; FU: follow-up; IMP: investigational medicinal product; IV: intravenously; QW: once weekly; Q2W: every 2 weeks; Q6W: every 6 weeks; Q8W: every 8 weeks; Q12W: every 12 weeks. Attorney Docket No.01183-0277-00PCT-SYN [0091] FIG.17A shows simulated (shaded areas and lines) and observed (dots) fold change in CD8+ T cell counts. FIG.17B shows simulated (shaded areas and lines) and observed (dots) fold change in NK cell counts. [0092] FIG.18A shows fold change in CD8+ T cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 μg/kgin accordance with the indicated dosing schedules. FIG.18B shows fold change in NK cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 μg/kg in accordance with the indicated dosing schedules. [0093] FIG.19A shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 8, 16, 24, or 32 μg/kg under the Q2W schedule. FIG. 19B shows fold change in Treg cell counts in blood collected from participants receiving the IL- 2 conjugate at a dose of 8, 16, 24, 32, or 40 μg/kg under the Q3W schedule. FIG.19C shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 or 24 μg/kg QW, as would be administered during part of the QW/Q2W schedule. DETAILED DESCRIPTION OF THE DISCLOSURE Definitions [0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. To the extent any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content controls. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless the context requires otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. [0095] Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the inventions. Attorney Docket No.01183-0277-00PCT-SYN [0096] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 µL” means “about 5 µL” and also “5 µL.” Generally, the term “about” includes an amount that would be expected to be within experimental error, such as for example, within 15%, 10%, or 5%. [0097] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [0098] As used herein, the terms “subject(s)” and “patient(s)” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent) of a health care worker (e.g., a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker). [0099] As used herein, the term “unnatural amino acid” refers to an amino acid other than one of the 20 naturally occurring amino acids. Exemplary unnatural amino acids are described in Young et al., “Beyond the canonical 20 amino acids: expanding the genetic lexicon,” J. of Biological Chemistry 285(15): 11039-11044 (2010), the disclosure of which is incorporated herein by reference. [0100] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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. [0101] As used herein, “nucleotide” refers to a compound comprising a nucleoside moiety and a phosphate moiety. Exemplary natural nucleotides include, without limitation, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), and guanosine monophosphate (GMP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine diphosphate (dADP), thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP), and Attorney Docket No.01183-0277-00PCT-SYN deoxyguanosine monophosphate (dGMP). Exemplary natural deoxyribonucleotides, which comprise a deoxyribose as the sugar moiety, include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural ribonucleotides, which comprise a ribose as the sugar moiety, include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP. [0102] As used herein, “base” or “nucleobase” refers to at least the nucleobase portion of a nucleoside or nucleotide (nucleoside and nucleotide encompass the ribo or deoxyribo variants), which may in some cases contain 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, a “base” may be incorporated by a DNA polymerase into DNA, or by an RNA polymerase into RNA). However, the term “base” should not be interpreted as necessarily representing the entire nucleoside or nucleotide unless required by the context. In the chemical structures provided herein of a base or nucleobase, only the base of the nucleoside or nucleotide is shown, with the sugar moiety and, optionally, any phosphate residues omitted for clarity. As used in the chemical structures provided herein of a base or nucleobase, the wavy line represents connection to a nucleoside or nucleotide, in which the sugar portion of the nucleoside or nucleotide may be further modified. In some embodiments, the wavy line represents attachment of the base or nucleobase to the sugar portion, such as a pentose, of the nucleoside or nucleotide. In some embodiments, the pentose is a ribose or a deoxyribose. [0103] In some embodiments, a nucleobase is generally the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring, may be modified, may bear no similarity to natural bases, and/or may be synthesized, e.g., by organic synthesis. In certain embodiments, a nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide, where the atom or group of atoms is capable of interacting with a base of another nucleic acid with or without the use of hydrogen bonds. In certain embodiments, an unnatural nucleobase is not derived from a natural nucleobase. It should be noted that unnatural nucleobases do not necessarily possess basic properties, however, they are referred to as nucleobases for simplicity. In some embodiments, when referring to a nucleobase, a “(d)” indicates that the nucleobase can be attached to a deoxyribose or a ribose, while “d” without parentheses indicates that the nucleobase is attached to deoxyribose. [0104] As used herein, a “nucleoside” is a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups. Nucleosides include nucleosides comprising any variety of Attorney Docket No.01183-0277-00PCT-SYN substituents. A nucleoside can be a glycoside compound formed through glycosidic linking between a nucleic acid base and a reducing group of a sugar. [0105] An “analog” of a chemical structure, as the term is used herein, refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure. In some embodiments, a nucleotide analog is an unnatural nucleotide. In some embodiments, a nucleoside analog is an unnatural nucleoside. A related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a “derivative.” [0106] As used herein, “advanced solid tumor” refers to a malignant solid neoplasm that has spread extensively to other anatomic sites or is no longer responding to treatment. [0107] “Line of treatment” or “line of therapy” describes a treatment or therapy given as part of a sequence or in order to a patient as the patient’s disease progresses. Initial treatment (first- line therapy) may not work or may stop working after a period. After first-line therapy is discontinued, a second different treatment (second-line therapy) may be given. Subsequent lines of therapy may be given when a second-line therapy does not work or stops working. Some patients may be administered multiple lines of therapy over the course of a disease. [0108] As used herein, “severe cytokine release syndrome” refers to level 4 or 5 cytokine release syndrome as described in Teachey et al., Cancer Discov.2016; 6(6); 664–79, the disclosure of which is incorporated herein by reference. [0109] As used herein, the assignment of amino acids in complementarity-determining regions (CDRs) to a heavy chain variable domain (VH) or a light chain variable domain (VL) is in accordance with the definitions of Kabat et al. 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); 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 Molecular Biology 262:732-745. [0110] The “percent identity” and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide Attorney Docket No.01183-0277-00PCT-SYN sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences. A comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. Expressions such as “comprises a sequence with at least X% identity to Y” with respect to a test sequence mean that, when aligned to sequence Y as described above, the test sequence comprises residues identical to at least X% of the residues of Y. [0111] Although various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment. IL-2 Conjugates [0112] Interleukin 2 (IL-2) is a pleiotropic type-1 cytokine whose structure comprises a 15.5 kDa four α-helix bundle. 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 produced primarily by CD4+ T cells post antigen stimulation and to a lesser extent, by CD8+ cells, Natural Killer (NK) cells, and Natural killer T (NKT) cells, activated dendritic cells (DCs), and mast cells. IL-2 signaling occurs through interaction with specific combinations of IL-2 receptor (IL-2R) subunits, IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132). Interaction of IL-2 with the IL-2Rα forms the “low- affinity” IL-2 receptor complex with a Kd of about 10-8 M. Interaction of IL-2 with IL-2Rβ and IL-2Rγ forms the “intermediate-affinity” IL-2 receptor complex with a Kd of about 10-9 M. Interaction of IL-2 with all three subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, forms the “high- affinity” IL-2 receptor complex with a Kd of about >10-11 M. [0113] In some instances, IL-2 signaling via the “high-affinity” IL-2Rαβγ complex modulates the activation and proliferation of regulatory T cells. Regulatory T cells, or CD4+CD25+Foxp3+ regulatory T (Treg) cells, mediate maintenance of immune homeostasis by suppression of effector cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells, and NKT cells. In some instances, Treg cells are generated from the thymus (tTreg cells) or are induced from naïve T cells in the periphery (pTreg cells). In some cases, Treg cells are considered as the mediator of Attorney Docket No.01183-0277-00PCT-SYN peripheral tolerance. Indeed, in one study, transfer of CD25-depleted peripheral CD4+ T cells produced a variety of autoimmune diseases in nude mice, whereas cotransfer of CD4+CD25+ T cells suppressed the development of autoimmunity (Sakaguchi, et al., “Immunologic self- tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25),” J. Immunol.155(3): 1151-1164 (1995), the disclosure of which is incorporated herein by reference). Augmentation of the Treg cell population down-regulates effector T cell proliferation and suppresses autoimmunity and T cell anti-tumor responses. [0114] IL-2 signaling via the “intermediate-affinity” IL-2Rβγ complex modulates the activation and proliferation of CD8+ effector T (Teff) cells, NK cells, and NKT cells. CD8+ Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, Tcon, or killer T cells) are T lymphocytes that recognize and kill damaged cells, cancerous cells, and pathogen-infected cells. NK and NKT cells are types of lymphocytes that, similar to CD8+ Teff cells, target cancerous cells and pathogen-infected cells. [0115] In some instances, IL-2 signaling is utilized to modulate certain immune cell responses (e.g., but not limited to T cell responses and/or NK cell responses) and subsequently for treatment of a cancer. For example, in some embodiments, IL-2 is administered in a high-dose form to induce expansion of Teff cell populations for treatment of a cancer. However, high-dose IL-2 further leads to concomitant stimulation of Treg cells that dampen anti-tumor immune responses. High-dose IL-2 also induces toxic adverse events mediated by the engagement of IL- 2R alpha chain-expressing cells in the vasculature, including type 2 innate immune cells (ILC- 2), eosinophils and endothelial cells. This leads to eosinophilia, capillary leak and vascular leak syndrome (VLS). [0116] In one aspect, provided herein are methods of stimulating and/or expanding certain immune cells that are useful for treatment of cancer, including, e.g., Teff cells, NK cells, and/or NKT cells, in a subject in need thereof, e.g., a subject having cancer, wherein such methods comprise administering to the subject an IL-2 conjugate as described herein. In some embodiments, such a cancer is a solid tumor. In some embodiments, such a cancer is a hematologic cancer. In another aspect, provided herein are methods of treating solid tumors, for example, melanoma, in a subject in need thereof, comprising administering to the subject an IL- 2 conjugate described herein. [0117] In some embodiments, the IL-2 sequence included in an IL-2 conjugate described herein comprises an amino acid sequence that is at least about 90% (including, e.g., 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 sequence of SEQ ID NO: 1: Attorney Docket No.01183-0277-00PCT-SYN PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFSQSIISTLT (SEQ ID NO: 1) wherein the amino acid at position P64 as set forth in SEQ ID NO: 1 is replaced by the structure of Formula (I):
Figure imgf000021_0001
– 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure:
Figure imgf000021_0002
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, the IL-2 sequence included in an IL-2 conjugate described herein comprises the Attorney Docket No.01183-0277-00PCT-SYN sequence of SEQ ID NO: 1, wherein the amino acid at position P64 as set forth in the SEQ ID NO: 1 is replaced by the structure of Formula (I) as described herein. [0118] In any of the embodiments or variations of Formula (I) described herein and pharmaceutical compositions comprising the same, average molecular weight encompasses both weight average molecular weight and number average molecular weight; in other words, for example, both a 30 kDa number average molecular weight and a 30 kDa weight average molecular weight qualify as a 30 kDa molecular weight. In some embodiments, the average molecular weight is weight average molecular weight. In other embodiments, the average molecular weight is number average molecular weight. It is understood that in the methods provided herein, administering an IL-2 conjugate as described herein to a subject comprises administering more than a single molecule of IL-2 conjugate; as such, use of the term “average” to describe the molecular weight of the PEG group refers to the average molecular weight of the PEG groups of the IL-2 conjugate molecules in a dose administered to the subject. [0119] In any of the embodiments or variations of Formula (I) described herein, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt. In some embodiments, the IL-2 conjugate is a solvate. In some embodiments, the IL-2 conjugate is a hydrate. [0120] In some embodiments of Formula (I), Z is CH2 and Y is .
Figure imgf000022_0001
In some embodiments of Formula (I), Y is CH2 and Z is
Figure imgf000022_0002
some embodiments of Formula
Figure imgf000022_0003
embodiments of Formula [0121] In some
Figure imgf000022_0004
(I), q is 2. In some embodiments of Formula (I), q is 3. [0122] 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. Attorney Docket No.01183-0277-00PCT-SYN [0123] In some embodiments of Formula (I), q is 1 and structure of Formula (I) is the structure of Formula (Ia):
Figure imgf000023_0001
– about 35 kDa; X is an L-amino acid having the structure:
Figure imgf000023_0002
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. [0124] In some embodiments of Formula (Ia), Z is CH2 and Y is
Figure imgf000023_0003
Attorney Docket No.01183-0277-00PCT-SYN
Figure imgf000024_0001
, of about 30 kDa. [0126] In some embodiments, the IL-2 conjugate comprises the sequence of SEQ ID NO: 2: PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELK[AzK_L1_PEG30kD]LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 2) wherein [AzK_L1_PEG30kD] is N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to PEG via DBCO-mediated click chemistry to form a compound comprising a structure of Formula (IV) or Formula (V), wherein q is 1 (such as Formula (IVa) or Formula (Va)), and wherein the PEG group has an average molecular weight of about 25-35 kiloDaltons (e.g., about 30 kDa), capped with a methoxy group. The term “DBCO” means a chemical moiety comprising a dibenzocyclooctyne group, such as comprising the mPEG-DBCO compound illustrated in Schemes 1 and 2 of Example 1. [0127] The ratio of regioisomers generated from the click reaction is about 1:1 or greater than 1:1. [0128] PEGs typically comprise a number of (OCH2CH2) monomers (or (CH2CH2O) monomers, depending on how the 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 about 30 kDa. [0129] In some instances, the PEG is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower C1-6 alkoxy group, or a hydroxyl group. In some embodiments, the PEG group is a methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG wherein one terminus of the polymer is a methoxy (-OCH3) group, and the other terminus is a hydroxyl or other functional group that can be optionally chemically modified. [0130] 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 Attorney Docket No.01183-0277-00PCT-SYN embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. For example, included within the scope of the present disclosure are IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da ± 3,000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 5,000 Da. [0131] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V):
Figure imgf000025_0001
wherein: W is a PEG group having an average molecular weight of about 25 kDa - 35kDa; q is 1, 2, or 3; and X has the structure:
Figure imgf000025_0002
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. In some embodiments, an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID Attorney Docket No. 01183-0277-00PCT-SYN NO: 1, in which the amino acid residue P64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V). [0132] 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. [0133] 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. [0134] In any of the embodiments 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). [0135] 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):
Figure imgf000026_0001
wherein: W is a PEG group having an average molecular weight of about 25 kDa – about 35kDa; and X has the structure: Attorney Docket No.01183-0277-00PCT-SYN
Figure imgf000027_0001
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. [0136] 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 about 30 kDa. [0137] In any of the embodiments 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). [0138] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII):
Figure imgf000027_0002
wherein: Attorney Docket No.01183-0277-00PCT-SYN n is is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 25 kDa – about 35 kDa; q is 1, 2, or 3; and the wavy lines indicate convalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. In some embodiments, an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue P64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII). [0139] 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. [0140] In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), n is is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 30 kDa. [0141] In any of the embodiments 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). [0142] 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):
Figure imgf000028_0001
Attorney Docket No.01183-0277-00PCT-SYN Formula (XIIIa); wherein: n is is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 25 kDa – about 35 kDa; and the wavy lines indicate convalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. [0143] In some embodiments of Formula (XIIa) or Formula (XIIIa), or a mixture of Formula (XIIa) and Formula (XIIIa), n is is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 30 kDa. [0144] In any of the embodiments 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). [0145] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV):
Figure imgf000029_0001
wherein: Attorney Docket No.01183-0277-00PCT-SYN m is an integer from 0 to 20; p is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa – about 35 kDa; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. In some embodiments, an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue P64 is replaced by the structure of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV). [0146] 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. [0147] 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. [0148] In some embodiments, p is an integer from 0 to 15. In some embodiments, p is an integer from 0 to 10. In some embodiments, p is an integer from 0 to 5. In some embodiments, p is an integer from 1 to 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. [0149] In some embodiments, m and p are each 2. [0150] In any of the embodiments 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). [0151] In some embodiments, the IL-2 conjugate comprises an amino acid sequence that is at least about 90% (including, e.g., 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 in which the amino acid residue P64 is replaced by the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII): Attorney Docket No.01183-0277-00PCT-SYN
Figure imgf000031_0001
wherein: m is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa - 35 kDa; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. In some embodiments, an IL-2 conjugate described herein comprises the amino acid sequence of SEQ ID NO: 1, in which the amino acid residue P64 is replaced by the structure of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII). [0152] 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. [0153] 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. [0154] In any of the embodiments 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). Attorney Docket No.01183-0277-00PCT-SYN Conjugation Chemistry [0155] In some embodiments, the IL-2 conjugates described herein can be prepared by a conjugation reaction comprising a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises reaction of an azide and an alkyne (“Click” reaction). In some embodiments, a conjugation reaction described herein comprises the reaction outlined in Scheme I, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1. Scheme I.
Figure imgf000032_0001
Figure imgf000032_0002
[0156] In some embodiments, the conjugating moiety comprises a PEG group as described herein. In some embodiments, a reactive group comprises an alkyne or azide. [0157] In some embodiments, a conjugation reaction described herein comprises the reaction outlined in Scheme II, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1. Scheme II.
Figure imgf000032_0003
Figure imgf000032_0004
[0158] In some embodiments, a conjugation reaction described herein comprises the reaction outlined in Scheme III, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1. Scheme III.
