WO2022174101A1 - Skin cancer combination therapy with il-2 conjugates and cemiplimab - Google Patents

Skin cancer combination therapy with il-2 conjugates and cemiplimab Download PDF

Info

Publication number
WO2022174101A1
WO2022174101A1 PCT/US2022/016216 US2022016216W WO2022174101A1 WO 2022174101 A1 WO2022174101 A1 WO 2022174101A1 US 2022016216 W US2022016216 W US 2022016216W WO 2022174101 A1 WO2022174101 A1 WO 2022174101A1
Authority
WO
WIPO (PCT)
Prior art keywords
conjugate
subject
skin cancer
formula
cemiplimab
Prior art date
Application number
PCT/US2022/016216
Other languages
French (fr)
Inventor
Giovanni Abbadessa
Carolina E. CAFFARO
Brigitte Demers
Joseph LEVEQUE
Wan-Ju MENG
Jerod PTACIN
Original Assignee
Synthorx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Synthorx, Inc. filed Critical Synthorx, Inc.
Publication of WO2022174101A1 publication Critical patent/WO2022174101A1/en

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Landscapes

  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Disclosed herein are methods for treating skin cancer in a subject in need thereof, comprising administering IL-2 conjugates in combination with cemiplimab.

Description

SKIN CANCER COMBINATION THERAPY WITH IL-2 CONJUGATES AND
CEMIPLIMAB
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 63/149,081, filed on February 12, 2021, and U.S. Provisional Application No. 63/276,955, filed on November 8, 2021, the disclosure of each of which is hereby incorporated by reference in its entirety.
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. Accordingly, in one aspect, provided herein are methods of treating cancer in a subject comprising administering an IL-2 conjugate in combination with the anti-PD-1 antibody cemiplimab.
SUMMARY OF THE DISCLOSURE
[0006] Described herein are methods of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate and (b) cemiplimab, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1 having an unnatural amino acid residue described herein at position 64, e.g., the amino acid sequence of SEQ ID NO: 2. [0007] In some embodiments, administration of the IL-2 conjugate and cemiplimab provides improved results in treatment of skin cancer or a subtype thereof relative to existing therapies. For example, improved results may be in terms of the frequency of favorable outcomes, such as complete responses, elimination of target lesions, reduction of the size of target lesions, partial responses, stable disease, or slowing the growth of target lesions. In some embodiments, administration of the IL-2 conjugate and cemiplimab provides improved safety relative to existing skin cancer or IL-2 therapies, or to monotherapy using an IL-2 conjugate or cemiplimab alone. For example, improved safety may be in terms of avoidance or reduced frequency of adverse events, such as Grade 4 adverse events; vascular leak syndrome (e.g., Grade 2, Grade 3, and/or Grade 4 vascular leak syndrome); capillary leak syndrome; extravasation of plasma proteins and fluid into the extravascular space in the subject; hypotension and/or reduced organ perfusion in the subject; impaired neutrophil function in the subject; a drop in mean arterial blood pressure in the subject following administration; a systolic blood pressure below 90 mm Hg or a 20 mm Hg drop from baseline systolic pressure; eosinophilia; edema or impairment of kidney or liver function; or reduced chemotaxis in the subject. In some embodiments, improved safety may be in terms of absence of increased risk of disseminated infection in the subject; absence of exacerbation of a pre-existing or initial presentation of an autoimmune disease or an inflammatory disorder in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab provides improved results in terms of a combination of one or more of the favorable outcomes discussed above or disclosed elsewhere herein or frequencies thereof and one or more of the improvements in safety discussed above or disclosed elsewhere herein. [0008] Exemplary embodiments include the following.
[0009] Embodiment 1. A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cemiplimab, 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 imgf000004_0001
wherein:
Figure imgf000004_0002
Figure imgf000005_0001
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000005_0002
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0010] Embodiment 2. A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cemiplimab, wherein: the skin cancer is unresectable skin cancer, locally advanced cutaneous squamous cell carcinoma, or metastatic skin cancer; and 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 imgf000005_0003
wherein:
Figure imgf000005_0004
Figure imgf000006_0001
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000006_0002
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0011] Embodiment 3. A method of treating skin cancer in a subject in need thereof, comprising: selecting a subject having skin cancer, wherein the subject is selected on the basis of one or more attributes comprising (i) the skin cancer being unresectable skin cancer; (ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma; or (iii) the skin cancer being metastatic skin cancer; and administering to the subject (a) an IL-2 conjugate, and (b) cemiplimab, 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_0003
wherein:
Figure imgf000007_0001
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000007_0002
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0012] Embodiment 4. A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate and (b) cemiplimab, 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 imgf000007_0003
wherein:
Figure imgf000008_0001
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000008_0002
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0013] Embodiment 5. The method of any one of embodiments 1-4, wherein the skin cancer is melanoma.
[0014] Embodiment 6. The method of any one of embodiments 1-4, wherein the skin cancer is cutaneous squamous cell carcinoma.
[0015] Embodiment 7. The method of any one of embodiments 1-4, wherein the skin cancer is locally advanced cutaneous squamous cell carcinoma.
[0016] Embodiment 8. The method of any one of embodiments 1-7, wherein the skin cancer is unresectable.
[0017] Embodiment 9. The method of any one of embodiments 1-8, wherein the skin cancer is metastatic.
[0018] Embodiment 10. The method of any one of embodiments 1-9, wherein the skin cancer is not amenable to local therapy. [0019] Embodiment 11. The method of any one of embodiments 1-10, wherein the skin cancer is advanced.
[0020] Embodiment 12. The method of any one of embodiments 1-11, wherein the skin cancer is immune checkpoint inhibitor-naive.
[0021] Embodiment 13. The method of any one of embodiments 1-12, comprising administering to the subject about 8 μg/kg of the IL-2 conjugate.
[0022] Embodiment 14. The method of any one of embodiments 1-12, comprising administering to the subject about 16 μg/kg of the IL-2 conjugate.
[0023] Embodiment 15. The method of any one of embodiments 1-12, comprising administering to the subject about 24 μg/kg of the IL-2 conjugate.
[0024] Embodiment 16. The method of any one of embodiments 1-15, wherein in the IL-2 conjugate the PEG group has an average molecular weight of about 30 kDa.
[0025] Embodiment 17. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate Z is CH2 and Y is
Figure imgf000009_0001
[0026] Embodiment 18. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate Y is CH2 and Z is
Figure imgf000009_0002
[0027] Embodiment 19. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate Z is CH2 and Y is
Figure imgf000009_0004
[0028] Embodiment 20. The method of any one of embodiments 1-16, wherein in the IL-2 conjugate Y is CH2 and Z is
Figure imgf000009_0003
[0029] Embodiment 21. The method of any one of embodiments 1-16, 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 imgf000009_0005
Figure imgf000010_0001
wherein:
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000010_0002
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0030] Embodiment 22. The method of any one of embodiments 1-16, 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 imgf000010_0003
wherein: n is an integer such that - has a molecular weight of about 30 kDa;
Figure imgf000010_0004
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.
[0031] Embodiment 23. The method of any one of embodiments 1-22, wherein q is 1.
[0032] Embodiment 24. The method of any one of embodiments 1-23, wherein q is 2.
[0033] Embodiment 25. The method of any one of embodiments 1-24, wherein q is 3.
[0034] Embodiment 26. The method of any one of embodiments 1-25, wherein the IL-2 conjugate is administered to the subject about once every two weeks, about once every three weeks, or about once every 4 weeks.