Figure imgf000032_0005
Figure imgf000032_0006
[0159] In some embodiments a conjugation reaction described herein comprises the reaction outlined in Scheme IV, wherein X is an unnatural amino acid at position P64 of SEQ ID NO: 1. Scheme IV. Attorney Docket No.01183-0277-00PCT-SYN
Figure imgf000033_0002
Figure imgf000033_0001
comprises a cycloaddition reaction between an azide moiety, such as that contained in a protein containing an amino acid residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as that derived from DBCO, which is a chemical moiety comprising a dibenzocyclooctyne group. PEG groups comprising a DBCO moiety are commercially available or may be prepared by methods known to those of ordinary skill in the art. Exemplary reactions are shown in Schemes V and VI.
Attorney Docket No. 01183-0277-00PCT-SYN Scheme V.
Attorney Docket No.01183-0277-00PCT-SYN Scheme VI.
Figure imgf000035_0001
[0161] Conjugation reactions such as a click reaction described herein may generate a single regioisomer, or a mixture of regioisomers. In some instances the ratio of regioisomers is about 1:1. In some instances the ratio of regioisomers is about 2:1. In some instances the ratio of regioisomers is about 1.5:1. In some instances the ratio of regioisomers is about 1.2:1. In some instances the ratio of regioisomers is about 1.1:1. In some instances the ratio of regioisomers is greater than 1:1. Attorney Docket No.01183-0277-00PCT-SYN IL-2 Polypeptide Production [0162] In some instances, the IL-2 conjugates described herein, either containing a natural amino acid mutation or an unnatural amino acid mutation, are generated recombinantly or are synthesized chemically. In some instances, IL-2 conjugates described herein are generated recombinantly, for example, either by a host cell system, or in a cell-free system. [0163] In some instances, IL-2 conjugates are generated recombinantly through a host cell system. In some cases, the host cell is a eukaryotic cell (e.g., mammalian cell, insect cells, yeast cells or plant cell) or a prokaryotic cell (e.g., Gram-positive bacterium or a Gram-negative bacterium). In some cases, a eukaryotic host cell is a mammalian host cell. In some cases, a mammalian host cell is a stable cell line, or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division. In other cases, a mammalian host cell is a transient cell line, or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division. [0164] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells , 293 H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO- K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™- 3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp- In™-Jurkat cell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™- 293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line. [0165] In some embodiments, a eukaryotic host cell is an insect host cell. Exemplary insect host cells include Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells. [0166] In some embodiments, a 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 strain such as INVSc1. [0167] In some embodiments, a eukaryotic host cell is a plant host cell. In some instances, the plant cells comprise a cell from algae. Exemplary plant cell lines include strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942. [0168] In some embodiments, a host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F’, INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™. Attorney Docket No.01183-0277-00PCT-SYN [0169] In some instances, suitable polynucleic acid molecules or vectors for the production of an IL-2 polypeptide described herein include any suitable vectors derived from either a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors from bacteria (e.g., E. coli), insects, yeast (e.g., Pichia pastoris, K. phaffii), algae, or mammalian source. Bacterial vectors include, for example, pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2. [0170] Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2. [0171] Yeast vectors include, for example, Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES- DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris (K. phaffii) vector, pGAPZA, B, & C Pichia pastoris (K. phaffii) vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector. [0172] Algae vectors include, for example, pChlamy-4 vector or MCS vector. [0173] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG- Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP- CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG- CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2. [0174] In some instances, a cell-free system is used for the production of an IL-2 polypeptide described herein. In some cases, a cell-free system comprises a mixture of cytoplasmic and/or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some instances, a cell-free system utilizes prokaryotic cell components. In other instances, a cell-free system utilizes eukaryotic cell components. Nucleic acid synthesis is obtained in a cell-free Attorney Docket No.01183-0277-00PCT-SYN system based on, for example, Drosophila cell, Xenopus egg, Archaea, or HeLa cells. Exemplary cell-free systems include E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®, XpressCF, and XpressCF+. [0175] Cell-free translation systems variously comprise components such as plasmids, mRNA, DNA, tRNAs, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and/or unnatural amino acids, and/or other components used for protein expression. Such components are optionally modified to improve yields, increase synthesis rate, increase protein product fidelity, or incorporate unnatural amino acids. In some embodiments, cytokines described herein are synthesized using cell-free translation systems described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or US 8,778,631, the disclosure of each of which is incorporated herein by reference. In some embodiments, cell-free translation systems comprise modified release factors, or even removal of one or more release factors from the system. In some embodiments, cell-free translation systems comprise a reduced protease concentration. In some embodiments, cell-free translation systems comprise modified tRNAs with re-assigned codons used to code for unnatural amino acids. In some embodiments, the synthetases described herein for the incorporation of unnatural amino acids are used in cell-free translation systems. In some embodiments, tRNAs are pre- loaded with unnatural amino acids using enzymatic or chemical methods before being added to a cell-free translation system. In some embodiments, components for a cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organism. [0176] In some embodiments, an IL-2 polypeptide is generated as a circularly permuted form, either via an expression host system or through a cell-free system. Production of Cytokine Polypeptide Comprising an Unnatural Amino Acid [0177] An orthogonal or expanded genetic code can be used in the present disclosure, in which one or more specific codons present in the nucleic acid sequence of an IL-2 polypeptide are allocated to encode the unnatural amino acid so that it can be genetically incorporated into the IL-2 by using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA synthetase/tRNA pair is capable of charging a tRNA with an unnatural amino acid and is capable of incorporating that unnatural amino acid into the polypeptide chain in response to the codon. [0178] In some instances, the codon is the codon amber, ochre, opal or a quadruplet codon. In some cases, the codon corresponds to the orthogonal tRNA which will be used to carry the unnatural amino acid. In some cases, the codon is amber. In other cases, the codon is an orthogonal codon. Attorney Docket No.01183-0277-00PCT-SYN [0179] In some instances, the codon is a quadruplet codon, which can be decoded by an orthogonal ribosome ribo-Q1. In some cases, the quadruplet codon is as illustrated in Neumann, et al., “Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome,” Nature, 464(7287): 441-444 (2010), the disclosure of which is incorporated herein by reference. [0180] In some instances, a codon used in the present disclosure is a recoded codon, e.g., a synonymous codon or a rare codon that is replaced with alternative codon. In some cases, the recoded codon is as described in Napolitano, et al., “Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli,” PNAS, 113(38): E5588-5597 (2016), the disclosure of which is incorporated herein by reference. In some cases, the recoded codon is as described in Ostrov et al., “Design, synthesis, and testing toward a 57-codon genome,” Science 353(6301): 819-822 (2016), the disclosure of which is incorporated herein by reference. [0181] In some instances, unnatural nucleic acids are utilized leading to incorporation of one or more unnatural amino acids into the IL-2. Exemplary unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5- halo particularly 5-bromo, 5-trifiuoromethyl 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 unnatural 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 duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5- methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5- halocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and Attorney Docket No.01183-0277-00PCT-SYN guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7- deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine( [5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g.9- (2- aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5- b]indol-2-one), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrrolo[2,3- d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced with other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, , azacytosine, 5- bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5- iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5- bromouracil, 5-chlorouracil, 5- fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8- azaguanine, 5- hydroxycytosine, 2’-deoxyuridine, 2-amino-2’-deoxyadenosine, and those described in 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; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096; WO 99/62923; Kandimalla et al., (2001) Bioorg. Med. Chem.9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858- 859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Additional base modifications can be found, for example, in U.S. Pat. No.3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993; the disclosure of each of which is incorporated herein by reference. [0182] Unnatural nucleic acids comprising various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acids in some cases include one or several heterocyclic bases other than the principal five base components of naturally- occurring nucleic acids. For example, the heterocyclic base includes, in some cases, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4- aminopyrrolo [2.3-d] pyrimidin-5-yl, 2-amino-4-oxopyrolo [2, 3-d] pyrimidin-5-yl, 2- amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the nucleic acid via Attorney Docket No.01183-0277-00PCT-SYN the 9-position, the pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1-position. [0183] In some embodiments, nucleotide analogs are also modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those with modification at the linkage between two nucleotides and contains, for example, a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It is understood that these phosphate or modified phosphate linkage between two nucleotides are through a 3’-5’ linkage or a 2’-5’ linkage, and the linkage contains inverted 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 United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 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 disclosure of each of which is incorporated herein by reference. [0184] In some embodiments, unnatural nucleic acids include 2’,3’-dideoxy-2’,3’-didehydro- nucleosides (PCT/US2002/006460), 5’-substituted DNA and RNA derivatives (PCT/US2011/033961; Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020; PCT/JP2000/004720; PCT/JP2003/002342; PCT/JP2004/013216; PCT/JP2005/020435; PCT/JP2006/315479; PCT/JP2006/324484; PCT/JP2009/056718; PCT/JP2010/067560), or 5’-substituted monomers made as the monophosphate with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337); the disclosure of each of which is incorporated herein by reference. [0185] In some embodiments, unnatural nucleic acids include modifications at the 5’-position and the 2’-position of the sugar ring (PCT/US94/02993), such as 5’-CH2-substituted 2’-O- protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem.1999, 10, 921-924). In some cases, unnatural nucleic acids include amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3’ linked nucleoside in the dimer (5’ to 3’) comprises a 2’-OCH3 and a 5’-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Unnatural nucleic acids can include 2’-substituted 5’-CH2 (or Attorney Docket No.01183-0277-00PCT-SYN O) modified nucleosides (PCT/US92/01020). Unnatural nucleic acids can 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). Unnatural nucleic acids can include 5’-phosphonate monomers having a 2’-substitution (US2006/0074035) and other modified 5’-phosphonate monomers (WO1997/35869). Unnatural nucleic acids can include 5’-modified methylenephosphonate monomers (EP614907 and EP629633). Unnatural nucleic acids can include analogs of 5’ or 6’-phosphonate ribonucleosides comprising a hydroxyl group at the 5’ and/or 6’-position (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033). Unnatural nucleic acids can include 5’-phosphonate deoxyribonucleoside monomers and dimers having a 5’-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Unnatural nucleic acids can include nucleosides having a 6’-phosphonate group wherein the 5’ or/and 6’-position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640; and Hampton et al., J. Am. Chem. Soc, 1973, 95(13), 4404-4414). The disclosure of each reference listed in this paragraph is incorporated herein by reference. [0186] In some embodiments, unnatural nucleic acids also include modifications of the sugar moiety. In some cases, nucleic acids contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, nucleic acids comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, without limitation, addition of substituent groups (including 5’ and/or 2’ substituent groups; bridging of two ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, C1-C12 alkyl or a protecting group); and combinations thereof. Examples of chemically modified sugars can be found in WO2008/101157, US2005/0130923, and WO2007/134181, the disclosure of each of which is incorporated herein by reference. [0187] In some instances, a modified nucleic acid comprises modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group. Attorney Docket No.01183-0277-00PCT-SYN The sugar can be in a pyranosyl or furanosyl form. The sugar moiety may be the furanoside of ribose, deoxyribose, arabinose or 2’-O-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2’-alkoxy-RNA analogs, 2’-amino-RNA analogs, 2’-fluoro-DNA, and 2’-alkoxy- or amino-RNA/DNA chimeras. For example, a sugar modification may include 2’-O-methyl-uridine or 2’-O-methyl-cytidine. Sugar modifications include 2’-O-alkyl-substituted deoxyribonucleosides and 2’-O-ethyleneglycol like ribonucleosides. The preparation of these sugars or sugar analogs and the respective “nucleosides” wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base) is known. Sugar modifications may also be made and combined with other modifications. [0188] Modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2’ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10, alkyl or C2 to C10 alkenyl and alkynyl.2’ sugar modifications also include but are not limited to -O[(CH2)nO]m CH3, -O(CH2)nOCH3, - O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)nONH2, and -O(CH2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10. [0189] Other modifications at the 2’ position include but are not limited to: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3’ position of the sugar on the 3’ terminal nucleotide or in 2’-5’ linked oligonucleotides and the 5’ position of the 5’ terminal nucleotide. Modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures and which detail and describe a range of base modifications, such as U.S. Patent Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; Attorney Docket No.01183-0277-00PCT-SYN 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,700,920, the disclosure of each of which is incorporated herein by reference. [0190] Examples of nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5’-vinyl, 5’-methyl (R or S), 4’-S, 2’-F, 2’-OCH3, and 2’- O(CH2)2OCH3 substituent groups. The substituent at the 2’ position can also be selected from allyl, amino, azido, thio, O-allyl, O-(C1-C1O alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-O- N(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. [0191] In certain embodiments, nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4’ and the 2’ ribosyl ring atoms. In certain embodiments, nucleic acids provided herein include one or more bicyclic nucleic acids wherein the bridge comprises a 4’ to 2’ bicyclic nucleic acid. Examples of such 4’ to 2’ bicyclic nucleic acids include, but are not limited to, one of the formulae: 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 analogs thereof (see, U.S. Patent No.7,399,845); 4’- C(CH3)(CH3)-O-2’and analogs thereof, (see WO2009/006478, WO2008/150729, US2004/0171570, U.S. Patent No.7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134, and WO2008/154401). Also see, 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. U. S. A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219- 2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239- 243; U.S. Patent Nos.4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; International Publication Nos. WO2004/106356, WO1994/14226, WO2005/021570, WO2007/090071, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. Provisional Application Nos.60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and International Applications Nos. PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922, and PCT/DK98/00393. The disclosure of each reference listed in this paragraph is incorporated herein by reference. [0192] In certain embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids can be linked together using any inter nucleic acid linkage. The two main classes of inter nucleic acid linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing inter nucleic acid linkages include, but are not limited to, Attorney Docket No.01183-0277-00PCT-SYN phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing inter nucleic acid linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates. Unnatural nucleic acids can contain a single modification. Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties. [0193] Backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in any combination. Other non- phosphate linkages may also be used. [0194] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and/or enhance their stability in vivo. [0195] In some instances, a phosphorous derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like. Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found in Peyrottes et al., 1996, Nucleic Acids Res.24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res.24:2318-2323; Schultz et al., (1996) Nucleic Acids Res.24:2966- 2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview” in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, “Oligonucleoside Phosphorothioates” in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp.165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem.27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Patent No.5,453,496; and Micklefield, 2001, Curr. Med. Chem.8: 1157-1179; the disclosure of each of which is incorporated herein by reference. [0196] In some cases, backbone modification comprises replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group. Examples of such modifications include: anionic internucleoside linkage; N3’ to P5’ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and Attorney Docket No.01183-0277-00PCT-SYN thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179, the disclosure of which is incorporated herein by reference). A modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g. a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages. [0197] Substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Numerous United States patents disclose how to make and use these types of phosphate replacements and include but are not limited to U.S. Patent Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. It is also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). United States Patent Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is herein incorporated by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., 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), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol 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), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O- hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651- Attorney Docket No.01183-0277-00PCT-SYN 3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous United States patents teach the preparation of such conjugates and include, but are not limited to U.S. Patent Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941. The disclosure of each reference listed in this paragraph is incorporated herein by reference. [0198] In some cases, the unnatural nucleic acids further form unnatural base pairs. Exemplary unnatural nucleotides capable of forming an unnatural DNA or RNA base pair (UBP) under conditions in vivo includes, but is not limited to, TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. In some embodiments, unnatural nucleotides include:
Figure imgf000047_0001
. Exemplary unnatural base pairs include: (d)TPT3-(d)NaM; (d)5SICS-(d)NaM; (d)CNMO- (d)TAT1; (d)NaM-(d)TAT1; (d)CNMO-(d)TPT3; and (d)5FM-(d)TAT1. [0199] Other examples of unnatural nucleotides capable of forming unnatural UBPs that may be used to prepare the IL-2 conjugates disclosed herein may be found in Dien et al., J Am Chem Soc., 2018, 140:16115–16123; Feldman et al., J Am Chem Soc, 2017, 139:11427–11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res.2014, 42:10235-10244; Malyshev et al., Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc.2013, 135:5408-5419; and Malyshev Attorney Docket No.01183-0277-00PCT-SYN et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010; the disclosure of each of which is incorporated herein by reference. In some embodiments, unnatural nucleotides include: . [0200] In some
Figure imgf000048_0001
to prepare the IL-2 conjugates disclosed herein may be derived from a compound of the formula
Figure imgf000048_0002
wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azido; and the wavy line indicates a bond to a ribosyl or 2’-deoxyribosyl, wherein the 5’-hydroxy group of the ribosyl or 2’-deoxyribosyl moiety is in free form, is connected to a monophosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or is included in an RNA or a DNA or in an RNA analog or a DNA analog. [0201] In some embodiments, the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein may be derived from a compound of the Formula Attorney Docket No.01183-0277-00PCT-SYN wherein: each X is independently carbon or nitrogen; R2 is absent when X is nitrogen, and 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 secondary amine; E is oxygen, sulfur, or selenium; and the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety or an analog thereof, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof is in free form, is connected to a mono-phosphate, diphosphate, triphosphate, α- thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or is included in an RNA or a DNA or in an RNA analog or a DNA analog. [0202] 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. [0203] 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. [0204] In some embodiments, E is sulfur. In some embodiments, E is oxygen. In some embodiments, E is selenium. [0205] In some embodiments, R2 is present when X is carbon. In some embodiments, R2 is absent when X is nitrogen. In some embodiments, each R2, where present, is hydrogen. In some embodiments, R2 is alkyl, such as methyl, ethyl, or propyl. In some embodiments, R2 is alkenyl, such as -CH2=CH2. In some embodiments, R2 is alkynyl, such as 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, such as chloro, bromo, or fluoro. In some embodiments, R2 is cyano. In some embodiments, R2 is azide. [0206] In some embodiments, E is sulfur, Y is sulfur, and each X is independently carbon or nitrogen. In some embodiments, E is sulfur, Y is sulfur, and each X is carbon. Attorney Docket No. 01183-0277-00PCT-SYN [0207] In some embodiments, the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein may be derived from ,
Figure imgf000050_0001
Figure imgf000050_0002
Attorney Docket No.01183-0277-00PCT-SYN CH3 CH3
Figure imgf000051_0001
[0208] In some embodiments, an unnatural base pair generate an unnatural amino acid described in Dumas et al., “Designing logical codon reassignment – Expanding the chemistry in biology,” Chemical Science, 6: 50-69 (2015), the disclosure of which is incorporated herein by reference. [0209] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a synthetic codon comprising an unnatural nucleic acid. In some instances, the unnatural amino acid is incorporated into the cytokine by an orthogonal, modified Attorney Docket No.01183-0277-00PCT-SYN synthetase/tRNA pair. Such orthogonal pairs comprise an unnatural synthetase that is capable of charging the unnatural tRNA with the unnatural amino acid, while minimizing charging of a) other endogenous amino acids onto the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs. Such orthogonal pairs comprise tRNAs that are capable of being charged by the unnatural synthetase, while avoiding being charged with a) other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, Archaea, or human sources. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from two different organisms. In some embodiments, an orthogonal synthetase/tRNA pair comprising components that prior to modification, promote translation of two different amino acids. In some embodiments, an orthogonal synthetase is a modified alanine synthetase. In some embodiments, an orthogonal synthetase is a modified arginine synthetase. In some embodiments, an orthogonal synthetase is a modified asparagine synthetase. In some embodiments, an orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, an orthogonal synthetase is a modified cysteine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamine synthetase. In some embodiments, an orthogonal synthetase is a modified glutamic acid synthetase. In some embodiments, an orthogonal synthetase is a modified alanine glycine. In some embodiments, an orthogonal synthetase is a modified histidine synthetase. In some embodiments, an orthogonal synthetase is a modified leucine synthetase. In some embodiments, an orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, an orthogonal synthetase is a modified lysine synthetase. In some embodiments, an orthogonal synthetase is a modified methionine synthetase. In some embodiments, an orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, an orthogonal synthetase is a modified proline synthetase. In some embodiments, an orthogonal synthetase is a modified serine synthetase. In some embodiments, an orthogonal synthetase is a modified threonine synthetase. In some embodiments, an orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, an orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, an orthogonal synthetase is a modified valine synthetase. In some embodiments, an orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, an orthogonal tRNA is a modified alanine tRNA. In some embodiments, an orthogonal tRNA is a modified arginine tRNA. In some embodiments, an orthogonal tRNA is a modified asparagine tRNA. In some embodiments, an orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, an orthogonal tRNA is a modified cysteine tRNA. In some embodiments, an orthogonal tRNA is a modified glutamine tRNA. In some embodiments, an orthogonal tRNA is Attorney Docket No.01183-0277-00PCT-SYN a modified glutamic acid tRNA. In some embodiments, an orthogonal tRNA is a modified alanine glycine. In some embodiments, an orthogonal tRNA is a modified histidine tRNA. In some embodiments, an orthogonal tRNA is a modified leucine tRNA. In some embodiments, an orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, an orthogonal tRNA is a modified lysine tRNA. In some embodiments, an orthogonal tRNA is a modified methionine tRNA. In some embodiments, an orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, an orthogonal tRNA is a modified proline tRNA. In some embodiments, an orthogonal tRNA is a modified serine tRNA. In some embodiments, an orthogonal tRNA is a modified threonine tRNA. In some embodiments, an orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, an orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, an orthogonal tRNA is a modified valine tRNA. In some embodiments, an orthogonal tRNA is a modified phosphoserine tRNA. [0210] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus tRNACUA pairs, E. coli LeuRS (Ec-Leu)/B. stearothermophilus tRNACUA pairs, and pyrrolysyl-tRNA pairs. In some instances, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Mj- TyrRS/tRNA pair. Exemplary UAAs that can be incorporated by a Mj-TyrRS/tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives such as p- aminophenylalanine and p-methoyphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronophenylalanine; and o-nitrobenzyltyrosine. [0211] In some instances, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a Ec-Tyr/tRNACUA or a Ec-Leu/tRNACUA pair. Exemplary UAAs that can be incorporated by a Ec-Tyr/tRNACUA or a Ec-Leu/tRNACUA pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O- propargyltyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3- (naphthalene-2-ylamino)-2-amino-propanoic acid. [0212] In some instances, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a pyrrolysyl-tRNA pair. In some cases, the PylRS is obtained from an archaebacterial, e.g., from a methanogenic archaebacterial. In some cases, the PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated by a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2- Attorney Docket No.01183-0277-00PCT-SYN carboxamido)hexanoic acid, N-ε-D-prolyl-L-lysine, and N-ε-cyclopentyloxycarbonyl-L-lysine; N- ε-Acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine; and N- ε-(1-methylcyclopro-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein may be prepared by use of M. mazei tRNA which is selectively charged with a non-natural amino acid such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) by the M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS). Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647, the disclosure of which is incorporated herein by reference. [0213] In some instances, an unnatural amino acid is incorporated into a cytokine described herein (e.g., the IL polypeptide) by a synthetase disclosed in US 9,988,619 and US 9,938,516, the disclosure of each of which is incorporated herein by reference. [0214] The host cell into which the constructs or vectors disclosed herein are introduced is cultured or maintained in a suitable medium such that the tRNA, the tRNA synthetase and the protein of interest are produced. The medium also comprises the unnatural amino acid(s) such that the protein of interest incorporates the unnatural amino acid(s). In some embodiments, a nucleoside triphosphate transporter (NTT) from bacteria, plant, or algae is also present in the host cell. In some embodiments, the IL-2 conjugates disclosed herein are prepared by use of a host cell that expresses a NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter used in the host cell may be selected from TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some embodiments, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, the NTT is PtNTT1. In some embodiments, the NTT is PtNTT2. In some embodiments, the NTT is PtNTT3. In some embodiments, the NTT is PtNTT4. In some embodiments, the NTT is PtNTT5. In some embodiments, the NTT is PtNTT6. 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. [0215] The orthogonal tRNA synthetase/tRNA pair charges a tRNA with an unnatural amino acid and incorporates the unnatural amino acid into the polypeptide chain in response to the codon. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus tRNACUA pairs, E. coli LeuRS (Ec-Leu)/B. stearothermophilus tRNACUA pairs, and pyrrolysyl-tRNA pairs. Other Attorney Docket No.01183-0277-00PCT-SYN aaRS-tRNA pairs that may be used according to the present disclosure include those derived from M. mazei those described in Feldman et al., J Am Chem Soc., 2018140:1447–1454; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317–1322; the disclosure of each of which is incorporated herein by reference. [0216] In some embodiments are provided methods of preparing the IL-2 conjugates disclosed herein in a cellular system that expresses a NTT and a 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, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, and M. mazei. In some embodiments, the NTT is PtNTT1 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT2 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT4 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT5 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr)/B. stearothermophilus, or M. mazei. [0217] In some embodiments, the IL-2 conjugates disclosed herein may be prepared in a cell, such as E. coli, comprising (a) nucleotide triphosphate transporter PtNTT2 (including a truncated variant in which the first 65 amino acid residues of the full-length protein are deleted), (b) a plasmid comprising a double-stranded oligonucleotide that encodes an IL-2 variant having a desired amino acid sequence and that contains a unnatural base pair comprising a first unnatural nucleotide and a second unnatural nucleotide to provide a codon at the desired position at which an unnatural amino acid, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), will be incorporated, (c) a plasmid encoding a tRNA derived from M. mazei and which comprises an unnatural nucleotide to provide a recognized anticodon (to the codon of the IL-2 variant) in place of its native sequence, and (d) a plasmid encoding a M. barkeri derived pyrrolysyl-tRNA synthetase (Mb PylRS), which may be the same plasmid that encodes the tRNA or a different plasmid. In some embodiments, the cell is further supplemented with Attorney Docket No. 01183-0277-00PCT-SYN deoxyribo triphosphates comprising one or more unnatural bases. In some embodiments, the cell is further supplemented with ribo triphosphates comprising one or more unnatural bases. In some embodiments, the cell is further supplemented with one or more unnatural amino acids, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the double- stranded oligonucleotide that encodes the amino acid sequence of the desired IL-2 variant contains a codon AXC at position 64 of the sequence that encodes the protein having SEQ ID NO: 1, wherein X is an unnatural nucleotide. In some embodiments, the cell further comprises a plasmid, which may be the protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene from M. mazei that comprises an AXC-matching anticodon GYT in place of its native sequence, wherein Y is an unnatural nucleotide that is complementary and may be the same or different as the unnatural nucleotide in the codon. In some embodiments, the unnatural nucleotide in the codon is different than and complimentary to the unnatural nucleotide in the anti-codon. In some embodiments, the unnatural nucleotide in the codon is the same as the unnatural nucleotide in the anti-codon. In some embodiments, the first and second unnatural nucleotides comprising the unnatural base pair in the double-stranded oligonucleotide the
Figure imgf000056_0001
stranded oligonucleotide may be derived from some embodiments, the triphosphates of the
Figure imgf000056_0002
Attorney Docket No. 01183-0277-00PCT-SYN CH3
Figure imgf000057_0001
salts thereof. In some embodiments, the mRNA
Figure imgf000057_0002
a first unnatural nucleotide and a second unnatural nucleotide may comprise a codon comprising an unnatural nucleotide derived from some embodiments, the
Figure imgf000057_0003
that recognizes the codon comprising the unnatural nucleotide of the mRNA. The anti-codon in the M. mazei Attorney Docket No. 01183-0277-00PCT-SYN tRNA may comprise an unnatural nucleotide derived from ,
Figure imgf000058_0001
Figure imgf000058_0002
Figure imgf000058_0005
nucleotide derived from some embodiments, the mRNA comprises an
Figure imgf000058_0003
unnatural nucleotide derived from some embodiments, the mRNA
Figure imgf000058_0004
Attorney Docket No. 01183-0277-00PCT-SYN comprises an unnatural nucleotide derived from
Figure imgf000059_0001
the mRNA comprises an unnatural nucleotide derived from
Figure imgf000059_0002
embodiments, the mRNA comprises an unnatural nucleotide derived from . In some embodiments, the tRNA comprises an unnatural nucleotide
Figure imgf000059_0003
Figure imgf000059_0005
nucleotide derived from some embodiments, the tRNA comprises an
Figure imgf000059_0004
Attorney Docket No. 01183-0277-00PCT-SYN unnatural nucleotide derived from some embodiments, the tRNA
Figure imgf000060_0001
comprises an unnatural nucleotide derived from
Figure imgf000060_0002
the tRNA comprises an unnatural nucleotide derived from embodiments, the mRNA comprises an unnatural
Figure imgf000060_0003
Figure imgf000060_0005
derived from the tRNA comprises an unnatural nucleotide derived from
Figure imgf000060_0004
Attorney Docket No.01183-0277-00PCT-SYN . In some embodiments, the mRNA comprises an unnatural nucleotide
Figure imgf000061_0001
derived from the tRNA comprises an unnatural nucleotide derived
Figure imgf000061_0002
from some embodiments, the mRNA comprises an unnatural nucleotide
Figure imgf000061_0003
derived from the tRNA comprises an unnatural nucleotide derived from
Figure imgf000061_0004
. The host cell is cultured in a medium containing appropriate nutrients,
Figure imgf000061_0005
(a) the triphosphates of the deoxyribo nucleosides comprising one or more unnatural bases that are necessary for replication of the plasmid(s) encoding the cytokine gene harboring the codon, (b) the triphosphates of the ribo nucleosides comprising one or more unnatural bases necessary for transcription of (i) the mRNA corresponding to the coding sequence of the cytokine and containing the codon comprising one or more unnatural bases, and (ii) the tRNA containing the anticodon comprising one or more unnatural bases, and (c) the unnatural amino acid(s) to be incorporated in to the polypeptide sequence of the cytokine of Attorney Docket No.01183-0277-00PCT-SYN interest. The host cells are then maintained under conditions which permit expression of the protein of interest. [0218] The resulting AzK-containing protein that is expressed may be purified by methods known to those of ordinary skill in the art and may then be allowed to react with an alkyne, such as DBCO comprising a PEG chain having a desired average molecular weight as disclosed herein, under conditions known to those of ordinary skill in the art, to afford the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO 2019014262; WO 2019014267; WO 2019028419; and WO2019/028425; the disclosure of each of which is incorporated herein by reference. [0219] The resulting protein comprising the one or more unnatural amino acids, Azk for example, that is expressed may be purified by methods known to those of ordinary skill in the art and may then be allowed to react with an alkyne, such as DBCO comprising a PEG chain having a desired average molecular weight as disclosed herein, under conditions known to those of ordinary skill in the art, to afford the IL-2 conjugates disclosed herein. Other methods are known to those of ordinary skill in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO 2019014262; WO 2019014267; WO 2019028419; and WO2019/028425; the disclosure of each of which is incorporated herein by reference. [0220] Alternatively, an IL-2 polypeptide comprising an unnatural amino acid(s) is prepared by introducing the nucleic acid constructs described herein comprising the tRNA and aminoacyl tRNA synthetase and comprising a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons into a host cell. The host cell is cultured in a medium containing appropriate nutrients, is supplemented with (a) the triphosphates of the deoxyribo nucleosides comprising one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene harboring the new codon and anticodon, (b) the triphosphates of the ribo nucleosides required for transcription of the mRNA corresponding to (i) the cytokine sequence containing the codon, and (ii) the orthogonal tRNA containing the anticodon, and (c) the unnatural amino acid(s). The host cells are then maintained under conditions which permit expression of the protein of interest. The unnatural amino acid(s) is incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more unnatural amino acids are incorporated into the IL-2 polypeptide. Alternatively, two or more unnatural amino acids may be incorporated into the IL-2 polypeptide at two or more sites in the protein. Attorney Docket No.01183-0277-00PCT-SYN [0221] Once the IL-2 polypeptide incorporating the unnatural amino acid(s) has been produced in the host cell it can be extracted therefrom by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. The IL-2 polypeptide can be purified by standard techniques known in the art such as preparative ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or any other suitable technique known to those of ordinary skill in the art. [0222] Suitable host cells may include bacterial cells (e.g., E. coli, BL21(DE3)), but most suitably host cells are eukaryotic cells, for example insect cells (e.g., Drosophila such as Drosophila melanogaster), yeast cells, nematodes (e.g., C. elegans), mice (e.g., Mus musculus), or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells) or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Suitably, the host cell is a mammalian cell - such as a human cell or an insect cell. In some embodiments, the suitable host cells comprise E. coli. [0223] Other suitable host cells which may be used generally in the embodiments of the invention are those mentioned in the examples section. Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of well-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art. [0224] When creating cell lines, it is generally preferred that stable cell lines are prepared. For stable transfection of mammalian cells for example, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (for example, for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, or methotrexate. Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (for example, cells that have incorporated the selectable marker gene will survive, while the other cells die). [0225] In one embodiment, the constructs described herein are integrated into the genome of the host cell. An advantage of stable integration is that the uniformity between individual cells Attorney Docket No.01183-0277-00PCT-SYN or clones is achieved. Another advantage is that selection of the best producers may be carried out. Accordingly, it is desirable to create stable cell lines. In another embodiment, the constructs described herein are transfected into a host cell. An advantage of transfecting the constructs into the host cell is that protein yields may be maximized. In one aspect, there is described a cell comprising the nucleic acid construct or the vector described herein. Methods of Treatment [0226] In one aspect, provided herein are methods of stimulating and/or expanding certain immune cells that are useful for treatment of cancer, including, e.g., Teff cells, NK cells, and/or NKT cells, in a subject in need thereof, e.g., a subject having cancer, wherein such methods comprise administering to the subject an IL-2 conjugate as described herein at a first dose (a) about once every week for a first plurality of weeks, followed by a second dose (b) about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a second plurality of weeks as described herein. In some embodiments, such a cancer is a solid tumor. In some embodiments, such a cancer is a hematologic cancer. [0227] In another aspect, provided herein is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an IL-2 conjugate as described herein at a first dose (a) about once every week for a first plurality of weeks, followed by a second dose (b) about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a second plurality of weeks as described herein. [0228] In some embodiments of various aspects described herein, the first dose and/or the second dose are the same. In some embodiments of various aspects described herein, the first dose and/or the second dose are different. In some embodiments of various aspects described herein, the second dose is lower than the first dose. In some embodiments of various aspects described herein, the second dose is higher than the first dose. [0229] In some embodiments of various aspects described herein, the first dose and/or the second dose is about 8 μg/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments of various aspects described herein, the first dose and/or the second dose is about 16 μg/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments of various aspects Attorney Docket No.01183-0277-00PCT-SYN described herein, the first dose and/or the second dose is about 24 μg/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments of various aspects described herein, the first dose and/or the second dose is about 32 μg/kg IL-2 as an IL-2 conjugate as described herein. Cancer Types [0230] In some embodiments, the cancer is a solid tumor as described herein. In some embodiments, the cancer is a hematologic cancer, including, e.g., but not limited to leukemia, lymphoma, and multiple myeloma. In some embodiments, the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (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, esophageal squamous cell carcinoma (ESCC), glioblastoma, mesothelioma, breast cancer, triple- negative breast cancer, prostate cancer, castrate-resistant prostate cancer, metastatic castrate- resistant prostate cancer, or metastatic castrate-resistant prostate cancer having DNA damage response (DDR) defects, bladder cancer, ovarian cancer, tumors of moderate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), squamous cell skin cancer (SCSC), low- to non-expressing PD-L1 tumors, PD-L1-expressing tumors, tumors disseminated systemically to the liver and CNS beyond their primary anatomic originating site. [0231] In one aspect, the cancer is a solid tumor. In one aspect, the cancer is melanoma. In one aspect, the cancer is renal cell carcinoma (RCC). In one aspect, the cancer is a carcinoma such as an ovarian, colorectal, pancreatic, or hepatocellular carcinoma. [0232] In one aspect, provided herein is a method of treating a cancer (e.g., solid tumor or a hematologic cancer) in a subject in need thereof, comprising administering to the subject an IL-2 conjugate as described herein. In some embodiments, the method of treating the cancer (e.g., solid tumor or hematologic cancer) in a subject in need thereof comprises administering to the subject about 8 μg/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating the cancer (e.g., solid tumor or hematologic cancer) in a subject in need thereof comprises administering to the subject about 16 μg/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating the cancer (e.g., solid tumor or hematologic cancer) in a subject in need thereof comprises administering to the subject about 24 μg/kg IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating the cancer (e.g., solid tumor or hematologic cancer) in a subject in need thereof Attorney Docket No.01183-0277-00PCT-SYN comprises administering to the subject about 32 μg/kg IL-2 as an IL-2 conjugate as described herein. [0233] In a further aspect, provided herein is use of an IL-2 conjugate for the manufacture of a medicament for a method disclosed herein of treating cancer, including a solid tumor, for example, melanoma or renal cell carcinoma (RCC), in a subject in need thereof. [0234] The embodiments described in the following sections apply to any of the foregoing aspects. Administration [0235] In some embodiments, the IL-2 conjugate is administered as at least a second or subsequent line of therapy. In some embodiments, the IL-2 conjugate is administered as at least a third or subsequent line of therapy. In some embodiments, the IL-2 conjugate is administered as at least a fourth or subsequent line of therapy. In some embodiments, the IL-2 conjugate is administered as at least a fifth or subsequent line of therapy. [0236] In some embodiments, the prior line of therapy comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a LAG-3 inhibitor. [0237] In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, or intramuscular administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous administration. In some embodiments, the IL-2 conjugate is administered to the subject by subcutaneous administration. In some embodiments, the IL-2 conjugate is administered to the subject by intramuscular administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous administration. [0238] The IL-2 conjugate may be administered more than once, e.g., twice, three times, four times, five times, or more. In some embodiments, the duration of the treatment is up to 24 months, such as 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months or 24 months. In some embodiments, the duration of treatment is further extended by up to another 24 months. [0239] In some embodiments, the IL-2 conjugate is administered to a subject in need thereof about once every week, about once every two weeks, about once every three weeks, or about once every 4 weeks. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once every week. In some embodiments, the IL-2 conjugate is administered to a Attorney Docket No.01183-0277-00PCT-SYN subject in need thereof once every two weeks. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once every three weeks. In some embodiments, the IL- 2 conjugate is administered to a subject in need thereof once every 4 weeks. In some embodiments, the IL-2 conjugate is administered about once every 14, 15, 16, 17, 18, 19, 20, or 21 days. [0240] Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof about once every week for a first plurality of weeks (induction period) followed by about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a second plurality of weeks (maintenance period). In some embodiments, the plurality of weeks of the induction period is about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In some embodiments the plurality of weeks of the maintenance period is about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 16 weeks, about 18 weeks, about 20 weeks, about 30 weeks, about 40 weeks, about 50 weeks, about 60 weeks, about 70 weeks, about 80 weeks, about 90 weeks, or about 100 weeks. In some embodiments the plurality of weeks of the maintenance period ranges from about 4 weeks to about 100 weeks, or from about 6 weeks to about 100 weeks, or from about 6 weeks to about 98 weeks, or from about 6 weeks to about 46 weeks. In some embodiments the plurality of weeks of the maintenance period 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. [0241] In some embodiments, the IL-2 conjugate is administered to a subject in need thereof about once every week for about six weeks followed by about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for a plurality of weeks (maintenance period). In some embodiments, the plurality of weeks for the maintenance period is about 6 weeks, about 12 weeks, 18 weeks, 24 Attorney Docket No.01183-0277-00PCT-SYN weeks, about 30 weeks, about 36 weeks, about 42 weeks, about 48 weeks, about 54 weeks, about 60 weeks or longer. [0242] In some embodiments, the IL-2 conjugate is administered to a subject in need thereof at a dose of about 8 μg/kg IL-2 as the IL-2 conjugate, about 16 μg/kg IL-2 as the IL-2 conjugate, about 24 μg/kg IL-2 as the IL-2 conjugate, about 32 μg/kg IL-2 as the IL-2 conjugate at a frequency of about once every week for the first plurality of weeks. [0243] In some embodiments, the IL-2 conjugate is administered to a subject in need thereof at a dose of about 8 μg/kg IL-2 as the IL-2 conjugate, about 16 μg/kg IL-2 as the IL-2 conjugate, about 24 μg/kg IL-2 as the IL-2 conjugate, about 32 μg/kg IL-2 as the IL-2 conjugate at a frequency of about once every at least two weeks (including, e.g., about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks or longer) for the second plurality of weeks. [0244] In some embodiments, the IL-2 conjugate is administered to a subject in need thereof on days 1, 8, 15, 22, 29, and 36 ±1 day of a first 6-week cycle, followed by administration on days 1, 15, and 29 ±1 day 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. [0245] In some instances, the desired doses are conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day. [0246] In some embodiments, a method described herein further comprises administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent comprises one or more chemotherapeutic agent. In some embodiments, the chemotherapeutic agent comprises pemetrexed. In some embodiments, the chemotherapeutic agent comprises a platinum agent, such as carboplatin. In some embodiments, the chemotherapeutic agent comprises cisplatin. In some embodiments, the chemotherapeutic agent comprises nab- paclitaxel. In some embodiments, the chemotherapeutic agent comprises pemetrexed and a platinum agent, such as carboplatin. In some embodiments, the chemotherapeutic agent comprises pemetrexed and cisplatin. In some embodiments, the additional therapeutic agent comprises one or more immunotherapeutics. In some embodiments, the additional therapeutic agent comprises an antihistamine, such as diphenhydramine. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent and an antihistamine, such as diphenhydramine. In some embodiments, the additional therapeutic agent comprises any one of the foregoing chemotherapeutic agents and an antihistamine, such as diphenhydramine. Attorney Docket No.01183-0277-00PCT-SYN [0247] In some embodiments, the additional therapeutic agent comprises an analgesic, such as acetaminophen. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent and an analgesic, such as acetaminophen. In some embodiments, the additional therapeutic agent comprises any one of the foregoing chemotherapeutic agents and an analgesic, such as acetaminophen. [0248] 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 any one of the foregoing chemotherapeutic agents and one or more vitamins, such as folic acid and/or vitamin B12. [0249] In some embodiments, the additional therapeutic agent comprises an antihistamine and an analgesic, such as diphenhydramine and acetaminophen. In some embodiments, the additional therapeutic agent comprises an antihistamine and one or more vitamins, such as 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, such as 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, such as diphenhydramine, acetaminophen, and one or both of folic acid and vitamin B12. In any of the foregoing embodiments, the additional therapeutic agent can further comprise a chemotherapeutic agent, such as any one of the foregoing chemotherapeutic agents. Subject [0250] In some embodiments, administration of the IL-2 conjugate is to an adult subject. In some embodiments, the adult subject is a male. In other embodiments, the adult subject is a female. In some embodiments, the adult subject is at least age 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 subject has relapsed or refractory cancer, such as a relapsed or refractory solid tumor or a relapsed or refractory hematologic cancer. In some embodiments, the subject has relapsed or refractory melanoma or relapsed or refractory renal cell carcinoma (RCC). In some embodiments, the subject has refractory melanoma. In some embodiments, the subject has relapsed melanoma. In some embodiments, the subject has an advanced solid tumor. In some embodiments, the subject has a metastatic solid tumor. In some embodiments, the metastatic solid tumor is metastatic melanoma or metastatic renal cell carcinoma (RCC). Attorney Docket No.01183-0277-00PCT-SYN [0251] In some embodiments, the subject is ≥ 18 years of age. In some embodiments, the subject has received prior anti-cancer therapy (eg, radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy treatment. In some embodiments, the subject has received prior anti-cancer therapy and any treatment related toxicity is (1) resolved to Grade 0 or 1 (alopecia excepted) according to NCI CTCAE v5.0, or (2) resolved to at least Grade 2 according to NCI CTCAE v5.0. In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. In some embodiments, the subject has been determined (e.g., by a physician) to have a life expectancy greater than or equal to 12 weeks. In some embodiments, the subject has histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors with at least one tumor lesion with location accessible to safely biopsy as determined by a physician. In some embodiments, the subject has received at least one prior line of therapy for metastatic melanoma and/or does not have any standard of care (SoC) treatment option or participant declines or is intolerant to be treated with SoC treatment. In some embodiments, the subject has advanced or metastatic solid tumors and has either refused standard of care; or for whom no reasonable standard of care exists that would confer clinical benefit; or for whom standard therapy is intolerable, not effective, or not accessible. In some embodiments, the subject has measurable disease per RECIST v1.1 and at least 1 measurable lesion. In some embodiments, the subject has adequate laboratory parameters including: (1) Absolute lymphocyte count ≥0.5 times lower limit of normal, (2) Platelet count ≥100 × 109/L (3) Hemoglobin ≥9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for erythropoiesis-stimulating agent (ESA) and colony-stimulating factor (CSF) administration is sufficient), (4) Absolute neutrophil count ≥1.5 × 109/L (absence of growth factors within 2 weeks), (5) Prothrombin time (PT) and partial thromboplastin time (PTT) ≤1.5 times ULN, (6) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤2.5 times ULN except if liver metastases are present may be ≤5 times ULN, and (7) Total bilirubin ≤1.5 × ULN. In some embodiments, the subject meets each of the foregoing criteria. [0252] In some embodiments, the subject did not receive radiotherapy ≤14 days prior to first dose of IL-2 conjugate, or palliative radiation or stereotactic radiosurgery within 7 days prior to first dose of IL-2 conjugate. In some embodiments, the subject did not receive systemic anti- cancer therapy or an investigational agent within 2 weeks prior to first dose of IL-2 conjugate, or within 4 weeks for immunotherapy and tyrosine kinase inhibitor therapy. In some embodiments, the subject did not experience a Grade 3 or higher immune-related toxicity from prior immuno- oncology therapy. In some embodiments, the subject did not have major surgery ≤30 days prior to first dose of IL-2 conjugate, or did not recover to at least Grade 1 from adverse effects from Attorney Docket No.01183-0277-00PCT-SYN such procedure, or anticipates the need for major surgery during treatment with the IL-2 conjugate. In some embodiments, the subject does not have active autoimmune disease requiring systemic treatment within the past 3 months or does not have a documented history of clinically severe autoimmune disease that requires systemic steroids or immunosuppressive agents; or did not receive corticosteroids at a dose >10 mg daily of prednisone or equivalent (except inhaled, intranasal, intraocular, and topical steroids, or steroids as prophylaxis against contrast reactions, for disease assessments). In some embodiments, the subject does not have primary central nervous system (CNS) disease or leptomeningeal disease, known CNS metastases unless the subject has been treated, is asymptomatic, is without evidence of radiological progression for at least 8 weeks and has had no requirement for steroids or enzyme inducing anticonvulsants in the last 14 days prior to screening; abnormal pulmonary function within the previous 6 months, including pneumonitis, active pneumonitis, interstitial lung disease requiring the use of steroids, idiopathic pulmonary fibrosis, confirmed pleural effusion, severe dyspnea at rest or requiring supplementary oxygen therapy. In some embodiments, the subject does not have a history of allogenic or solid organ transplant. In some embodiments, the subject does not have uncontrolled diabetes mellitus or other uncontrolled immune-related endocrinopathies in the opinion of a physician; In some embodiments, the subject did not receive parenteral antibiotics ≤14 days prior to first dose of IL-2 conjugate, and does not have any serious systemic fungal, bacterial, viral, or other infection that is not controlled or requires IV antibiotics. In some embodiments, the subject does not have known human immunodeficiency virus (HIV) infection or active infection with hepatitis C, or a known uncontrolled hepatitis B virus (HBV) infection (unless the subject has received anti-HBV therapy before initiation of first dose of IL-2 conjugate and the subject has an HBV viral load <2000 IU/mL (104 copies/mL), or the subject has positive anti-HBc, positive anti HBs, negative HBsAg, and HBV virus load without HBV therapy). In some embodiments, the subject did not receive a live or live attenuated virus vaccination (except seasonal flu vaccines or SARS-CoV-2 vaccines that do not contain live virus) ≤14 days of treatment. In some embodiments, the subject did not experience clinically significant bleeding within 2 weeks prior to the initial IL-2 conjugate dose (eg, gastrointestinal bleeding, intracranial hemorrhage). In some embodiments, the subject did not have a prior diagnosis of deep vein thrombosis or pulmonary embolism within 3 months of screening. In some embodiments, the subject did not have a severe or unstable cardiac condition within 6 months prior to first dose of IL-2 conjugate, such as congestive heart failure (New York Heart Association Class III or IV), cardiac bypass surgery or coronary artery stent placement, angioplasty, cardiac ejection fraction below the lower limit of normal, unstable angina, medically uncontrolled hypertension (eg, ≥160 mm Hg systolic or ≥100 mm Hg diastolic), Attorney Docket No.01183-0277-00PCT-SYN uncontrolled cardiac arrhythmia requiring medication (≥ Grade 2, according to NCI CTCAE v5.0), or myocardial infarction. In some embodiments, the subject did not have a history of non- pharmacologically induced prolonged corrected QT