[0035] Embodiment 27. The method of any one of embodiments 1-26, wherein the IL-2 conjugate and cemiplimab are administered to the subject about once every two weeks, about once every three weeks, or about once every 4 weeks.
[0036] Embodiment 28. The method of any one of embodiments 1-27, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.
[0037] Embodiment 29. The method of any one of embodiments 1-28, wherein cemiplimab is administered at a dose of about 350 mg every 3 weeks.
[0038] Embodiment 30. The method of any one of embodiments 1-29, wherein the IL-2 conjugate and cemiplimab are administered separately.
[0039] Embodiment 31. The method of embodiment 30, wherein the IL-2 conjugate and cemiplimab are administered sequentially.
[0040] Embodiment 32. The method of embodiment 30 or 31, wherein the IL-2 conjugate is administered before cemiplimab.
[0041] Embodiment 33. The method of embodiment 30 or 31, wherein the IL-2 conjugate is administered after cemiplimab.
[0042] Embodiment 34. The method of any one of embodiments 1-33, wherein the IL-2 conjugate is administered to the subject by intravenous administration.
[0043] Embodiment 35. The method of any one of embodiments 1-34, wherein the IL-2 conjugate and cemiplimab are administered to the subject by intravenous administration.
[0044] Embodiment 36. The method of any one of embodiments 1-35, further comprising administering acetaminophen to the subject.
[0045] Embodiment 37. The method of any one of embodiments 1-36, further comprising administering diphenhydramine to the subject.
[0046] Embodiment 38. The method of any one of embodiments 1-37, further comprising administering ondansetron to the subject. [0047] Embodiment 39. The method of any one of embodiments 36-38, wherein the acetaminophen, diphenhydramine, and/or ondansetron is administered to the subject before administering the IL-2 conjugate.
[0048] Embodiment 40. The method of any one of embodiments 1-39, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being unresectable skin cancer.
[0049] Embodiment 41. The method of any one of embodiments 1-40, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being the skin cancer being locally advanced cutaneous squamous cell carcinoma.
[0050] Embodiment 42. The method of any one of embodiments 1-41, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being metastatic skin cancer.
[0051] Embodiment 43. The method of any one of embodiments 1-42, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being not amenable to local therapy.
[0052] Embodiment 44. The method of any one of embodiments 1-43, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being immune checkpoint inhibitor-naive.
[0053] Embodiment 45. An IL-2 conjugate for use in the method of any one of embodiments 1-44.
[0054] Embodiment 46. Use of an IL-2 conjugate for the manufacture of a medicament for the method of any one of embodiments 1-45.
[0055] Embodiment 47. The method, IL-2 conjugate for use, or use of any one of the preceding embodiments, wherein the IL-2 conjugate is pegenzileukin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] 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:
[0057] FIG. 1A shows the change in peripheral CD8+ Teff counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. Here and elsewhere, designations such as “C1D1” indicate the treatment cycle and day (e.g., treatment cycle 1, day 1). “PRE” indicates the baseline measurement before administration; 24HR indicates 24 hours after administration; and so on.
[0058] FIG. IB shows the peak peripheral CD8+ Teff cell expansion following administration of the first dose of 24 μg/kg [Q3W] of the IL-2 conjugate. Data is normalized to pre-treatment (C1D1) CD8+ T cell count.
[0059] FIG. 1C shows the peripheral CD8+ Teff cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0060] FIG. 2 shows the percentage of CD8+ Teff cells expressing Ki67 in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0061] FIG. 3A shows the change in CD8+ memory cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0062] FIG. 3B shows CD8+ memory cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0063] FIG. 4A shows the change in peripheral natural killer (NK) cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0064] FIG. 4B shows the peak peripheral NK cell expansion following administration of the first dose of 24 μg/kg [Q3W] of the IL-2 conjugate. Data is normalized to pre-treatment (C1D1) NK cell count.
[0065] FIG. 4C shows the change in peripheral natural killer (NK) cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0066] FIG. 4D shows peripheral natural killer (NK) cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0067] FIG. 5 shows the percentage of NK cells expressing Ki67 in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0068] FIG. 6A shows the change in peripheral CD4+ Treg counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0069] FIG. 6B shows the peak peripheral CD4+ Treg cell expansion following administration of the first dose of 24 μg/kg [Q3W] of the IL-2 conjugate. Data is normalized to pre-treatment (C1D1) CD4+ T cell count.
[0070] FIG. 6C shows the peripheral CD4+ Treg cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0071] FIG. 7 shows the percentage of CD4+ Treg cells expressing Ki67 in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0072] FIG. 8A shows the change in eosinophil cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0073] FIG. 8B shows the peak peripheral eosinophil cell expansion following administration of the first dose of 24 μg/kg [Q3W] of the IL-2 conjugate. Data is normalized to pre-treatment (C1D1) eosinophil cell count.
[0074] FIG. 8C shows eosinophil cell counts in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate. [0075] FIG. 9A shows serum levels of IFN-g, IL-5, and IL-6 in the indicated subjects treated with 24 μg/kg [Q3W] of the IL-2 conjugate at specified times following administration of IL-2 conjugate.
[0076] FIG. 9B shows the serum level of IL-5 following administration of 24 μg/kg [Q3W] of IL-2 conjugate. BLQ = below limit of quantification. Data is plotted as mean (range BLQ to maximum value).
[0077] FIG. 9C shows the serum level of IL-6 following administration of 24 μg/kg [Q3W] of IL-2 conjugate. BLQ = below limit of quantification. Data is plotted as mean (range BLQ to maximum value).
[0078] FIG. 10A shows the change in peripheral CD8+ Teff counts in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab.
[0079] FIG. 10B shows the change in peak peripheral CD8+ Teff cell expansion following administration of the first dose of IL-2 conjugate and pembrolizumab. Data is normalized to pre- treatment (C1D1) CD8+ T cell count. Listed values indicate median fold changes.
[0080] FIG. IOC shows the change in peripheral CD8+ Teff counts in the indicated subjects at specified times following administration of 16 μg/kg IL-2 conjugate and pembrolizumab. [0081] FIG. 11 shows the percentage of CD8+ Teff cells expressing Ki67 in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab.
[0082] FIG. 12A shows the change in peripheral natural killer (NK) cell counts in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab.
[0083] FIG. 12B shows the change in peak peripheral NK cell expansion following administration of the first dose of IL-2 conjugate and pembrolizumab. Data is normalized to pre- treatment (C1D1) NK cell count. Listed values indicate median fold changes.
[0084] FIG. 12C shows the change in peripheral natural killer (NK) cell counts in the indicated subjects at specified times following administration of 16 μg/kg IL-2 conjugate and pembrolizumab.
[0085] FIG. 13 shows the percentage of NK cells expressing Ki67 in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab.
[0086] FIG. 14A shows the change in peripheral CD4+ Treg counts in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab.
[0087] FIG. 14B shows the change in peak peripheral CD4+ Treg cell expansion following administration of the first dose of IL-2 conjugate and pembrolizumab. Data is normalized to pre- treatment (C1D1) CD4+ T cell count. Listed values indicate median fold changes.
[0088] FIG. 14C shows the change in peripheral CD4+ Treg counts in the indicated subjects at specified times following administration of 16 μg/kg IL-2 conjugate and pembrolizumab.
[0089] FIG. 15 shows the percentage of CD4+ Treg cells expressing Ki67 in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab.
[0090] FIG. 16A shows the change in eosinophil cell counts in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab.
[0091] FIG. 16B shows the change in peak peripheral eosinophil cell expansion following administration of the first dose of IL-2 conjugate and pembrolizumab. Data is normalized to pre- treatment (C1D1) eosinophil cell count. Listed values indicate median fold changes.
[0092] FIG. 16C shows the change in eosinophil cell counts in the indicated subjects at specified times following administration of 16 μg/kg IL-2 conjugate and pembrolizumab.
[0093] FIG. 17A shows serum levels of IFN-g, IL-5, and IL-6 in the indicated subjects at specified times following administration of 8 μg/kg IL-2 conjugate and pembrolizumab. [0094] FIG. 17B shows the serum level of IL-5 following administration of 8 μg/kg IL-2 conjugate and pembrolizumab. BLQ = below limit of quantification. Data is ploted as mean (range BLQ to maximum value).
[0095] FIG. 17C shows the serum level of IL-6 following administration of 8 μg/kg IL-2 conjugate and pembrolizumab. BLQ = below limit of quantification. Data is ploted as mean (range BLQ to maximum value).
[0096] FIG. 17D shows serum levels of IFN-g, IL-5, and IL-6 in the indicated subjects at specified times following administration of 16 μg/kg IL-2 conjugate and pembrolizumab.
[0097] FIG. 18A and FIG. 18B show mean concentrations of the IL-2 conjugate, administered at a dose of 8 μg/kg with pembrolizumab, after 1 and 2 cycles, respectively.
[0098] FIG. 18C and FIG. 18D show mean concentrations of the IL-2 conjugate, administered at a dose of 16 μg/kg with pembrolizumab, after 1 and 2 cycles, respectively.
[0099] FIG. 19 shows the peripheral CD8+ Teff cell counts in the indicated subjects at specified times following administration of 24 μg/kg IL-2 conjugate and pembrolizumab.
[0100] FIG. 20 shows the peripheral NK cell counts in the indicated subjects at specified times following administration of 24 μg/kg IL-2 conjugate and pembrolizumab.
[0101] FIG. 21 shows the change in peripheral CD4+ Treg cell counts in the indicated subjects at specified times following administration of 24 μg/kg IL-2 conjugate and pembrolizumab. [0102] FIG. 22 shows the peripheral eosinophil cell counts in the indicated subjects at specified times following administration of 24 μg/kg IL-2 conjugate and pembrolizumab.
[0103] FIG. 23A and FIG. 23B show mean concentrations of the IL-2 conjugate, administered at a dose of 24 μg/kg with pembrolizumab, after 1 and 2 cycles, respectively.
[0104] FIG. 24 shows the levels of IFN-g, IL-6, and IL-5 in the indicated subjects treated with 24 μg/kg of the IL-2 conjugate and pembrolizumab at specified times following administration of the IL-2 conjugate.
[0105] FIG. 25A shows serum levels of IFN-g, IL-5, and IL-6 in the indicated melanoma subjects at specified times following administration of IL-2 conjugate. BLQ = below limit of quantification.
[0106] FIG. 25B shows serum levels of IFN-g, IL-5, and IL-6 in the indicated basal cell carcinoma (BCC) subjects at specified times following administration of IL-2 conjugate. BLQ = below limit of quantification.
[0107] FIG. 26 shows a graph of anti-tumor activity of Compound A dosed IV on a QWx3 Schedule from Study 1 in Example 5. Black arrows denote days of dosing with Compound A. [0108] FIG. 27 shows a graph of tumor volumes with Compound A dosed IV on a QWx3 Schedule from Study 1 in Example 5. [0109] FIG. 28 shows tumor volumes on Day 15 post treatment for each animal treated QWx3 dosing with Compound A from Study 1 in Example 5. Black arrows denote days of dosing with Compound A.
[0110] FIG. 29 shows tumor volumes on Day 15 post treatment for each animal with Q2Wx2 dosing with Compound A from Study 1 in Example 5.
[0111] FIG. 30 shows mean tumor growth curves from treatment of mice with vehicle, 6 mg/kg Compound A as a single agent, anti-PD-1 antibody as a single agent, and the combination of 6 mg/kg Compound A and anti-PD-1 antibody from Study 2 of Example 5. Black arrows denote days of dosing with Compound A.
[0112] FIG. 31 shows a graph of %TGI data on Day 15 post treatment in the group treated with the combination of Compound A and anti-PD-1 antibody, compared to the groups treated with vehicle, Compound A alone or the anti-PD-1 antibody alone from Study 2 of Example 5. *p<0.05, **p<0.01, and ***p<0.01; vs. vehicle control. vs. anti-PD-1 antibody.
Figure imgf000017_0002
#p<0.05 vs. Compound A. Data represent mean tumor volume ± SEM (14 mice/group).
[0113] FIG. 32 shows a graph of Kaplan-Meier survival curves for treatment groups from Study 2 of Example 5. *p<0.05 vs. vehicle control vs. anti-PD-1 antibody. #p<0.05 vs.
Figure imgf000017_0001
Compound A.
[0114] FIG. 33 represents mean tumor growth curves when Compound A was dosed a single agent at 1 mg/kg, 3 mg/kg, 6 mg/kg, and 9 mg/kg in Study 3 of Example 5. Data represent mean tumor volume ± SEM (14 mice/group; except 12 mice/group for 9 mg/kg Compound A). Black arrows denote days of Compound A dosing.
[0115] FIG. 34 represent individual tumor volumes on Day 15 post-treatment from Study 3 of Example 5. Data represent individual tumor volumes; the mean ± SEM and %TGI compared to the vehicle control are also displayed. ***p<0.01 vs. vehicle control.
[0116] FIG. 35 shows a graph of Kaplan-Meier survival curves for treatment groups treated with vehicle (control), anti-PD-1 antibody alone, Compound A alone, and the combination of Compound A and anti-PD-1 antibody. *p<0.05 vs. vehicle control from Study 3 of Example 5. "Lp<0.05 vs. anti-PD-1 antibody. #p<0.05 vs. Compound A.
[0117] Fig. 36 shows serum levels of the indicated cytokines in the indicated subjects treated with 8 μg/kg [Q3W] at specified times following IL-2 conjugate administration.
[0118] Fig. 37 shows serum levels of the indicated cytokines in the indicated subjects treated with 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration.
[0119] Figs. 38A-D show eosinophil cell counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration as measured by cytometry or CBC (complete blood count). [0120] Figs. 39A-D show lymphocyte counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration as measured by cytometry or CBC.
[0121] Figs. 40A-D show peripheral CD8+ Teff counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration. [0122] Figs. 41A-B show the percentage of CD8+ Teff cells expressing Ki67 in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration.
[0123] Figs. 42A-B show peripheral memory CD8+ counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration.
[0124] Figs. 43A-D show peripheral natural killer (NK) cell counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration.
[0125] Figs. 44A-B show the percentage of NK cells expressing Ki67 in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration.
[0126] Figs. 45A-B show peripheral CD4+ Treg counts in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration. [0127] Figs. 46A-B show the percentage of CD4+ Treg cells expressing Ki67 in the indicated subjects treated with 8 μg/kg [Q3W] or 16 μg/kg [Q3W] at specified times following IL-2 conjugate administration.
DETAILED DESCRIPTION OF THE DISCLOSURE
Definitions
[0128] 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.
[0129] 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.
[0130] 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%.
[0131] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[0132] 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).
[0133] 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.
[0134] 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.
[0135] 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), deoxy cytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine diphosphate (dADP), thymidine diphosphate (dTDP), deoxy cytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxy cytidine monophosphate (dCMP), and 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.
[0136] 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.
[0137] 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.