interval determined using Fridericia's formula (QTcF) (>450 milliseconds (msec) for a male subject or >470 msec for a female subject. In some embodiments, the subject did not have known hypersensitivity or contraindications to any components of the IL-2 conjugate, PEG, pegylated drugs and E. coli derived-protein. In some embodiments, the subject did not have an active second malignancy, or history of previous malignancy that would impact the assessment of any study endpoints (except subjects with non-melanomatous skin cancer or cervical cancer that has been curatively surgically resected). In some embodiments, the subject did not have a serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concurrent medical illness that in the opinion of a physician would preclude protocol therapy or would make the subject inappropriate for the study, for example subjects with active SARS-CoV-2 (COVID-19) infections with clinically significant symptoms. In some embodiments, the subject is not pregnant or breast- feeding, or expecting to conceive or father children within the projected duration of the trial, starting with the screening visit through for at least 7 days for female subjects, and for at least 3 days for male subjects after the last dose of IL-2 conjugate. In some embodiments, the subject did not receive concurrent therapy with any other investigational agent, vaccine, or device (except concomitant participation in observational studies). In some embodiments, the subject did not have baseline oxygen saturation of <92%. In some embodiments, the subject does not have uveal or ocular or desmoplastic metastatic melanoma. In some embodiments, the subject does not have any of the characteristics listed in this paragraph. In some embodiments, the subject does not have any of the characteristics listed in this paragraph and meets each of the criteria in the preceding paragraph. [0253] In some embodiments, the subject is a female of childbearing potential and has (1) agreed to use a medically accepted method of birth control during the treatment and for at least 7 days after the last dose of the IL-2 conjugate, and (2) has a negative serum pregnancy test within 7 days prior to first dose of IL-2 conjugate. In some embodiments, the subject is a male who is not surgically sterile, and (1) has agreed to use medically accepted method of birth control during the treatment and for at least 3 days after the last dose of the IL-2 conjugate, and (2) has agreed to refrain from donating or banking sperm during the treatment period and for at least 3 days after last dose of the IL-2 conjugate. [0254] In some embodiments, the subject has no known hypersensitivity or contraindications to the IL-2 conjugate being administered or to PEG. In some embodiments, the subject has no Attorney Docket No.01183-0277-00PCT-SYN known hypersensitivity or contraindications to the IL-2 conjugate being administered, PEG, or pegylated drugs. [0255] In some embodiments, the subject does not have any serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concurrent medical illness that would preclude treatment or would make treatment inappropriate. [0256] In some embodiments, the subject is not pregnant or breastfeeding. In some embodiments, the subject is not expecting to conceive or father children during the course of the treatment and following up to 1, 2, 3, 4, 5, 6, or 7 months after administration of the final treatment dose. [0257] In some embodiments, the subject is not receiving a concurrent therapy with any investigational agent, vaccine, or device during the course of treatment. In some embodiments, the subject is receiving concurrent therapy during the course of treatment with an IL-2 conjugate described herein. In some embodiments, the subject is receiving concurrent therapy with an investigational agent, vaccine, or device during the course of treatment with an IL-2 conjugate described herein after physician approval. Effects of Administration In Accordance with Methods Described Herein [0258] In some embodiments, administration of the IL-2 conjugate provides a complete response, a partial response, or stable disease. [0259] In some embodiments, following administration of the IL-2 conjugate, the subject experiences a complete response (CR) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1. In some embodiments, following administration of the IL-2 conjugate, the subject experiences a partial response (PR) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1 In some embodiments, following administration of the IL-2 conjugate, the subject experiences stable disease (SD) as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1. [0260] In some embodiments, administration of the IL-2 conjugate to the subject does not cause vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2, Grade 3, or Grade 4 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 3 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade Attorney Docket No.01183-0277-00PCT-SYN 4 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause loss of vascular tone in the subject. [0261] In some embodiments, administration of the IL-2 conjugate to the subject does not cause extravasation of plasma proteins and fluid into the extravascular space in the subject. [0262] In some embodiments, administration of the IL-2 conjugate to the subject does not cause hypotension and reduced organ perfusion in the subject. [0263] In some embodiments, administration of the IL-2 conjugate to the subject does not cause impaired neutrophil function in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause reduced chemotaxis in the subject. [0264] In some embodiments, administration of the IL-2 conjugate to the subject is not associated with an increased risk of disseminated infection in the subject. In some embodiments, the disseminated infection is sepsis or bacterial endocarditis. In some embodiments, the disseminated infection is sepsis. In some embodiments, the disseminated infection is bacterial endocarditis. In some embodiments, the subject is treated for any preexisting bacterial infections prior to administration of the IL-2 conjugate. In some embodiments, the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate. [0265] In some embodiments, administration of the IL-2 conjugate to the subject does not exacerbate a pre-existing or initial presentation of an autoimmune disease or an inflammatory disorder in the subject. In some embodiments, the administration of the IL-2 conjugate to the subject does not exacerbate a pre-existing or initial presentation of an autoimmune disease in the subject. In some embodiments, the administration of the IL-2 conjugate to the subject does not exacerbate a pre-existing or initial presentation of an 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, oculo- bulbar myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome and bullous pemphigoid. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is Crohn’s disease. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is scleroderma. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is thyroiditis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is inflammatory arthritis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is diabetes mellitus. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is oculo-bulbar myasthenia gravis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is crescentic IgA Attorney Docket No.01183-0277-00PCT-SYN glomerulonephritis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cholecystitis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cerebral vasculitis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is Stevens-Johnson syndrome. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is bullous pemphigoid. [0266] In some embodiments, administration of the IL-2 conjugate to the subject does not cause changes in mental status, speech difficulties, cortical blindness, limb or gait ataxia, hallucinations, agitation, obtundation, or coma in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause seizures in the subject. In some embodiments, administration of the IL-2 conjugate to the subject is not contraindicated in subjects having a known seizure disorder. [0267] In some embodiments, administration of the IL-2 conjugate to the subject does not cause capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2, Grade 3, or Grade 4 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 2 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 3 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause Grade 4 capillary leak syndrome in the subject. [0268] In some embodiments, administration of the IL-2 conjugate to the subject does not cause a drop in mean arterial blood pressure in the subject following administration. In some embodiments, administration of the IL-2 conjugate to the subject does cause hypotension in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause the subject to experience a systolic blood pressure below 90 mm Hg or a 20 mm Hg drop from baseline systolic pressure. [0269] In some embodiments, administration of the IL-2 conjugate to the subject does not cause edema or impairment of kidney or liver function in the subject. [0270] In some embodiments, administration of the IL-2 conjugate to the subject does not cause eosinophilia in the subject. In some embodiments, administration of the IL-2 conjugate to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 500 per μL. In some embodiments, administration of the IL-2 conjugate to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 500 µL to 1,500 per μL. In some embodiments, administration of the the IL-2 conjugate to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 1,500 per μL to 5,000 per μL. In some embodiments, administration of the IL-2 conjugate to the subject does not cause the Attorney Docket No.01183-0277-00PCT-SYN eosinophil count in the peripheral blood of the subject to exceed 5,000 per μL. In some embodiments, administration of the IL-2 conjugate to the subject is not contraindicated in subjects on an existing regimen of psychotropic drugs. [0271] In some embodiments, administration of the IL-2 conjugate to the subject is not contraindicated in subjects on an existing regimen of nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs. In some embodiments, administration of the IL-2 conjugate to the subject is not contraindicated in subjects on an existing regimen of aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase. In some embodiments, administration of the IL-2 conjugate to the subject 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. [0272] In some embodiments, administration of the IL-2 conjugate to the subject does not cause one or more Grade 4 adverse events in the subject following administration. In some embodiments, Grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystoles; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; AV block second degree; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function tests abnormal; gastrointestinal hemorrhage; hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; alkaline phosphatase increase; blood urea nitrogen (BUN) increase; hyperuricemia; non-protein nitrogen (NPN) increase; respiratory acidosis; somnolence; agitation; neuropathy; paranoid reaction; convulsion; grand mal convulsion; delirium; asthma, lung edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; pupillary disorder; kidney function abnormal; kidney failure; and acute tubular necrosis. In some embodiments, administration of the IL-2 conjugate to a group of subjects does not cause one or more Grade 4 adverse events in greater than 1% of the subjects following administration. In some embodiments, Grade 4 adverse events are selected from hypothermia; shock; bradycardia; ventricular extrasystoles; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; AV block second degree; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function tests abnormal; gastrointestinal hemorrhage; hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; alkaline phosphatase increase; blood urea nitrogen (BUN) increase; hyperuricemia; non-protein nitrogen (NPN) increase; respiratory acidosis; somnolence; agitation; neuropathy; paranoid reaction; convulsion; grand mal convulsion; delirium; asthma, lung edema; hyperventilation; hypoxia; hemoptysis; Attorney Docket No.01183-0277-00PCT-SYN hypoventilation; pneumothorax; mydriasis; pupillary disorder; kidney function abnormal; kidney failure; and acute tubular necrosis. [0273] In some embodiments, administration of the IL-2 conjugate to a group of subjects does not cause one or more adverse events in greater than 1% of the subjects following administration, wherein the one or more adverse events is selected from duodenal ulceration; bowel necrosis; myocarditis; supraventricular tachycardia; permanent or transient blindness secondary to optic neuritis; transient ischemic attacks; meningitis; cerebral edema; pericarditis; allergic interstitial nephritis; and tracheo-esophageal fistula. [0274] In some embodiments, administration of the IL-2 conjugate to a group of subjects does not cause one or more adverse events in greater than 1% of the subjects following administration, wherein the one or more adverse events is selected from malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary emboli; stroke; intestinal perforation; liver or renal failure; severe depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure. [0275] In some embodiments, administration of the IL-2 conjugate to the subject stimulates CD8+ cells in a subject. In some embodiments, administration of the IL-2 conjugate to the subject stimulates NK cells in a subject. Stimulation may comprise an increase in the number of CD8+ cells in the subject, e.g., 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 comprise memory CD8+ cells. In some embodiments, the CD8+ cells comprise effector CD8+ cells. Stimulation may comprise an increase in the proportion of CD8+ cells that are Ki67 positive in the subject, e.g., about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. Stimulation may comprise an increase in the number of NK cells in the subject, e.g., about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. [0276] In some embodiments, CD8+ cells are expanded in the subject following administration of the IL-2 conjugate by at least 1.5-fold, such as by at least 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, or 2.5-fold. In some embodiments, NK cells are expanded in the subject following administration of the IL-2 conjugate by at least 3.5-fold, such as by at least 4.0-fold, 4.5-fold, 5.5-fold, 6-fold, 6.5-fold, 7.0-fold, or 7.5-fold. In some embodiments, eosinophils are expanded in the subject following administration of the IL-2 conjugate by no more than about 2-fold, such as no more than about 1.5-fold, 1.4-fold, or 1.3-fold. In some embodiments, CD4+ cells are expanded in the subject following administration of the IL-2 conjugate by no more than about 2-fold, such as no more than about 1.8-fold, 1.7-fold, or 1.6-fold. In some embodiments, the expansion of CD8+ cells and/or NK cells in the subject following administration of the IL-2 conjugate is greater than the Attorney Docket No.01183-0277-00PCT-SYN expansion of CD4+ cells and/or eosinophils. In some embodiments, the expansion of CD8+ cells is greater than the expansion of CD4+ cells. In some embodiments, the expansion of NK cells is greater than the expansion of CD4+ cells. In some embodiments, the expansion of CD8+ cells is greater than the expansion of eosinophils. In some embodiments, the expansion of NK cells is greater than the expansion of eosinophils. Fold expansion is determined relative to a baseline value measured before administration of the IL-2 conjugate. In some embodiments, fold expansion is determined at any of the times after administration, such as about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. [0277] In some embodiments, administration of the IL-2 conjugate to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral CD4+ regulatory T cells in the subject. In some embodiments, administration of the IL-2 conjugate to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject. In some embodiments, administration of the IL-2 conjugate to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of intratumoral CD8+ T and NK cells in the subject and without increasing the number of intratumoral CD4+ regulatory T cells in the subject. [0278] In some embodiments, administration of the IL-2 conjugate to the subject does not require the availability of an intensive care facility or skilled specialists in cardiopulmonary or intensive care medicine. In some embodiments, administration of the IL-2 conjugate to the subject does not require the availability of an intensive care facility or skilled specialists in cardiopulmonary or intensive care medicine. In some embodiments, administration of the IL-2 conjugate to the subject does not require the availability of an intensive care facility. In some embodiments, administration of the IL-2 conjugate to the subject does not require the availability of skilled specialists in cardiopulmonary or intensive care medicine. [0279] In some embodiments, administration of the IL-2 conjugate does not cause dose- limiting toxicity (DLT). In some embodiments, administration of the IL-2 conjugate causes dose-limiting toxicity (DLT). In some embodiments, a DLT is defined as an adverse event occurring within Day 1 through Day 42 (6 week-cycle) (inclusive) ±1 day of a treatment cycle that was not clearly or incontrovertibly solely related to an extraneous cause and that meets the criteria set forth in Example 4 for DLT. [0280] In some embodiments, administration of the IL-2 conjugate does not cause severe cytokine release syndrome. In some embodiments, the IL-2 conjugate does not induce anti-drug antibodies (ADAs), i.e., antibodies against the IL-2 conjugate. In some embodiments, a lack of induction of ADAs is determined by direct immunoassay for antibodies against PEG and/or Attorney Docket No.01183-0277-00PCT-SYN ELISA for antibodies against the IL-2 conjugate. An IL-2 conjugate is considered not to induce ADAs if a measured level of ADAs is statistically indistinguishable from a baseline (pre- treatment) level or from a level in an untreated control. Kits/Article of Manufacture [0281] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods and compositions described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic. [0282] A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions isalso typically be included. [0283] In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein. [0284] In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal 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 is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for drugs, or the approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Attorney Docket No.01183-0277-00PCT-SYN EXAMPLES [0285] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. Example 1. Preparation of pegylated IL-2 conjugates. [0286] An exemplary method with details for preparing IL-2 conjugates described herein is provided in this Example. [0287] IL-2 employed for bioconjugation was expressed as inclusion bodies in E. coli using methods disclosed herein, using: (a) an expression plasmid encoding (i) the protein with the desired amino acid sequence, which gene contains a first unnatural base pair to provide a codon at the desired position at which an unnatural amino acid N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) was incorporated and (ii) a tRNA derived from M. mazei Pyl, which gene comprises a second unnatural nucleotide to provide a matching anticodon in place of its native sequence; (b) a plasmid encoding a M. barkeri derived pyrrolysyl-tRNA synthetase (Mb PylRS), (c) N6-((2- azidoethoxy)-carbonyl)-L-lysine (AzK); and (d) a truncated variant of nucleotide triphosphate transporter PtNTT2 in which the first 65 amino acid residues of the full-length protein were deleted. The double-stranded oligonucleotide that encodes the amino acid sequence of the desired IL-2 variant contained a codon AXC as codon 64 of the sequence that encodes the protein having SEQ ID NO: 1 in which P64 is replaced with an unnatural amino acid described herein. The plasmid encoding an orthogonal tRNA gene from M. mazei comprised an AXC- matching anticodon GYT in place of its native sequence, wherein Y is an unnatural nucleotide as disclosed herein. X and Y were selected from unnatural nucleotides dTPT3 and dNaM as disclosed herein. The expressed protein was extracted from inclusion bodies and re-folded using standard procedures before site-specifically pegylating the AzK-containing IL-2 product using DBCO-mediated copper-free click chemistry to attach stable, covalent mPEG moieties to the AzK. Examplary reactions are shown in Schemes 1 and 2 (wherein n indicates the number of repeating PEG units). The reaction of the AzK moiety with the DBCO alkynyl moiety may afford one regioisomeric product or a mixture of regioisomeric products. Attorney Docket No.01183-0277-00PCT-SYN Scheme 1.
Attorney Docket No.01183-0277-00PCT-SYN Scheme 2.