[0138] 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 substituents. A nucleoside can be a glycoside compound formed through glycosidic linking between a nucleic acid base and a reducing group of a sugar.
[0139] 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.”
[0140] As used herein, “dose-limiting toxicity” (DLT) is defined as an adverse event occurring within Day 1 through Day 29 (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 2 for DLT.
[0141] 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.
[0142] As used herein, “cemiplimab” refers to the human anti-PD-1 antibody marketed under the brand name “Libtayo” by Regeneron Pharmaceuticals, Inc. and Sanofi-Aventis.
[0143] 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
[0144] Interleukin 2 (IL-2) is a pleiotropic type-1 cytokine whose structure comprises a 15.5 kDa four a-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 CD 122), and IL-2Ry (also known as CD 132). 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-2Ry, forms the “high- affinity” IL-2 receptor complex with a Kd of about >10-11 M.
[0145] In some instances, IL-2 signaling via the “high-affinity” IL-2Rαβy 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 naive T cells in the periphery (pTreg cells). In some cases, Treg cells are considered as the mediator of 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.
[0146] IL-2 signaling via the “intermediate-affinity” IL-2Rβy 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.
[0147] In some instances, IL-2 signaling is utilized to modulate T cell responses and subsequently for treatment of a cancer. For example, 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).
[0148] Adoptive cell therapy enables physicians to effectively harness a patient’s own immune cells to fight diseases such as proliferative disease (e.g., cancer) as well as infectious disease. The effect of IL-2 signaling may be further enhanced by the presence of additional agents or methods in combination therapy. For example, programmed cell death protein 1, also known as PD-1 or CD279, is a cell surface receptor expressed on T cells and pro-B cells which plays a role in regulating the immune system’s response to the cells of the human body. PD-1 down-regulates the immune system and promotes self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases but can also prevent the immune system from killing cancer cells. PD-1 guards against autoimmunity through two mechanisms. First, PD-1 promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, PD-1 reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). Cemiplimab is a human anti-PD-1 antibody that can block PD-1, activate the immune system to attack tumors, and is approved for treatment of certain squamous cell skin cancers. [0149] Provided herein are methods of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cemiplimab.
[0150] In some embodiments, the IL-2 sequence comprises the sequence of SEQ ID NO: 1: PTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFSQSIISTLT (SEQ ID NO: 1) wherein the amino acid at position P64 is replaced by the structure of Formula (I):
Figure imgf000023_0001
wherein:
Figure imgf000023_0002
Y is CH2 and Z is
Figure imgf000024_0001
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000024_0002
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0151] 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.
[0152] 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.
[0153] In some embodiments of Formula (I), Z is CH2 and Y is
Figure imgf000024_0003
In some embodiments of Formula (I), Y is CH2 and Z is
In some embodiments of Formula (I), Z is CH2 and Y is
Figure imgf000024_0004
Figure imgf000025_0001
. In some embodiments of Formula (I), Y is CH2 and Z is
Figure imgf000025_0002
[0154] In some embodiments of Formula (I), q is 1. In some embodiments of Formula (I), q is 2. In some embodiments of Formula (I), q is 3.
[0155] 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.
[0156] In some embodiments of Formula (I), q is 1 and structure of Formula (I) is the structure of Formula (la):
Figure imgf000025_0003
wherein:
Figure imgf000025_0004
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; X is an L-amino acid having the structure:
Figure imgf000026_0001
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue. [0157] In some embodiments of Formula (la), Z is CH2 and Y is
. In some embodiments of Formula (la), Y is CEB and Z is
In other embodiments of Formula (la), Z is CH2 and Y is
. In some embodiments of Formula (la), Y is CH2 and Z is
Figure imgf000026_0002
[0158] In some embodiments of Formula (la), the PEG group has an average molecular weight of about 30 kDa.
[0159] In some embodiments, the IL-2 conjugate comprises the sequence of SEQ ID NO: 2: PTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKIAzK LI PEG30kDlLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 2) wherein [AzK_Ll_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 (IV a) 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.
[0160] The ratio of regioisomers generated from the click reaction is about 1 : 1 or greater than 1:1.
[0161] PEGs will 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.
[0162] 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 Ci-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.
[0163] In some embodiments, the PEG group is a linear or branched PEG group. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. For example, 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.
[0164] In some embodiments, the IL-2 conjugate comprises 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 imgf000027_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 imgf000028_0001
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0165] 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.
[0166] 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.
[0167] 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).
[0168] 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 imgf000028_0002
Formula (Va); wherein:
W is a PEG group having an average molecular weight of about 25 kDa - 35kDa; and X has the structure:
Figure imgf000029_0001
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
[0169] In some embodiments of Formula (IV a) or Formula (Va), or a mixture of Formula (IV a) and Formula (Va), the PEG group has an average molecular weight of about 30 kDa. [0170] 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 (IV a) 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). [0171] In some embodiments, the IL-2 conjugate 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):
Figure imgf000029_0002
wherein: h is is an integer such that has a molecular weight of about 25 kDa - 35 kDa;
Figure imgf000030_0003
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.
[0172] 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.
[0173] 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 has a molecular
Figure imgf000030_0002
weight of about 30 kDa.
[0174] 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). [0175] 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 (Xlla), and the structure of Formula (XIII) is the structure of Formula (XIIIa):
Figure imgf000030_0001
wherein: n is is an integer such that -(OCFECH2VOCFE has a molecular weight of about 25 kDa - 35 kDa; and the wavy lines indicate convalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced.
[0176] In some embodiments of Formula (Xlla) or Formula (XIIIa), or a mixture of Formula (Xlla) and Formula (XIIIa), n is is an integer such that
Figure imgf000031_0002
has a molecular weight of about 30 kDa.
[0177] In any of the embodiments described herein, the structure of Formula (I) has the structure of Formula (Xlla) or Formula (XIIIa), or is a mixture of Formula (Xlla) and Formula (Xllla). In some embodiments, the structure of Formula (I) has the structure of Formula (Xlla). In some embodiments, the structure of Formula (I) has the structure of Formula (Xllla). In some embodiments, the structure of Formula (I) is a mixture of Formula (Xlla) and Formula (Xllla). [0178] In some embodiments, the IL-2 conjugate 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):
Figure imgf000031_0001
wherein: 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 - 35 kDa; and the wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not replaced.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] In some embodiments, m and p are each 2.
[0183] 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). [0184] In some embodiments, the IL-2 conjugate 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):
Figure imgf000032_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.
[0185] 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.
[0186] 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.
[0187] 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).
Conjugation Chemistry
[0188] 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 imgf000033_0001
[0189] In some embodiments, the conjugating moiety comprises a PEG group as described herein. In some embodiments, a reactive group comprises an alkyne or azide. [0190] 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 imgf000034_0001
[0191] 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 imgf000034_0002
[0192] 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.
Figure imgf000034_0003
[0193] In some embodiments, a conjugation reaction described herein comprises a cycloaddition reaction between an azide moiety, such as that contained in a protein containing an amino acid residue derived from
Figure imgf000034_0004
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. Scheme V.
Figure imgf000035_0001
Scheme VI.
Figure imgf000036_0001
[0194] 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. IL-2 Polypeptide Production
[0195] 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.
[0196] 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.
[0197] 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-l 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.
[0198] 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.
[0199] In some embodiments, a eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris ( K phaffli) yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strain such as INVScl.
[0200] 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.
[0201] In some embodiments, a host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Machl™, DH10B™, TOP10, DH5a, DHlOBac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F’, INVaF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™. [0202] 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-l, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift- 12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.