Figure imgf000082_0002
Figure imgf000082_0001
Example 2. Preclinical Safety, PK, and PDy study of an IL-2 conjugate [0288] A study in cynomolgus monkeys was performed to evaluate the safety, PK, and PDy of IL-2 conjugate. The animals were dosed IV with IL-2 conjugate at 0.1 mg/kg on a QW, Q2W, Q3W, or Q4W for a total of 3 doses. The IL-2 conjugate comprised SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. This IL-2 conjugate can also be described as an IL-2 conjugate comprising Attorney Docket No.01183-0277-00PCT-SYN SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. The IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain. This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin. [0289] Expansion of peripheral CD8+ T cells peaked at Day 6. Peripheral CD8+ T cell proliferation was comparable across all dose schedules as of Day 6. With the QW dosing schedule the CD8+ T cell remained elevated. With the less frequent dosing schedules, the CD8+ T cells gradually returned to pre-dose levels by Day 11 and remained at this level until the subsequent dose (see Figure 1). There were no significant changes in clinical signs, body weight, blood pressure, and hematology. Troponin I levels in IL-2 conjugate-treated groups were comparable to that of the vehicle control group. These results support a conclusion that a QW regimen increases the likelihood of more sustained CD8+ T cell increases compared to Q2W and Q3W regimens. Example 3. Clinical Study of Biomarker Effects Following IL-2 Conjugate Administration (8, 16, 24, and 32 μg/kg [Q2W]; 8, 16, 24, 32, and 40 μg/kg [Q3W]). [0290] Studies were performed to characterize immunological effects of in vivo administration of the IL-2 conjugate. The IL-2 conjugate comprised SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. The IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain. This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin. The IL-2 conjugate was administered via IV infusion at a dose of 8, 16, 24, or 32 μg/kg (e.g., for about 30 minutes) every 2 weeks [Q2W], or was administered via IV infusion at a dose of 8, 16, 24, 32, or 40 μg/kg (e.g., for 30 minutes) every 3 weeks [Q3W]. [0291] Effects on the following biomarkers were analyzed as surrogate predictors of safety and/or efficacy: Attorney Docket No.01183-0277-00PCT-SYN Eosinophilia (elevated peripheral eosinophil count): Cell surrogate marker for IL-2-induced proliferation of cells (eosinophils) linked to vascular leak syndrome (VLS); Interleukin 5 (IL-5): Cytokine surrogate marker for IL-2 induced activation of type 2 innate lymphoid cells and release of this chemoattractant that leads to eosinophilia and potentially VLS; Interleukin 6 (IL-6): Cytokine surrogate marker for IL-2 induced cytokine release syndrome (CRS); and Interferon γ (IFN- γ): Cytokine surrogate marker for IL-2 induced activation of CD8+ cytotoxic T lymphocytes. [0292] Effects on the following biomarkers were analyzed as surrogate predictors of anti- tumor immune activity: Peripheral CD8+ Effector Cells: Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing a potentially latent therapeutic response; Peripheral CD8+ Memory Cells: Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing a potentially durable latent therapeutic and maintenance of the memory population; Peripheral NK Cells: Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing a potentially rapid therapeutic response; and Peripheral CD4+ Regulatory Cells: Marker for IL-2-induced proliferation of these target cells in the periphery that upon infiltration become a surrogate marker of inducing an immunosuppressive TME and offsetting of an effector-based therapeutic effect. [0293] Subjects were human males or females aged ≥18 years at screening. All subjects had been previously treated with an anti-cancer therapy and met at least one of the following: Treatment related toxicity resolved to grade 0 or 1 (alopecia excepted) according to NCI CTCAE v5.0; or Treatment related toxicity resolved to at least grade 2 according to NCI CTCAE v5.0 with prior approval of the Medical Monitor. The most common tumors were colorectal or melanoma. [0294] Subjects also met the following criteria: Provided informed consent. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Life expectancy greater than or equal to 12 weeks as determined by the Investigator. Histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors. Subjects with advanced or metastatic solid tumors who have refused standard of care; or for whom no reasonable standard of care exists that would confer clinical benefit; or for whom standard therapy is intolerable, not Attorney Docket No.01183-0277-00PCT-SYN effective, or not accessible. Measurable disease per RECIST v1.1. Adequate laboratory parameters including: Absolute lymphocyte count > 0.5 times lower limit of normal; Platelet count ≥ 100 × 109/L; Hemoglobin ≥ 9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for ESA and CSF administration is sufficient); Absolute neutrophil count ≥ 1.5 × 109/L (absence of growth factors within 2 weeks); Prothrombin time (PT) and partial thromboplastin time (PTT) ≤ 1.5 times upper limit of normal (ULN); Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 times ULN except if liver metastases are present may be ≤ 5 times ULN; Total bilirubin ≤ 1.5 × ULN. Premenopausal women and women less than 12 months after menopause had a negative serum pregnancy test within 7 days prior to initiating study treatment. [0295] Data regarding treatment emergent adverse events (TEAE) and efficacy biomarkers for the 8, 16, and 24 μg/kg [Q2W] cohorts are described as follows. First Cohort Using 8 μg/kg Dose ([Q2W]) [0296] Four individuals (4 [100%] male, 0 [0%] female, Caucasian, median age of 64 years, ranging from 49-70 years) having advanced or metastatic solid tumors received the IL-2 conjugate at a 8 μg/kg dose Q2W (1 dose per cycle). Tumor types included colorectal, pancreactic, and sarcoma. Here and throughout the cohorts of Example 3, drug mass per kg subject (e.g., 8 μg/kg) refers to IL-2 mass exclusive of PEG and linker mass. Treatment duration ranged from 1.4-9.0 months (2.0 months, median), and subjects received from 4-20 total doses (5.0 doses, median). [0297] Three of the subjects (75%) experienced at least one TEAE, all of which were Grade 1 or 2. No drug discontinuations resulted from TEAE, and there were no dose-limiting toxicities. One subject died as a result of disease progression (Grade 5 AE). No cumulative toxicity, end organ toxicity, or QTc prolongation or other cardiac toxicity was observed. In addition, there were no meaningful elevations in IL-5. TEAEs are detailed in Table 1. Table 1. Treatment Emergent Adverse Events (TEAE), 8 μg/kg [Q2W] (n=4) System Organ Class Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
Figure imgf000085_0001
Attorney Docket No.01183-0277-00PCT-SYN Inf ti n nd inf t ti n 1/4 (25%) 0/4 (0%) 1/4 (25%) 0/4 (0%) 0/4 (0%) [
Figure imgf000086_0001
] ff cacy omar ers. erp era + eff ce counts were measure ( . ), an peripheral NK cell counts are shown in FIG.3. Peripheral CD4+ Treg cell counts are shown in FIG.4. Peripheral lymphocyte cell counts are shown in FIG.5, and peripheral eosinophil cell counts are shown in FIG.6. [0299] Mean concentrations of the IL-2 conjugate after 1 and 2 cycles are shown in FIG.7A and FIG.7B, respectively. [0300] Cytokine levels (IFN-γ, IL-6, and IL-5) are shown in FIG.8. [0301] Accordingly, the IL-2 conjugate was generally well-tolerated. Overall, the results are considered to support non-alpha preferential activity of the IL-2 conjugate, with a tolerable safety profile, and preliminary evidence of activity in patients with immune-sensitive tumors. Second Cohort Using 16 μg/kg Dose ([Q2W]) Attorney Docket No.01183-0277-00PCT-SYN [0302] Four individuals having advanced or metastatic solid tumors received the IL-2 conjugate at a 16 μg/kg dose Q2W (1 dose per cycle). Tumor types included melanoma, prostate, and colon cancer. [0303] All 4 (100%) subjects experienced at least one TEAE; 3 of 4 (75%) patients experienced at least 1 Grade 3-4 related TEAEs (1 Grade 3 and 2 Grade 4). One subject experienced a Grade 3 lymphocyte count decrease, and 2 subjects experienced a Grade 4 lymphocyte count decrease (one with Grade 3 hypophosphatemia); the lymphocyte count decrease lasted 2 days. There were no related SAEs these subjects (one unrelated SAE of bowel obstruction). No drug discontinuations resulted from the TEAEs. No DLTs were observed. One patient was not evaluable for DLT since 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. Table 2. Treatment Emergent Adverse Events (TEAE), 16 μg/kg [Q2W] (n=4) System Organ Class Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 [
Figure imgf000087_0001
] ff cacy omar ers. erp era + eff ce counts were measure ( . ). e CD8+ expansion was about 2-fold, similar to the observed expansion of the first [Q2W] cohort (8 µg/kg dose). Peripheral NK cell counts are shown in FIG.10. The NK cell expansion was about 1- to 20-fold higher than the first [Q2W] cohort (8 µg/kg dose). Peripheral CD4+ Treg cell counts are shown in FIG.11. Peripheral eosinophil cell counts are shown in FIG.12. The CD4+ Attorney Docket No.01183-0277-00PCT-SYN Treg and eosinophil cell expansions were similar to the expansion of the first [Q2W] cohort (8 µg/kg dose). [0305] Cytokine levels (IFN-γ, IL-6, and IL-5) are shown in FIG.13. [0306] Mean concentrations of the IL-2 conjugate after 1 and 2 cycles are shown in FIG.14A and FIG.14B, respectively. [0307] Accordingly, the IL-2 conjugate demonstrated encouraging PD data and was generally well-tolerated. Overall, the results are considered to support non-alpha preferential activity of the IL-2 conjugate, with a tolerable safety profile, encouraging PD and preliminary evidence of activity in patients with immune-sensitive tumors. Third Cohort Using 24 μg/kg Dose ([Q2W]) [0308] Three individuals having advanced or metastatic solid tumors received the IL-2 conjugate at a 16 μg/kg dose Q2W (1 dose per cycle). Tumor types included melanoma and lung. [0309] All 3 (100%) subjects experienced at least one TEAE; 2 (33.3%) of 3 subjects experienced at least one Grade 3-4 related TEAEs (2 Grade 4). There were two instances of Grade 4 lymphocyte count decrease (one subject with Grade 1 transaminitis and Grade 1 decrease TSH). There were no DLTs. There were also no related SAEs. One subject required a dose hold to receive treatment for an adverse event of special interest (COVID-19 infection), and subsequent IL-2 conjugate treatment was discontinued as a result of PD. There were no drug discontinuations from TEAEs. One subject had a dose hold for C2D1 from GI bleed (gastric ulcer) unrelated to IL-2 conjugate treatment. TEAEs are detailed in Table 3. Table 3. Treatment Emergent Adverse Events (TEAE), 24 μg/kg [Q2W] (n=3) System Organ Class Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
Figure imgf000088_0001
Attorney Docket No.01183-0277-00PCT-SYN Metabolism and nutrition 1/3 33% 1/3 33% 0/3 0% 0/3 0% 0/3 0% [
Figure imgf000089_0001
] ccor ng y, t e - conjugate emonstrate encourag ng ata an was genera y well-tolerated. Overall, the results are considered to support non-alpha preferential activity of the IL-2 conjugate, with a tolerable safety profile, encouraging PD and preliminary evidence of activity in patients with immune-sensitive tumors. Example 4. Clinical Study of Solid Tumor Treatment Using an IL-2 Conjugate [0311] An open-label, multi-center Phase 1/2, dose escalation study assessing the clinical benefit of the IL-2 conjugate as a single agent in adult subjects with advanced or metastatic solid tumors is undertaken. [0312] The IL-2 conjugate comprises SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. The IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain. This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin. [0313] Participants received the IL-2 conjugate monotherapy once weekly (QW) for 6 weeks (induction period), and then every 2 weeks (Q2W) thereafter (maintenance period), which is also referred to as QW/Q2W. The dose regimen was IV infusion over 30 minutes QW on Day 1 of each week for 6 weeks (i.e., at D1, D8, D15, D22, D29 and D36) for dose escalation (induction), and Q2W of each cycle (Cycle 2 onwards, each cycle = 6 weeks) for dose expansion (maintenance). See FIG.15. [0314] During Cycle 1 (6 weekly doses), each subject was continuously monitored in the clinic for a minimum of 24 hours after study drug administration. The first subject enrolled into a dose level for each cohort was observed for a minimum of 72 hours before the second subject is enrolled at the same dose level. [0315] Dose-limiting toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) Version 5.0. Neurologic events occurring in the context of cytokine release syndrome were evaluated using CTCAE Attorney Docket No.01183-0277-00PCT-SYN Version 5.0 supplemented with the American Society for Transplantation and Cellular Therapy (ASTCT) criteria. [0316] The DLT assessment period consists of Day 1 through Day 42 (6 week-cycle) (inclusive) ±1 day. The administration of Cycle 2 Day 1 was excluded from the DLT observation period. If the patient doses early, the DLT observation period will end prior to C2D1 administration. [0317] A DLT is defined as any of the following events occurring during the first treatment cycle for which causal relationship to the IL-2 conjugate is determined to be at least possibly related: [0318] Hematologic toxicities • Grade 3 neutropenia (absolute neutrophil count [ANC] <1000/mm3 >500/mm3) lasting ≥7 days, or Grade 4 neutropenia of any duration • Grade 3+ febrile neutropenia • Grade 4+ thrombocytopenia (platelet count <25000/mm3) • Grade 3+ thrombocytopenia (platelet count <50000-25000/mm3) lasting ≥5 days, or associated with clinically significant bleeding or requiring platelet transfusion • Failure to meet recovery criteria of an ANC of at least 1000 cells/mm3 and a platelet count of at least 75000 cells/mm3 within 10 days • Any other Grade 4+ hematologic toxicity lasting ≥5 days [0319] Non-hematologic toxicities • Grade 3+ ALT or AST in combination with a bilirubin >2 times upper limit of normal (ULN) with no evidence of cholestasis or another cause such as viral infection or other drugs (ie, Hy’s law) • Grade 3 infusion-related reaction that occurs with premedication: Grade 4 infusion- related reaction • Grade 3 Vascular Leak Syndrome (VLS) defined as hypotension associated with fluid retention and pulmonary edema • Grade 3+ anaphylaxis • Grade 3+ hypotension • Grade 3+ cytokine release syndrome • Grade 3+ AE that does not resolve to Grade ≤2 within 7 days of starting accepted standard of care medical management The following Grade 3 non-hematologic AEs, are exceptions: Attorney Docket No.01183-0277-00PCT-SYN • Grade 3 fatigue, nausea, vomiting, or diarrhea that resolves to Grade ≤2 with optimal medical management in ≤3 days • Grade 3 fever (as defined by >40°C for ≤24 hours) • Grade 3 infusion-related reaction that occurs without premedication; subsequent doses should use premedication and if reaction recurs then it will be a DLT • Grade 3 arthralgia or rash that resolves to Grade ≤2 within 7 days of starting accepted standard of care medical management (eg, systemic corticosteroid therapy) For subjects with Grade 1 or 2 ALT or AST elevation at baseline considered secondary to liver metastases, a Grade 3 elevation must also be ≥3 times baseline and last >7 days. [0320] Participants received the IL-2 conjugate monotherapy QW/Q2W. The starting dose of the IL-2 conjugate was 16 µg/kg (DL1). Provided that DLT does not occur and escalation is permissible in view of an overall assessment of safety, dosage was escalated to 24 µg/kg (DL2) and subsequently to 32 µg/kg (DL3). A dose of 8 µg/kg (dose level -1) may be utilized if DL1 is determined to be unsafe. Here and throughout the examples and the specification described herein, drug mass per kg subject (e.g., 16 μg/kg) refers to IL-2 mass exclusive of PEG and linker mass. In other words, the presentation of drug mass per kg subject (e.g., “16 μg/kg” or “16 μg/kg IL-2 as the IL-2 conjugate” or “16 μg/kg of the IL-2 conjugate”) throughout the specification including the Examples described herein refers to the dose based on the IL-2 protein part of an IL-2 conjugate described herein. [0321] The following inclusion criteria apply. The participant must be willing and able to provide informed consent and comply with protocol requirements for the duration of the study. The participant must be ≥ 18 years of age. Prior anti-cancer therapy is allowed (eg, radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy treatment. If previously treated with any anti-cancer therapy, at least one of the following must be met: (1) treatment related toxicity resolved to Grade 0 or 1 (alopecia excepted) according to NCI CTCAE v5.0, and (2) treatment related toxicity resolved to at least Grade 2 according to NCI CTCAE v5.0 with prior approval of the Medical Monitor. Participants must have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Participants must have a life expectancy greater than or equal to 12 weeks as determined by the Investigator. Participants must have histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors with at least one tumor lesion with location accessible to safely biopsy per clinical judgment of the Investigator. Subjects with advanced or metastatic solid tumors who have refused standard of care; or for whom no reasonable standard of care exists that would confer clinical benefit; or for whom standard therapy is intolerable, not effective, or not accessible. Measurable disease per RECIST v1.1. Participants must have at least 1 measurable lesion. On-treatment biopsy may be Attorney Docket No.01183-0277-00PCT-SYN omitted upon written request from the Investigators considering biopsy clinical feasibility and upon written approval by the Sponsor. [0322] Participants must have adequate laboratory parameters including: (1) Absolute lymphocyte count ≥0.5 times lower limit of normal, (2) Platelet count ≥100 × 109/L (3) Hemoglobin ≥9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for erythropoiesis-stimulating agent (ESA) and colony-stimulating factor (CSF) administration is sufficient), (4) Absolute neutrophil count ≥1.5 × 109/L (absence of growth factors within 2 weeks), (5) Prothrombin time (PT) and partial thromboplastin time (PTT) ≤1.5 times ULN, (6) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤2.5 times ULN except if liver metastases are present may be ≤5 times ULN, and (7) Total bilirubin ≤1.5 × ULN. Females of childbearing potential and men who are not surgically sterile must agree to use medically accepted method of birth control during the study and for at least 7 days for females, and for at least 3 days for males after the last dose of study intervention. Female participants must have a negative serum pregnancy test within 7 days prior to initiating study treatment in premenopausal women and women less than 12 months after menopause. Male participants must agree to refrain from donating or banking sperm during the treatment period and for at least 3 days after last dose of study treatment. [0323] Participants are excluded from the study if any of the following criteria applies: – Radiotherapy ≤14 days prior to first dose of study drug (palliative radiation or stereotactic radiosurgery within 7 days prior to start of study treatment). – Treated with systemic anti-cancer therapy or an investigational agent within 2 weeks prior to start of study drug treatment (within 4 weeks for immunotherapy and tyrosine kinase inhibitor therapy). – Participants who experienced Grade 3 or higher immune-related toxicity from prior immuno- oncology therapy. – Major surgery ≤30 days prior to first dose of study drug, or has not recovered to at least Grade 1 from adverse effects from such procedure, or anticipation of the need for major surgery during study treatment. – Active autoimmune disease requiring systemic treatment within the past 3 months or have a documented history of clinically severe autoimmune disease that requires systemic steroids or immunosuppressive agents; subjects on corticosteroids are excluded for a dose >10 mg daily of prednisone or equivalent. Inhaled, intranasal, intraocular, and topical steroids are allowed. Steroids as prophylaxis against contrast reactions (for disease assessments) are also allowed. Attorney Docket No.01183-0277-00PCT-SYN – Primary central nervous system (CNS) disease or leptomeningeal disease; known CNS metastases unless treated, are