[0203] Insect vectors include, for example, pFastBacl, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 Mil, pVL1393 M12, FLAG vectors such as pPolh-FLAGl or pPolh-MAT 2, or MAT vectors such as pPolh-MATl, or pPolh-MAT2. [0204] 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, pA0815 Pichia vector, pFLDl 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, pTEFl/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.
[0205] Algae vectors include, for example, pChlamy-4 vector or MCS vector.
[0206] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG- Myc-CMV 24, pCMV -FLAG-MAT 1 , pCMV -FLAG-MAT2, pBICEP-CMV 3, or pBICEP- CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG- CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.
[0207] 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 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+.
[0208] 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. [0209] 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
[0210] 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.
[0211] 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. [0212] In some instances, the codon is a quadruplet codon, which can be decoded by an orthogonal ribosome ribo-Ql. 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.
[0213] 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.
[0214] 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 (-CºC-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 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- hydroxy cytosine, 2 ’-deoxy uridine, 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.
[0215] 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 the 9-position, the pyrimidines via the 1 -position, the pyrrolopyrimi dines via the 7-position and the pyrazolopyrimidines via the 1-position.
[0216] 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. [0217] 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. [0218] 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 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 (WO 1997/35869). 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 thereol); a methyleneamino group (CH2NH2) (and analogs thereol) or a cyano group (CN) (and analogs thereol) (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.
[0219] 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 W02008/101157, US2005/0130923, and W02007/134181, the disclosure of each of which is incorporated herein by reference.
[0220] 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. The sugar can be in a pyranosyl or furanosyl form. The sugar moiety may be the furanoside of ribose, deoxyribose, arabinose or 2’ -O-alky lribose, 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. [0221] 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
Figure imgf000044_0001
alkyl or alkenyl and alkynyl. 2’ sugar
Figure imgf000044_0002
modifications also include but are not limited to
Figure imgf000044_0006
- where n and m
Figure imgf000044_0003
are from 1 to about 10.
[0222] Other modifications at the 2’ position include but are not limited to: Ci to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, heterocycloalkyl, heterocycloalkaryl,
Figure imgf000044_0004
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
Figure imgf000044_0005
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; 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.
[0223] 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’- substituent groups. The substituent at the 2’ position can also be selected from
Figure imgf000045_0007
allyl, amino, azido, thio
Figure imgf000045_0008
Figure imgf000045_0005
where each is, independently, H or
Figure imgf000045_0009
substituted or unsubstituted alkyl.
Figure imgf000045_0006
[0224] 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
Figure imgf000045_0003
’ and analogs thereof (see, U.S. Patent No. 7,399,845); 4’-
Figure imgf000045_0004
Figure imgf000045_0002
Figure imgf000045_0001
and analogs thereof, (see W02009/006478, W02008/150729, US2004/0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al, J. Org. Chem., 209, 74, 118-134, and W02008/154401). 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. W02004/106356, W01994/14226, W02005/021570, W02007/090071, and W02007/134181; 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.
[0225] 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, 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. [0226] 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. [0227] 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. [0228] 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. [0229] 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 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.
[0230] Substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside 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 CEL 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 ak, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, 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, Nuck Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EM50J, 1991, 10, 1111-1118; Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al, Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or tri ethyl ammonium 1-di-O- hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651- 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-oxy cholesterol 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.
[0231] 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, TATI, dTATl, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. In some embodiments, unnatural nucleotides include:
Figure imgf000048_0001
Exemplary unnatural base pairs include: (d)TPT3-(d)NaM; (d)5SICS-(d)NaM; (d)CNMO- (d)TATl; (d)NaM-(d)TATl ; (d)CNMO-(d)TPT3; and (d)5FM-(d)TATl.
[0232] 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 et al. ProcNatl Acad Sci USA, 2012, 109:12005-12010; the disclosure of each of which is incorporated herein by reference. In some embodiments, unnatural nucleotides include:
Figure imgf000049_0001
[0233] 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
Figure imgf000049_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, a-thiotriphosphate, b-thiotriphosphate, or g-thiotriphosphate group, or is included in an RNA or a DNA or in an RNA analog or a DNA analog.
[0234] 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
Figure imgf000049_0003
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, a- thiotriphosphate, b-thiotriphosphate, or g-thiotriphosphate group, or is included in an RNA or a DNA or in an RNA analog or a DNA analog.
[0235] 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.
[0236] 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.
[0237] In some embodiments, E is sulfur. In some embodiments, E is oxygen. In some embodiments, E is selenium.
[0238] 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.
[0239] 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. [0240] In some embodiments, the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein may be derived from
Figure imgf000051_0003
Figure imgf000051_0001
Figure imgf000051_0002
, in some embodiments, the unnatural nucleotides that may be used to prepare the IL-2 conjugates disclosed herein include
Figure imgf000051_0004
Figure imgf000052_0001
thereof.
[0241] 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.
[0242] 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 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 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.
[0243] 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)IB . stearothermophilus tRNAcuA pairs, E. coli LeuRS ( Ec-Leu)IB . 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- '/VrRS/tRNA pair. Exemplary UAAs that can be incorporated by a
Figure imgf000054_0005
pair include, but are not limited to, para-substituted phenylalanine derivatives such as p- aminophenylalanine and p-methoy phenyl alanine: meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3, 4-dihydroxy phenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronophenylalanine: and o-nitrobenzyltyrosine.
[0244] In some instances, the unnatural amino acid is incorporated into the cytokine (e.g., the IL polypeptide) by a or a
Figure imgf000054_0002
pair. Exemplary UAAs that can be
Figure imgf000054_0001
incorporated by a or a E pair include, but are not limited to,
Figure imgf000054_0003
Figure imgf000054_0004
phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O- propargyltyrosine; a-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3- (naphthalene-2-ylamino)-2-amino-propanoic acid.
[0245] 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 Methanes arcina 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- carboxamido)hexanoic acid, /V-s-D-prolyl-L-lysine, and /V-s-cyclopentyloxycarbonyl-L-lysine; N- e-Acryloyl-L-lysine; /V-s-[(l-(6-nitrobenzo[d][l,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine; and N- E-(l-methylcyclopro-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein may be prepared by use ofM mazei tRNA which is selectively charged with a non-natural amino acid such as M>-((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.
[0246] 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.
[0247] 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 TpNTTl, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTTl, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricomutum), GsNTT (Galdieria sulphuraria), AtNTTl, AtNTT2 (Arabidopsis thaliana), CtNTTl, CtNTT2 (Chlamydia trachomatis), PamNTTl, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTTl (Rickettsia prowazekii). In some embodiments, the NTT is selected from PtNTTl, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, the NTT is PtNTTl. 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.
[0248] 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)IB . stearothermophilus tRNAcuA pairs, and pyrrolysyl-tRNA pairs. Other 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., 2018 140:1447-1454; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322; the disclosure of each of which is incorporated herein by reference.
[0249] 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 PtNTTl, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii, E. coli TyrRS ( Ec-Tyr)IB . stearothermophilus , andM mazei. In some embodiments, the NTT is PtNTTl and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS ( Ec-Tyr)IB . 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)IB . 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)IB . 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)IB . 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)IB . 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)IB . 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)IB . stearothermophilus, or M. mazei.