asymptomatic, are without evidence of radiological progression for at least 8 weeks and have had no requirement for steroids or enzyme inducing anticonvulsants in the last 14 days prior to Screening. – Abnormal pulmonary function within the previous 6 months, including pneumonitis, active pneumonitis, interstitial lung disease requiring the use of steroids, idiopathic pulmonary fibrosis, confirmed pleural effusion, severe dyspnea at rest or requiring supplementary oxygen therapy. – History of allogenic or solid organ transplant. – Uncontrolled diabetes mellitus or other uncontrolled immune-related endocrinopathies in the opinion of the Investigator. – Parenteral antibiotic use ≤14 days prior to first dose of study drug, or any serious systemic fungal, bacterial, viral, or other infection that is not controlled or requires IV antibiotics. – Known human immunodeficiency virus (HIV) infection or active infection with hepatitis C: (a) known uncontrolled hepatitis B virus (HBV) infection: (i) anti-HBV therapy started before initiation of IMP and HBV viral load <2000 IU/mL (104 copies/mL) are eligible. The anti-HBV therapy should continue throughout the treatment period; (ii) positive anti-HBc, positive anti HBs, negative HbsAg, and HBV virus load without HBV therapy are eligible. – Received a live-virus vaccination ≤14 days prior to first dose of study drug. Seasonal flu and other inactivated vaccines that do not contain live virus are permitted. – Clinically significant bleeding within 2 weeks prior to the initial IL-2 conjugate dose (eg, gastrointestinal bleeding, intracranial hemorrhage). – Prior diagnosis of deep vein thrombosis or pulmonary embolism within 3 months. – Severe or unstable cardiac condition within 6 months prior to starting study treatment, such as congestive heart failure (New York Heart Association Class III or IV), cardiac bypass surgery or coronary artery stent placement, angioplasty, cardiac ejection fraction below the lower limit of normal, unstable angina, medically uncontrolled hypertension (eg, ≥160 mm Hg systolic or ≥100 mm Hg diastolic), uncontrolled cardiac arrhythmia requiring medication (≥ Grade 2, according to NCI CTCAE v5.0), or myocardial infarction. – History of non-pharmacologically induced prolonged corrected QT interval determined using Fridericia’s formula (QTcF) >450 milliseconds (msec) in males or >470 msec in females. – Known hypersensitivity or contraindications to any components of the IL-2 conjugate, PEG, pegylated drugs and E. coli derived-protein. Attorney Docket No.01183-0277-00PCT-SYN – Active second malignancy, or history of previous malignancy that would impact the assessment of any study endpoints. Subjects with non-melanomatous skin cancer or cervical cancer that has been curatively surgically resected are eligible. – Any serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concurrent medical illness that in the opinion of the Investigator would preclude protocol therapy or would make the subject inappropriate for the study: (a) patients with active SARS-CoV-2 (COVID-19) infections with clinically significant symptoms. – Is pregnant or breast-feeding, or expecting to conceive or father children within the projected duration of the trial, starting with the screening visit through for at least 7 days for females, and for at least 3 days for males after the last dose of study intervention. – Concurrent therapy with any other investigational agent, vaccine, or device. Concomitant participation in observational studies is acceptable after Sponsor approval. – Subjects with baseline oxygen saturation <92% are not eligible for enrollment. [0324] Safety Assessments: Safety was assessed throughout the study by the following: • Aes (type, incidence, severity, timing, seriousness, and relatedness) • Vital sign measurements (blood pressure, heart rate, respiratory rate, body temperature, pulse oximetry) • Physical examinations • Clinical laboratory tests • Electrocardiogram(s) • Concomitant medications and procedures, including all supportive care provided. [0325] Adverse events were assessed until approximately 90 days after the last dose of study drug(s) to monitor for delayed immune toxicities that have been associated with immunotherapy products, or until a subsequent anti-cancer treatment is administered (whichever comes first). [0326] Efficacy Assessments: Anti-tumor activity was determined by repeat radiological imaging and response assessed using RECIST 1.1 and iRECIST. All subjects must have scans performed using a modality appropriate for the subject’s tumor (eg, PET-CT scan, CT scans, MRI). All anatomical areas that are imaged during Screening should be assessed at every on- study time point using the same imaging modality. [0327] Study Endpoints [0328] Primary Endpoints • DLTs were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. Attorney Docket No.01183-0277-00PCT-SYN • The MTD of the IL-2 conjugate as a single agent was the highest tested dose level that has <33% probability of causing a DLT (ie, a dose level at which 0/6 or 1/6 subjects experience first-cycle DLT and at least 2/3 or 2/6 subjects experience first-cycle DLT at the next higher dose level). • The RP2D of the IL-2 conjugate as a single agent was based on available safety, tolerability, PK, and Pdy (which may include, but is not limited to, Ki67 expression in lymphocytes) data from different dose levels and schedules tested, but was no higher than the MTD. [0329] Secondary Endpoints: Clinical anti-tumor activity was assessed according to RECIST v1.1, as determined by the Investigator. • ORR, defined as the proportion of subjects with confirmed complete response (CR) or partial response (PR); a confirmed response is a response that persists on repeat-imaging ≥4 weeks after initial documentation of response. • DOR, defined as time from date of first objective response (either CR or PR) to first documentation of radiographic disease progression or death due to any cause, whichever occurs first. • PFS, defined as the time from first dose of the IL-2 conjugate to first documentation of radiographic disease progression or death due to any cause, whichever occurs first. • OS, defined as the time from first dose of the IL-2 conjugate to the date of death due to any cause. • TTR, defined as the time from first dose of the IL-2 conjugate to first documentation of objective response (either CR or PR). • DCR, defined as the proportion of subjects who have achieved CR, PR, or stable disease (duration of stable disease should be ≥ 3 months). • Percentage of subjects with no disease progression at 6 months post-treatment. Example 5. Clinical Study of Melanoma Treatment Using an IL-2 Conjugate. [0330] An open-label, multi-center Phase 1/2, dose expansion study assessing the clinical benefit of the IL-2 conjugate as a single agent in adult subjects with late-line metastatic melanoma is undertaken. [0331] The IL-2 conjugate comprises SEQ ID NO: 2, wherein position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), and a 30 kDa, linear mPEG chain. The IL-2 conjugate can also Attorney Docket No.01183-0277-00PCT-SYN be described as an IL-2 conjugate comprising SEQ ID NO: 1, wherein position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), and a 30 kDa, linear mPEG chain. This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegenzileukin. [0332] Based on 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 opened within the dose expansion phase. The goal will be to evaluate the anti-tumor activity, safety and PK/Pdy of the IL-2 conjugate monotherapy once weekly (QW) for 6 weeks (induction period), and then every 2 weeks (Q2W) thereafter (maintenance period). See FIG.16. Example 5 will include approximately 40 participants with late-line metastatic melanoma. DLTs will not apply. [0333] The following inclusion criteria apply. The participant must be willing and able to provide informed consent and comply with protocol requirements for the duration of the study. The participant must be ≥ 18 years of age. Prior anti-cancer therapy is allowed (eg, radiotherapy, chemotherapy, surgery, targeted therapy), including prior immunotherapy treatment. If previously treated with any anti-cancer therapy, at least one of the following must be met: (1) treatment related toxicity resolved to Grade 0 or 1 (alopecia excepted) according to NCI CTCAE v5.0, and (2) treatment related toxicity resolved to at least Grade 2 according to NCI CTCAE v5.0 with prior approval of the Medical Monitor. Participants must have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Participants must have a life expectancy greater than or equal to 12 weeks as determined by the Investigator. Participants must have histologically or cytologically confirmed diagnosis of advanced and/or metastatic solid tumors with at least one tumor lesion with location accessible to safely biopsy per clinical judgment of the Investigator. The participant must have received at least one prior line of therapy for metastatic melanoma and/or does not have any standard of care (SoC) treatment option or participant declines or is intolerant to be treated with SoC treatment. Subjects with advanced or metastatic solid tumors who have refused standard of care; or for whom no reasonable standard of care exists that would confer clinical benefit; or for whom standard therapy is intolerable, not effective, or not accessible. Measurable disease per RECIST v1.1. Participants must have at least 2 measurable lesions to safely perform mandatory pre & on-treatment biopsy. On-treatment biopsy may be omitted upon written request from the Investigators considering biopsy clinical feasibility and upon written approval by the Sponsor. [0334] Participants must have adequate laboratory parameters including: (1) Absolute lymphocyte count ≥0.5 times lower limit of normal, (2) Platelet count ≥100 × 109/L (3) Hemoglobin ≥9.0 g/dL (absence of growth factors or transfusions within 2 weeks; 1-week washout for erythropoiesis-stimulating agent (ESA) and colony-stimulating factor (CSF) Attorney Docket No.01183-0277-00PCT-SYN administration is sufficient), (4) Absolute neutrophil count ≥1.5 × 109/L (absence of growth factors within 2 weeks), (5) Prothrombin time (PT) and partial thromboplastin time (PTT) ≤1.5 times ULN, (6) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤2.5 times ULN except if liver metastases are present may be ≤5 times ULN, and (7) Total bilirubin ≤1.5 × ULN. Females of childbearing potential and men who are not surgically sterile must agree to use medically accepted method of birth control during the study and for at least 7 days for females, and for at least 3 days for males after the last dose of study intervention. Female participants must have a negative serum pregnancy test within 7 days prior to initiating study treatment in premenopausal women and women less than 12 months after menopause. Male participants must agree to refrain from donating or banking sperm during the treatment period and for at least 3 days after last dose of study treatment. [0335] Participants are excluded from the study if any of the following criteria applies: – Radiotherapy ≤14 days prior to first dose of study drug (palliative radiation or stereotactic radiosurgery within 7 days prior to start of study treatment). – Treated with systemic anti-cancer therapy or an investigational agent within 2 weeks prior to start of study drug treatment (within 4 weeks for immunotherapy and tyrosine kinase inhibitor therapy). – Participants who experienced Grade 3 or higher immune-related toxicity from prior immuno- oncology therapy. – Major surgery ≤30 days prior to first dose of study drug, or has not recovered to at least Grade 1 from adverse effects from such procedure, or anticipation of the need for major surgery during study treatment. – Active autoimmune disease requiring systemic treatment within the past 3 months or have a documented history of clinically severe autoimmune disease that requires systemic steroids or immunosuppressive agents; subjects on corticosteroids are excluded for a dose >10 mg daily of prednisone or equivalent. Inhaled, intranasal, intraocular, and topical steroids are allowed. Steroids as prophylaxis against contrast reactions (for disease assessments) are also allowed. – Primary central nervous system (CNS) disease or leptomeningeal disease; known CNS metastases unless treated, are asymptomatic, are without evidence of radiological progression for at least 8 weeks and have had no requirement for steroids or enzyme inducing anticonvulsants in the last 14 days prior to Screening. – Abnormal pulmonary function within the previous 6 months, including pneumonitis, active pneumonitis, interstitial lung disease requiring the use of steroids, idiopathic pulmonary Attorney Docket No.01183-0277-00PCT-SYN fibrosis, confirmed pleural effusion, severe dyspnea at rest or requiring supplementary oxygen therapy. – History of allogenic or solid organ transplant. – Uncontrolled diabetes mellitus or other uncontrolled immune-related endocrinopathies in the opinion of the Investigator. – Parenteral antibiotic use ≤14 days prior to first dose of study drug, or any serious systemic fungal, bacterial, viral, or other infection that is not controlled or requires IV antibiotics. – Known human immunodeficiency virus (HIV) infection or active infection with hepatitis C: (a) known uncontrolled hepatitis B virus (HBV) infection: (i) anti-HBV therapy started before initiation of IMP and HBV viral load <2000 IU/mL (104 copies/mL) are eligible. The anti-HBV therapy should continue throughout the treatment period; (ii) positive anti-HBc, positive anti HBs, negative HBsAg, and HBV virus load without HBV therapy are eligible. – Received a live-virus vaccination ≤14 days prior to first dose of study drug. Seasonal flu and other inactivated vaccines that do not contain live virus are permitted. – Clinically significant bleeding within 2 weeks prior to the initial IL-2 conjugate dose (eg, gastrointestinal bleeding, intracranial hemorrhage). – Prior diagnosis of deep vein thrombosis or pulmonary embolism within 3 months. – Severe or unstable cardiac condition within 6 months prior to starting study treatment, such as congestive heart failure (New York Heart Association Class III or IV), cardiac bypass surgery or coronary artery stent placement, angioplasty, cardiac ejection fraction below the lower limit of normal, unstable angina, medically uncontrolled hypertension (eg, ≥160 mm Hg systolic or ≥100 mm Hg diastolic), uncontrolled cardiac arrhythmia requiring medication (≥ Grade 2, according to NCI CTCAE v5.0), or myocardial infarction. – History of non-pharmacologically induced prolonged corrected QT interval determined using Fridericia's formula (QTcF) >450 milliseconds (msec) in males or >470 msec in females. – Known hypersensitivity or contraindications to any components of the IL-2 conjugate, PEG, pegylated drugs and E. coli derived-protein. – Active second malignancy, or history of previous malignancy that would impact the assessment of any study endpoints. Subjects with non-melanomatous skin cancer or cervical cancer that has been curatively surgically resected are eligible. Attorney Docket No.01183-0277-00PCT-SYN – Any serious medical condition (including pre-existing autoimmune disease or inflammatory disorder), laboratory abnormality, psychiatric condition, or any other significant or unstable concurrent medical illness that in the opinion of the Investigator would preclude protocol therapy or would make the subject inappropriate for the study: (a) patients with active SARS-CoV-2 (COVID-19) infections with clinically significant symptoms. – Is pregnant or breast-feeding, or expecting to conceive or father children within the projected duration of the trial, starting with the screening visit through for at least 7 days for females, and for at least 3 days for males after the last dose of study intervention. – Concurrent therapy with any other investigational agent, vaccine, or device. Concomitant participation in observational studies is acceptable after Sponsor approval. – Subjects with baseline oxygen saturation <92% are not eligible for enrollment. – Participants with uveal or ocular or desmoplastic metastatic melanoma. [0336] Participants will receive the IL-2 conjugate monotherapy QW/Q2W. The starting dose of the IL-2 conjugate will be the RP2D from Example 4. [0337] Safety Assessments: Safety will be assessed throughout the study by the following: • AEs (type, incidence, severity, timing, seriousness, and relatedness) • Vital sign measurements (blood pressure, heart rate, respiratory rate, body temperature, pulse oximetry) • Physical examinations • Clinical laboratory tests • Electrocardiogram(s) • Concomitant medications and procedures, including all supportive care provided. [0338] Adverse events will be assessed until approximately 90 days after the last dose of study drug(s) to monitor for delayed immune toxicities that have been associated with immunotherapy products, or until a subsequent anti-cancer treatment is administered (whichever comes first). [0339] Efficacy Assessments: Anti-tumor activity will be determined by repeat radiological imaging and response assessed using RECIST 1.1 and iRECIST. All subjects must have scans performed using a modality appropriate for the subject’s tumor (eg, PET-CT scan, CT scans, MRI). All anatomical areas that are imaged during Screening should be assessed at every on- study time point using the same imaging modality. [0340] Study Endpoints [0341] Primary Endpoints Attorney Docket No.01183-0277-00PCT-SYN [0342] Objective Response Rate (ORR), defined as the proportion of subjects with confirmed complete response (CR) or partial response (PR); a confirmed response is a response that persists on repeat-imaging ≥4 weeks after initial documentation of response. [0343] Secondary Endpoints • DOR, defined as time from date of first objective response (either CR or PR) to first documentation of radiographic disease progression or death due to any cause, whichever occurs first. • PFS, defined as the time from first dose of the IL-2 conjugate to first documentation of radiographic disease progression or death due to any cause, whichever occurs first. • OS, defined as the time from first dose of the IL-2 conjugate to the date of death due to any cause. • TTR, defined as the time from first dose of the IL-2 conjugate to first documentation of objective response (either CR or PR). • DCR, defined as the proportion of subjects who have achieved CR, PR, or stable disease (duration of stable disease should be ≥ 3 months). • Percentage of subjects with no disease progression at 6 months post-treatment. • Incidence of TEAEs, SAEs, laboratory abnormalities according to NCI CTCAE v 5.0 and ASTCT consensus gradings. Example 6. TEAE(s) by Preferred Term and Adverse Events of Specific Interest [0344] For participants receiving the IL-2 conjugate at a dose of 16, 24, or 32 μg/kg under the Q2W, Q3W, or QW/Q2W schedules, as described in Examples 3 and 4, data regarding TEAE(s) by preferred term (PT) (worst grade by participant) are summarized in Tables 4-6, respectively. [0345] In Tables 4-6, the adverse events of specific interest (AESI) and their data are underlined. If an adverse event is shown in a line in one table, but there is no corresponding line in another table, then that adverse event was not observed at any of the dose levels described 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 CLS of any grade was observed at any of the dose levels described in the Tables 4 and 5. [0346] As shown in Tables 4-6, incidences of Grade 3 or higher AESI are not high among the different schedules, and there was no observable increase in the TEAE profile among the different schedules. For example, as shown in Tables 4-6, there are no observable increase in incidences of the following adverse events of Grade 3 or higher: cytokine release syndrome, increased aspartate aminotransferase, increased alanine aminotransferase, increased blood bilirubin, infusion related reaction, hyperbilirubinaemia, and capillary leak syndrome, in the Attorney Docket No.01183-0277-00PCT-SYN QW/Q2W dosing schedule (at all tested dose levels), relative to the Q2W dosing schedule (at all tested dose levels), or the Q3W dosing schedule (at all tested dose levels). That is, while the QW/Q2W dosing schedule is more aggressive than the Q2W dosing schedule or the Q3W dosing schedule, surprisingly the QW/Q2W dosing schedule is relatively safe (e.g., with no observable increase in incidences of any of the aforementioned adverse events of Grade 3 or higher).