[0250] In some embodiments, the IL-2 conjugates disclosed herein may be prepared in a cell, such as E. coli, comprising (a) nucleotide triphosphate transporter P/NTT2 (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 fromM 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. harkeri 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 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 M>-((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 fromM 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
Figure imgf000057_0001
Figure imgf000057_0002
In some embodiments, the first and second unnatural nucleotides comprising the unnatural base pair in the double-stranded oligonucleotide may be derived
Figure imgf000057_0004
In some
Figure imgf000057_0003
embodiments, the triphosphates of the first and second unnatural nucleotides include,
Figure imgf000058_0001
, or salts thereof. In some embodiments, the triphosphates
Figure imgf000058_0002
of the first and second unnatural nucleotides include,
Figure imgf000058_0003
Figure imgf000058_0004
, or salts thereof. In some embodiments, the mRNA derived the double-stranded oligonucleotide comprising a first unnatural nucleotide and a second unnatural nucleotide may comprise a codon comprising an unnatural nucleotide derived from
Figure imgf000058_0005
In some embodiments, the M. mazei tRNA may comprise an anti-codon comprising an unnatural nucleotide that recognizes the codon comprising the unnatural nucleotide of the mRNA. The anti-codon in the M. mazei tRNA may comprise an unnatural nucleotide derived from
Figure imgf000059_0001
Figure imgf000059_0002
Figure imgf000059_0003
In some embodiments, the mRNA comprises an unnatural nucleotide derived from
Figure imgf000059_0004
. In some embodiments, the mRNA comprises an unnatural nucleotide derived from
Figure imgf000059_0005
some embodiments, the mRNA comprises an unnatural nucleotide derived from in some embodiments, the mRNA
Figure imgf000059_0006
comprises an unnatural nucleotide derived from
Figure imgf000060_0001
the mRNA comprises an unnatural nucleotide derived from
Figure imgf000060_0002
. In some embodiments, the mRNA comprises an unnatural nucleotide derived from
Figure imgf000060_0003
In some embodiments, the tRNA comprises an unnatural nucleotide derived from
. In some embodiments, the tRNA comprises an unnatural nucleotide
Figure imgf000060_0005
derived from
Figure imgf000060_0006
in some embodiments, the tRNA comprises an unnatural nucleotide derived from
Figure imgf000060_0004
. in some embodiments, the tRNA comprises an unnatural nucleotide derived from
Figure imgf000061_0001
jn some embodiments, the tRNA comprises an unnatural nucleotide derived from
Figure imgf000061_0002
. In some embodiments 5 the tRNA comprises an unnatural nucleotide derived from
Figure imgf000061_0003
. In some embodiments, the mRNA comprises an unnatural nucleotide derived from
Figure imgf000061_0005
and the tRNA comprises an unnatural nucleotide derived from
Figure imgf000061_0006
In some embodiments, the mRNA comprises an unnatural nucleotide derived from
Figure imgf000061_0004
the tRNA comprises an unnatural nucleotide derived from
Figure imgf000062_0004
derived from
Figure imgf000062_0005
and the tRNA comprises an unnatural nucleotide derived from
Figure imgf000062_0001
In some embodiments, the mRNA comprises an unnatural nucleotide derived from
Figure imgf000062_0002
the tRNA comprises an unnatural nucleotide derived from
Figure imgf000062_0003
The host cell is cultured in a medium containing appropriate nutrients, and is supplemented with (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 interest. The host cells are then maintained under conditions which permit expression of the protein of interest.
[0251] 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.
[0252] 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.
[0253] 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. [0254] 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.
[0255] 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.
[0256] 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.
[0257] 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).
[0258] 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 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
[0259] In one aspect, provided herein is a method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate as described herein, and (b) cemiplimab. In one aspect, provided herein is a method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method of treating skin cancer in a subject in need thereof comprises administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method of treating skin cancer in a subject in need thereof comprises administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method of treating skin cancer in a subject in need thereof comprises administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab.
[0260] In another aspect, provided herein is an IL-2 conjugate for use in a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) an IL-2 conjugate as described herein, and (b) cemiplimab. In another aspect, provided herein is an IL-2 conjugate for use in a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab.
[0261] In a further aspect, provided herein is use of an IL-2 conjugate for the manufacture of a medicament for a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) an IL-2 conjugate as described herein, and (b) cemiplimab. In a further aspect, provided herein is use of an IL-2 conjugate for the manufacture of a medicament for a method of treating skin cancer in a subject in need thereof, the method comprising administering to the subject (a) about 8 μg/kg, 16 μg/kg. or 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab.
[0262] In still another aspect, the methods described herein further comprise selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being unresectable skin cancer. In some aspects, the methods described herein further comprise selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being locally advanced cutaneous squamous cell carcinoma. In some aspects, the methods described herein further comprise selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being metastatic skin cancer. In some aspects, the methods described herein further comprise selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being not amenable to local therapy. In some aspects, the methods described herein further comprise selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being immune checkpoint inhibitor-naive.
[0263] In some aspects, provided herein is a method of treating skin cancer in a subject in need thereof, comprising selecting a subject having skin cancer, wherein the subject is selected on the basis of one or more attributes comprising (i) the skin cancer being unresectable skin cancer; (ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma; or (iii) the skin cancer being metastatic skin cancer; and administering to the subject (a) an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab.
[0264] In some aspects, provided herein is use of an IL-2 conjugate for stimulating CD8+ and/or NK cells in a subject in need thereof, the method comprising administering to the subject (a) an IL-2 conjugate as described herein, and (b) cemiplimab. In some aspects, provided herein is use of an IL-2 conjugate for stimulating CD8+ and/or NK cells in a subject in need thereof, the method comprising administering to the subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 8 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 16 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab. In some embodiments, the method comprises administering to the subject (a) about 24 μg/kg of an IL-2 conjugate as described herein, and (b) cemiplimab.
Cancer Types
[0265] In some embodiments, the skin cancer is squamous cell skin cancer. In some embodiments, the skin cancer is cutaneous squamous cell carcinoma. In some embodiments, the skin cancer is unresectable skin cancer, locally advanced cutaneous squamous cell carcinoma, or metastatic skin cancer. In some embodiments, the skin cancer is unresectable skin cancer. In some embodiments, the skin cancer is locally advanced cutaneous squamous cell carcinoma. In some embodiments, the skin cancer is metastatic skin cancer. In some embodiments, the skin cancer is melanoma. In some embodiments, the skin cancer is basal cell carcinoma.
[0266] In some embodiments, the skin cancer is metastatic cutaneous squamous cell carcinoma (mCSCC) or locally advanced cutaneous squamous cell carcinoma (laCSCC) in a subject who is not a candidate for curative surgery or curative radiation. In some embodiments, the skin cancer is metastatic cutaneous squamous cell carcinoma (mCSCC) in a subject who is not a candidate for curative surgery or curative radiation. In some embodiments, the skin cancer is locally advanced cutaneous squamous cell carcinoma (laCSCC) in a subject who is not a candidate for curative surgery or curative radiation. In some embodiments, the skin cancer is immune checkpoint inhibitor (ICI)-nai've metastatic cutaneous squamous cell carcinoma (CSCC).
[0267] In some embodiments, the skin cancer is refractory skin cancer. In some embodiments, the skin cancer is relapsed skin cancer. In some embodiments, the skin cancer is unresectable. In some embodiments, the skin cancer is metastatic. In some embodiments, the skin cancer is not amenable to local therapy. In some embodiments, the skin cancer is advanced. In some embodiments, the skin cancer is immune checkpoint inhibitor (ICI)-nai've.
Administration
[0268] 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 and cemiplimab are administered to the subject by intravenous administration.
[0269] 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.
[0270] In some embodiments, the IL-2 conjugate is administered to the subject separately from the administration of cemplimab. In some embodiments, the IL-2 conjugate and cemiplimab are administered to the subject sequentially. In some embodiments, the IL-2 conjugate is administered to the subject prior to the administration to the subject of cemiplimab. In some embodiments, the IL-2 conjugate is administered to the subject after the administration to the subject of cemiplimab. In some embodiments, the IL-2 conjugate and cemiplimab are administered to the subject simultaneously.
[0271] In some embodiments, the IL-2 conjugate is administered to a subject in need thereof 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 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.
[0272] In some embodiments, cemiplimab is administered to a subject in need thereof about once every two weeks, about once every three weeks, or about once every 4 weeks. In some embodiments, cemiplimab is administered to a subject in need thereof once every two weeks. In some embodiments, cemiplimab is administered to a subject in need thereof once every three weeks. In some embodiments, cemiplimab is administered to a subject in need thereof once every 4 weeks. In some embodiments, cemiplimab is administered about once every 14, 15, 16, 17, 18, 19, 20, or 21 days. [0273] In some embodiments, the IL-2 conjugate and cemiplimab are administered to a subject in need thereof about once every two weeks, about once every three weeks, or about once every 4 weeks. In some embodiments, the IL-2 conjugate and cemiplimab are administered to a subject in need thereof once every two weeks. In some embodiments, the IL-2 conjugate and cemiplimab are administered to a subject in need thereof once every three weeks. In some embodiments, the IL-2 conjugate and cemiplimab are administered to a subject in need thereof once every 4 weeks. In some embodiments, the IL-2 conjugate and cemiplimab are administered about once every 14, 15, 16, 17, 18, 19, 20, or 21 days.
[0274] 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.
[0275] In some embodiments, cemiplimab is administered at a dose of about 350 mg every 3 weeks.
Subject
[0276] In some embodiments, administration of the IL-2 conjugate and cemiplimab is to an adult. In some embodiments, the adult is a male. In other embodiments, the adult is a female.
In some embodiments, the subject is 18 years of age or older. In some embodiments, the adult is at least age 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age. In some embodiments, administration of the IL-2 conjugate and cemiplimab is to an infant, child, or adolescent. In some embodiments, the subject is at least 1 month, 2 months, 3 months, 6 months, 9 months or 12 months of age. In some embodiments, the subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 years of age.
[0277] In some embodiments, the subject is a female who is not pregnant or breastfeeding. In some embodiments, the subject is a female who is not of childbearing potential. In some embodiments, the subject is a female who is of childbearing potential and uses an effective contraception method for at least 180 days after discontinuing treatment. In some embodiments, the subject is a male who does not donate sperm, abstains from heterosexual intercourse, or uses an effective contraception during treatment and for at least 210 days after discontinuing treatment.
[0278] In some embodiments, the subject does not have an Eastern Cooperative Oncology Group (ECOG) performance status of >2. In some embodiments, the subject does not have central nervous system (CNS) disease or leptomeningeal disease. In some embodiments, the subject does not have a history of allogenic or solid organ transplant. [0279] In some embodiments, the subject has not been treated with prior immune checkpoint inhibitors except in the context of adjuvant or neoadjuvant. In some embodiments, the subject has not received adjuvant or neoadjuvant therapy during the 6 months prior to development of skin cancer. In some embodiments, the subject is under anti -hypertensive treatment and has temporarily (for 12 to 48 hours) withheld antihypertensive medications prior to treatment.
[0280] In some embodiments, the subject has received no prior systemic treatment for advanced/metastatic disease (i.e., skin cancer). In some embodiments, the subject has not received more than 2 prior lines of any systemic treatment for advanced/metastatic disease (i.e., skin cancer).
[0281] In some embodiments, the subject can undergo contrast-enhanced radiologic response assessment prior, during, and following treatment.
[0282] In some embodiments, the subject has measurable disease (i.e., skin cancer) as determined by RECIST vl.l. In some embodiments, the subject has at least one measurable lesion per RECIST vl.l. In some embodiments, the subject has been determined to have Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. In some embodiments, the subject has adequate cardiovascular, hematological, liver, renal function, and laboratory parameters, as determined by a physician. 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 had prior anti-cancer therapy before administration of the first treatment dose.
[0283] In some embodiments, the subject has squamous cell skin cancer. In some embodiments, the subject has cutaneous squamous cell carcinoma. In some embodiments, the subject has unresectable skin cancer, locally advanced cutaneous squamous cell carcinoma, or metastatic skin cancer. In some embodiments, the subject has unresectable skin cancer. In some embodiments, the subject has locally advanced cutaneous squamous cell carcinoma. In some embodiments, the subject has metastatic skin cancer. In some embodiments, the subject has melanoma. In some embodiments, the subject has basal cell carcinoma.
[0284] In some embodiments, the subject has metastatic cutaneous squamous cell carcinoma (mCSCC) or locally advanced cutaneous squamous cell carcinoma (laCSCC) and is not a candidate for curative surgery or curative radiation. In some embodiments, the subject has metastatic cutaneous squamous cell carcinoma (mCSCC) and is not a candidate for curative surgery or curative radiation. In some embodiments, the subject has locally advanced cutaneous squamous cell carcinoma (laCSCC) and is not a candidate for curative surgery or curative radiation. In some embodiments, the subject has received no more than 2 prior lines of systemic therapy. In some embodiments, the subject has immune checkpoint inhibitor (ICI)-nai've metastatic cutaneous squamous cell carcinoma (CSCC).
[0285] In some embodiments, the subject has refractory skin cancer. In some embodiments, the subject has relapsed skin cancer. In some embodiments, the subject has unresectable skin cancer. In some embodiments, the subject has skin cancer that is not amenable to local therapy. In some embodiments, the subject has advanced skin cancer. In some embodiments, the subject has skin cancer that is immune checkpoint inhibitor (ICI)-nai've.
[0286] In some embodiments, the subject is immune checkpoint inhibitor (ICI)-nai've, has locally advanced, unresectable or metastatic melanoma, and has not received prior treatment (i.e., the IL-2 conjugate treatment is 1L or first-line therapy; the subject is treatment-naive).
That is, the subject will receive the IL-2 conjugate treatment as 1L or first-line therapy. In some embodiments, the subject is a 1L melanoma subject. In some embodiments, the subject is a treatment-naive melanoma subject. In some embodiments, the subject has a histologically - confirmed diagnosis of unresectable locally advanced or metastatic melanoma not amenable to local therapy. In some embodiments, the subject does not have uveal or ocular or desmoplastic melanoma. In some embodiments, the subject has received no prior systemic treatment for advanced/metastatic skin cancer. In some embodiments, the subject has not received a live-virus vaccination within 28 days of starting IL-2 conjugate treatment.
[0287] In some embodiments, the subject is immune checkpoint inhibitor (ICI)-nai've, has metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC, is not a candidate for curative surgery or curative radiation, and has received no more than 2 prior lines of systemic therapy. That is, the subject will receive the IL-2 conjugate treatment as 1L, 2L, or 3L therapy. In some embodiments, the subject is a 1L, 2L, or 3L metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC subject. In some embodiments, the subject is a 1L CSCC subject. In some embodiments, the subject is a treatment-naive CSCC subject. In some embodiments, the subject is a 2L CSCC subject. In some embodiments, the subject is a 3L CSCC subject. In some embodiments, the subject has a histologically-confirmed diagnosis of locally advanced or metastatic cutaneous squamous cell carcinoma (CSCC). In some embodiments, the subject does not have dry red lip (vermillion) or anogenital area as the primary site of CSCC and mixed CSCC histologies (e.g., sarcomatoid, adenosquamous). In some embodiments, the subject has not received more than 2 prior lines of any systemic treatment for advanced/metastatic skin cancer.