N Y S-T 3 = C > ) 8 ) 9 ) 3 ) ) P 6 3 ) 6 ) 6 ) 6 0 0-7 7 2 0-3 8 1 1 0. o N t e k c o D y e n r ot t A
Figure imgf000102_0001
p f c e d ) ) 5 ) 0 ) ) ) ) o m ) 6 a r 8 = . 2 . 5 6 5 . 0 2 2 1 0 . 5 5 . 5 2 0 2 . 1 2 1 0 0 0 % 1 Gl N ( ( ( ( ( ( ( ( l 5 2 1 2 1 1 r A eb mu N. 4 e d l e b s a d e a e T r s c a n e i r c e e m s n i d e o e s a r a r d e s e n f a s r r d e c e s a n d a i oi e ys n a f s e d t e r me t s a c a a e e r t n sa o a n r n c n s e r c d t u i a o o n i n r i d e e d e s e l i n c b m t t n a e t e n r m a i e m t y u ri a s n o a i l e u r c ) e v e e n e i t a c o l i n a r s n r o n c e k a t r e n t d i h b d t r e t oi e l t h %( n y n o t a p n p a m o e o s o p p u e t g i e A y s l C A A y L l y y f a B H H n I l P W N Y S-T C P 0 0-7 7 2 0-3 8 1 1 0. o N t e k c o D y e n r ot t A
Figure imgf000103_0001
8 n W2 e d = Q a r N 0 0 0 0 0 0 0 0 0 e c ( r e g p k G d / n gc s a e r d ) ) e m a 5 1 G = 0 0 0 0 0 . b 6 r 8 2 0 0 0 m u ll N ( 1 ( 1 n A = ) d % ( es n a e ; r 0 c . e 5 d n e o i n s o r e m v r o h d E e g s d A C n a e i t e a r s a T l n u si s a c i n e i r CI o t c e C i m o t i c t b m n d m e a u t N n , u s rt d i o r n i o s o b r h t b n c u 1 o . 6 2 n u r o i i e s t c y li A ) o y y h t i e g l i n % r d v n i b e c t o h p si t R ( ai h r r h p or i b D l o o p e s e p e p m t u e l d e n i B l e u B D l y y F H H y e L N h P M N Y S-T C P 0 0-7 7 2 0-3 8 1 1 0. o N t e k c o D y e n r ot t A
Figure imgf000104_0001
p f g o k / ) g c s e % ( m d ) ) 0 ) ) ) ) ) r 6 a r 6 = . 7 0 . 3 . 7 . 7 7 5 6 1 0 3 3 6 0 . 6 . 6 0 0 0 e 1 G b ll N ( ( ( ( 1 ( 1 ( 1 ( 3 1 2 1 1 1 m A u N d . e s d 5 a el e e r s b c a n e r a i d T e c s e e s n i ar m a e e e d d e l f o s r n d r c s e a r e s n s a oi a e a m a n r y d e t s t f s e t n r c c a r c n r o on e n i s u a n e a o r t i r i n d e b n a el c o n i e n i b n e t a i n s i i s t m n a e r t y m u ri o i s l e t a n oi y l ) e e v t e % e a t r n c i o a k h p e l i ni b n d e r t r n e r s o n a n i c d e t a ( e n y a n p s o t y my n a l o e s o l p y u f o o o l p y ni r A A C L A B H n I B H U N Y S-T C P 0 0-7 7 2 0-3 8 1 1 0. o N t e k c o D y e n r ot t A
Figure imgf000105_0001
/ g c s e m d ) ) 7 6 a 1 r 6 G = 0 0 0 0 0 0 . 6 1 0 0 0 0 0 l N ( ( l 1 A de s a e r c n i e d l n s e a s a d oi t a s a e h r d m e r e o s a d d e u l p c c s e s a n e e b r e o d r a c e s a s e a c o h p s u c e t s d r e c r c a v e r n n o i e r u e t t n e d e d a c il h a p r s u t s c s y r a n r o u o t n e f a f i t c n c li u i l e k l o h e r g u j s u n h o f p c o v r ) a p p n i n i r u e f o t e y t oi % d ( o d o d o h s d r e rt l e il r e n o l o o l l u a e m u v u e t a a u p B l B l B a F l F H H i L N l P Q u S NY S-T C P 0 0-7 7 2 0-3 8 1 1 0. o N t e k c o D y e n
Figure imgf000106_0001
r Q o r 1 W t t g k G A p y Q G A / E g c s T e e b d ) ) 7 n e g o d k / s a g e c d ) ) ) a ) 4 ) 0 0 0 ) . 0 . 0 . 0 .
Figure imgf000106_0002
m d 6 a ) 6 ) c a r 5 . 0 . 0 . 0 1 r G = 0 0 % ( % ( m G = N 4 ( 4 ( 0 0 2 ( ll N ( n; r e 6 1 ll ( 2 2 1 A 0 . b A 5 n m o i u sr N d e . 6 d e e s v e s a a e E l e r A b r c c n i d C a T T n i e e s e es a C n s e I o r it a a r r m c C e e f o r d N c a f s s n n n i d e , e n a r y s se s 1 . r a r t t o e s a a e 6 d e o n i s a n r i c 2 t e A a n t l e i m e l m a e r ) m d a t R n % s h D e r a e v n e t a t e n i ( n g n a i r e d e ) M % e oi ( y s n i n r a k n u f a p o t T W n l s A n I A A y C N Y S-T C P 0 0-7 7 2 0-3 8 1 1 0. o N t e k c o D y e n r ot t A
Figure imgf000107_0001
3 r W = 2 > ) e p e 5 = 0 0 0 0 0 0 d Q / d 0 0 0 a N n W r ( a r G e Q b g / s m k g e u c d a ) n m r 5 = = 0 0 0 0 0 0 0 0 0 ) 6 G % 1 ll N ( ( A n; 0 d . de d s e e 5 n a s s e a a o e e r i c s r e d d r e c r c ni n n i v i o E e s e it A d s e a a s e e e s s a a r r c a t e r a e r d C T e c r e n i a h p m f o c d r s n e C a si l I n t d i n e t s o u a g h d p n a r y t i ly m a C e m N a r , n i o u e s m n o n 1 . b c j n n k r a a t i l 6 u e il t o il e u b u a 2 i y c c a k l l l y g- h ril n o i A ) b o n i a r a s i d h b d al u l b r t a R D % p ( o u m r i o l i m p m ft e p n r e d e n o l B y l L i o B l B a a o y t C G H H n I M Attorney Docket No.01183-0277-00PCT-SYN Example 7. Modeling of Lymphocyte Dynamics [0347] For participants receiving the IL-2 conjugate at a dose of 16 or 24 μg/kg under the Q2W, Q3W, or QW schedules (where the QW schedule corresponds to the induction period of a QW/Q2W schedule), as described in Examples 3 and 4, a semi-mechanistic population pharmacokinetic/pharmacodynamic (PK/PD) model was developed to illustrate the intricate dynamics of the IL-2 conjugate-induced lymphocyte trafficking from blood to expansion sites immediately following administration, as well as the subsequent reappearance of expanded lymphocytes in the blood. [0348] The results of the model are shown in Figs.17A and 17B, which also include observed data. Fig.17A shows simulated (shaded areas and lines) and observed (dots) fold change in CD8+ T cell counts. Fig.17B shows simulated (shaded areas and lines) and observed (dots) fold change in NK cell counts. [0349] The model was developed to sufficiently capture the complexities inherent in the IL- 2 conjugate pharmacokinetics and pharmacodynamics. Notably, body weight emerged as a statistically significant covariate exerting influence on both clearance and volume terms. [0350] The lymphocyte re-circulation and expansion component of the model utilized the fold-change of the respective immune cells to explain the dynamics at play. Leveraging plasma concentrations to drive cell margination and migration, and individual area under the curve (AUC) values to dictate the exposure-response relationship governing cell expansion, the model adeptly captured the interplay between drug exposure and immune cell dynamics. [0351] Simulation activities showed the dose-dependent effects of the IL-2 conjugate on CD8+ T-cell and NK cell expansion dynamics. Notably, the IL-2 conjugate exhibited the ability to stimulate expansion of these immune cell populations. Also, it was observed that QW dosing schedule induced increased expansion of CD8+ T cells and NK cells over multiple cycles compared to the Q2W and Q3W schedules. Such data show that QW dosing schedule as the induction period (which is then followed by a maintenance period, e.g., every at least two weeks) has a higher chance to trigger a therapeutic effect compared to the Q2W and Q3W schedules as the induction period. Example 8. Observation of Lymphocyte Dynamics [0352] Blood was collected from participants receiving an IL-2 conjugate described herein (i) at a dose of 8, 16, 24, 32 μg/kg under the Q2W schedule, (ii) at a dose of 8, 16, 24, 32, or 40 μg/kg under the Q3W schedule, or (iii) at a dose of 16 or 24 μg/kg under the QW schedule (where the QW schedule corresponds to the induction period of a QW/Q2W schedule), as described in Examples 3 and 4. Specific antibodies against CD8 (for CD8+ T cells), CD56 (for Attorney Docket No.01183-0277-00PCT-SYN NK cells), FoxP3 (for regulatory T (Treg) cells) were used to recognize these cells in the collected blood by flow cytometry. [0353] Fig.18A shows fold change in CD8+ T cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 μg/kg in accordance with the indicated dosing schedule. Fig.18B shows fold change in NK cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 μg/kg in accordance with the indicated dosing schedule. [0354] Figures 19A-19C show flat modulation of peripheral CD4+ Treg cells across different tested dosing schedules. Fig.19A shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 8, 16, 24, or 32 μg/kg conjugate under the Q2W schedule. Fig.19B shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 8, 16, 24, 32, or 40 μg/kg under the Q3W schedule. Fig.19C shows fold change in Treg cell counts in blood collected from participants receiving the IL-2 conjugate at a dose of 16 or 24 μg/kg QW, as would be administered during part of the QW/Q2W schedule. [0355] As shown in these figures, administration of the IL-2 conjugate stimulated the expansion of peripheral CD8+ T and NK cell counts without significantly expanding CD4+ Treg cells. More frequent dosing during the induction period (e.g., using a QW/Q2W schedule) can induce increased CD8+ T cell expansion and NK cell expansion, as compared to a dosing schedule without a QW induction period (e.g., a Q2W or Q3W dosing schedule as described in Example 3). In addition, no increased Treg modulation was observed when dosing more frequently during the induction period (e.g., using a QW/Q2W schedule), as compared to a dosing schedule without a QW induction period (e.g., a Q2W or Q3W dosing schedule as described in Example 3). TABLE OF SEQUENCES [0356] The following Table of Sequences provide the sequences referenced in the claims and specification. SEQ ID Description Sequence
Figure imgf000109_0001
Attorney Docket No.01183-0277-00PCT-SYN [AzK_L1_PEG30kD] is N6-((2- NFHLRPRDLISNINVIVLELKGSETTFMCEY
Figure imgf000110_0001

Claims

Attorney Docket No.01183-0277-00PCT-SYN CLAIMS WHAT IS CLAIMED IS: 1. A method of treating a cancer in a subject in need thereof, comprising administering to the subject an IL-2 conjugate, wherein: the IL-2 conjugate comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 wherein the amino acid at position P64 is replaced by the structure of Formula (I):
Figure imgf000111_0001
– about 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure:
Figure imgf000111_0002
point of attachment to the preceding amino acid residue; and Attorney Docket No.01183-0277-00PCT-SYN X+1 indicates the point of attachment to the following amino acid residue; wherein the IL-2 conjugate is administered to the subject (a) about once every week for a first plurality of weeks, followed by (b) about once every at least two weeks for a second plurality of weeks. 2. The method of claim 1, wherein in the IL-2 conjugate the PEG group has an average molecular weight of about 30 kDa. 3. The method of claim 1 or 2, wherein in the IL-2 conjugate Z is CH2 and Y is
Figure imgf000112_0002
5. The method of claim 1 or 2, wherein in the IL-2 conjugate Z is CH2 and Y is
Figure imgf000112_0003
7. The method of claim 1 or 2, wherein the structure of Formula (I) has the structure of Formula (IV) or Formula (V), or is a mixture of Formula (IV) and Formula (V):
Figure imgf000112_0001
Attorney Docket No.01183-0277-00PCT-SYN
Figure imgf000113_0001
wherein: q is 1, 2, or 3; X is an L-amino acid having the structure:
Figure imgf000113_0002
point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. 8. The method of claim 1 or 2, wherein the structure of Formula (I) has the structure of Formula (XII) or Formula (XIII), or is a mixture of Formula (XII) and Formula (XIII):
Figure imgf000113_0003
wherein: Attorney Docket No.01183-0277-00PCT-SYN n is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 30 kDa; q is 1, 2, or 3; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced. 9. The method of any one of claims 1-8, wherein q is 1. 10. The method of any one of claims 1-8, wherein q is 2. 11. The method of any one of claims 1-8, wherein q is 3. 12. The method of any one of claims 1-11, wherein the cancer is a solid tumor. 13. The method of claim 12, wherein the solid tumor is an advanced or metastatic solid tumor. 14. The method of claim 12, wherein the solid tumor is a relapsed or refractory solid tumor, or the solid tumor has relapsed after one or more prior lines of systemic therapy for the solid tumor. 15. The method of any one of claims 12-14, further comprising selecting the subject to whom the IL-2 conjugate is administered at least in part on the basis of the subject having received one or more prior lines of systemic therapy for the solid tumor. 16. The method of any one of claims 12-15, wherein the subject has received one or more lines of systemic therapy for the solid tumor. 17. The method of claim 16, wherein the subject has received two or more lines of systemic therapy for the solid tumor. 18. The method of claim 17, wherein the subject has received three or more lines of systemic therapy for the solid tumor. 19. The method of claim 12, wherein the solid tumor is melanoma. 20. The method of claim 19, wherein the melanoma is metastatic melanoma. 21. The method of claim 19, wherein the melanoma is a relapsed or refractory melanoma, or the melanoma has relapsed after one or more prior lines of systemic therapy for the melanoma. 22. The method of any one of claims 19-21, further comprising selecting the subject to Attorney Docket No.01183-0277-00PCT-SYN whom the IL-2 conjugate is administered at least in part on the basis of the subject having received one or more prior lines of systemic therapy for the melanoma. 23. The method of any one of claims 19-22, wherein the subject has received one or more lines of systemic therapy for the melanoma. 24. The method of claim 23, wherein the subject has received two or more lines of systemic therapy for the melanoma. 25. The method of claim 24, wherein the subject has received three or more lines of systemic therapy for the melanoma. 26. The method of any one of claims 16-18 and 23-25, wherein the one or more prior lines of systemic therapy for the solid tumor or melanoma comprises an immune checkpoint inhibitor. 27. The method of claim 26, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a LAG-3 inhibitor. 28. The method of any one of claims 1-27, wherein a dose of about 8 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. 29. The method of any one of claims 1-27, wherein a dose of about 16 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. 30. The method of any one of claims 1-27, wherein a dose of about 24 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. 31. The method of any one of claims 1-27, wherein a dose of about 32 μg/kg IL-2 as the IL-2 conjugate is administered to the subject. 32. The method of any one of claim 1-27, comprising administering to the subject about 8 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. 33. The method of any one of claim 1-27, comprising administering to the subject about 16 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. 34. The method of any one of claim 1-27, comprising administering to the subject about 24 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. Attorney Docket No.01183-0277-00PCT-SYN 35. The method of any one of claim 1-27, comprising administering to the subject about 32 μg/kg IL-2 as the IL-2 conjugate about once every week for the first plurality of weeks. 36. The method of any one of claims 32-35, comprising administering to the subject about 8 μg/kg IL-2 as the IL-2 conjugate about once every at least two weeks for the second plurality of weeks. 37. The method of any one of claims 32-35, comprising administering to the subject about 16 μg/kg IL-2 as the IL-2 conjugate about once every at least two weeks for the second plurality of weeks. 38. The method of any one of claims 32-35, comprising administering to the subject about 24 μg/kg IL-2 as the IL-2 conjugate about once every at least two weeks for the second plurality of weeks. 39. The method of any one of claims 32-35, comprising administering to the subject about 32 μg/kg IL-2 as the IL-2 conjugate about once every at least two weeks for the second plurality of weeks. 40. The method of any one of claims 32-39, wherein the first plurality of weeks is about 5 weeks, about 6 weeks, or about 7 weeks. 41. The method of claim 40, wherein the first plurality of weeks is about 6 weeks. 42. The method of any one of claims 32-40, wherein the second plurality of weeks is at least about 6 weeks. 43. The method of any one of claims 32-40, wherein the second plurality of weeks is at least about 12 weeks. 44. The method of any one of claims 32-40, wherein the second plurality of weeks ranges from about 6 weeks to about 46 weeks. 45. The method of any one of claims 32-40, wherein the second plurality of weeks ranges from about 6 weeks to about 98 weeks. 46. The method of any one of claims 1-45, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. 47. The method of any one of claims 1-46, wherein the IL-2 conjugate is administered to the Attorney Docket No.01183-0277-00PCT-SYN subject by intravenous administration. 48. The method of any one of claims 1-46, wherein the IL-2 conjugate is administered to the subject by subcutaneous administration. 49. The method of any one of claims 1-48, further comprising administering acetaminophen to the subject. 50. The method of any one of claims 1-49, further comprising administering diphenhydramine to the subject. 51. The method of 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 of any one of claims 1-51. 53. Use of an IL-2 conjugate for the manufacture of a medicament for the method of any one of claims 1-51.
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