[0288] In some embodiments, the subject has an ECOG performance status of less than 2. In some embodiments, the subject does not have a history of allogenic tissue or solid organ transplant. In some embodiments, the subject does not have immune-mediated/related toxicity from prior immuno-oncology therapy of Grade 4 or leading to discontinuation. In some embodiments, the subject does not have ongoing AEs caused by any prior anti-cancer therapy >Grade 2. In some embodiments, the subject does not have baseline oxygen saturation (Sp02) <92% (without oxygen therapy). In some embodiments, the subject does not have active brain metastases or leptomeningeal disease. In some embodiments, the subject does not have lung disease. In some embodiments, the subject does not have a comorbidity requiring corticosteroid therapy. In some embodiments, the subject can temporarily (for at least 36 hours) withhold antihypertensive medications prior to each IL-2 conjugate dosing. In some embodiments, the subject does not have any medical or clinical condition, laboratory abnormality, or any specific situation as judged by the supervising physician that would preclude protocol therapy or would make the subject inappropriate for the study. In some embodiments, the subject has not received antibiotics, excluding topical antibiotics, within 14 days of administering the first dose of the IL- 2 conjugate. In some embodiments, the subject has not had severe or unstable cardiac condition within 6 months of starting IL-2 conjugate treatment. In some embodiments, the subject does not have active, known, or suspected autoimmune disease that has required systemic treatment within 2 years of starting IL-2 conjugate treatment. In some embodiments, the subject does not have a known second malignancy either progressing or requiring active treatment within 3 years of starting IL-2 conjugate treatment.
[0289] In some embodiments, the subject having skin cancer is selected for treatment on the basis of one or more attributes comprising: (i) the skin cancer being unresectable skin cancer;
(ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma; or (iii) the skin cancer being metastatic skin cancer. In some embodiments, the subject having skin cancer is selected for treatment on the basis of (i) the skin cancer being unresectable skin cancer. In some embodiments, the subject having skin cancer is selected for treatment on the basis of (ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma. In some embodiments, the subject having skin cancer is selected for treatment on the basis of (iii) the skin cancer being metastatic skin cancer. In some embodiments, the subject having skin cancer is selected for treatment on the basis of (i) the skin cancer being unresectable skin cancer, and (ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma. In some embodiments, the subject having skin cancer is selected for treatment on the basis of (i) the skin cancer being unresectable skin cancer and (iii) the skin cancer being metastatic skin cancer. In some embodiments, the subject having skin cancer is selected for treatment on the basis of (ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma, and (iii) the skin cancer being metastatic skin cancer. In some embodiments, the subject having skin cancer is selected for treatment on the basis of (i) the skin cancer being unresectable skin cancer, (ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma, and (iii) the skin cancer being metastatic skin cancer.
[0290] In some embodiments, the subject having skin cancer is selected for treatment at least in part on the basis of the skin cancer being unresectable skin cancer. In some embodiments, the subject having skin cancer is selected for treatment at least in part on the basis of the skin cancer being the skin cancer being locally advanced cutaneous squamous cell carcinoma. In some embodiments, the subject having skin cancer is selected for treatment at least in part on the basis of the skin cancer being metastatic skin cancer. In some embodiments, the subject having skin cancer is selected for treatment at least in part on the basis of the skin cancer being not amenable to local therapy. In some embodiments, the subject having skin cancer is selected for treatment at least in part on the basis of the skin cancer being immune checkpoint inhibitor-naive.
[0291] In some embodiments, the subject has no known hypersensitivity or contraindications to any of the IL-2 conjugates disclosed herein, PEG, pegylated drugs, or cemiplimab.
Effects of Administration
[0292] In some embodiments, administration of the IL-2 conjugate and cemiplimab provides a complete response, a partial response, or stable disease. In some embodiments, administration of the IL-2 conjugate and cemiplimab provides a complete response. In some embodiments, administration of the IL-2 conjugate and cemiplimab provides a partial response. In some embodiments, administration of the IL-2 conjugate and cemiplimab provides stable disease. [0293] In some embodiments, administration of the IL-2 conjugate and cemiplimab provides a decrease in the size of target lesions. In some embodiments, administration of the IL-2 conjugate and cemiplimab stabilizes the size of target lesions. In some variations, administration of the IL-2 conjugate and cemiplimab slows down the growth rate of target lesions. In some variations, administration of the IL-2 conjugate and cemiplimab stops the growth of target lesions. In some embodiments, administration of the IL-2 conjugate and cemiplimab eliminates the target lesions.
[0294] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab to the subject does not cause Grade 2 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause Grade 3 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause Grade 4 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause loss of vascular tone in the subject.
[0295] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause extravasation of plasma proteins and fluid into the extravascular space in the subject.
[0296] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause hypotension and reduced organ perfusion in the subject.
[0297] In some embodiments, administration of the IL-2 conjugate and a cemiplimab to the subject does not cause impaired neutrophil function in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause reduced chemotaxis in the subject.
[0298] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab. In some embodiments, the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate and cemiplimab.
[0299] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab 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 and cemiplimab 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 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.
[0300] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab to the subject does not cause seizures in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject is not contraindicated in subjects having a known seizure disorder.
[0301] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab to the subject does not cause Grade 2 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause Grade 3 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause Grade 4 capillary leak syndrome in the subject.
[0302] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab to the subject does cause hypotension in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab 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.
[0303] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause edema or impairment of kidney or liver function in the subject.
[0304] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause eosinophiba in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 500 μL to 1500 per μL. In some embodiments, administration of the the IL-2 conjugate and cemiplimab to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 1500 per μL to 5000 per μL. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not cause the eosinophil count in the peripheral blood of the subject to exceed 5000 per μL. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject is not contraindicated in subjects on an existing regimen of psychotropic drugs.
[0305] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab 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 and cemiplimab 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.
[0306] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab 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; hypoventilation; pneumothorax; mydriasis; pupillary disorder; kidney function abnormal; kidney failure; and acute tubular necrosis.
[0307] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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.
[0308] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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.
[0309] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject stimulates CD8+ cells in a subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab 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. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject expands the number of CD8+ cells in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject expands the number of NK cells in the subject.
[0310] In some embodiments, eosinophils are expanded in the subject following administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab 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 and cemiplimab is greater than the 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.
[0311] In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral CD4+ regulatory T cells in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject. In some embodiments, administration of the IL-2 conjugate and cemiplimab 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.
[0312] In some embodiments, administration of the IL-2 conjugate and cemiplimab 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 and cemiplimab 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 and cemiplimab to the subject does not require the availability of an intensive care facility. In some embodiments, administration of the IL-2 conjugate and cemiplimab to the subject does not require the availability of skilled specialists in cardiopulmonary or intensive care medicine. [0313] In some embodiments, administration of the IL-2 conjugate and cemiplimab does not cause dose-limiting toxicity. In some embodiments, administration of the IL-2 conjugate and cemiplimab does not cause severe cytokine release syndrome. In some embodiments, the IL-2 conjugate does not induce anti-drug antibodies (AD As), i.e., antibodies against the IL-2 conjugate. In some embodiments, a lack of induction of AD As is determined by direct immunoassay for antibodies against PEG and/or ELISA for antibodies against the IL-2 conjugate. An IL-2 conjugate is considered not to induce AD As if a measured level of AD As is statistically indistinguishable from a baseline (pre-treatment) level or from a level in an untreated control.
Additional Agents
[0314] In some embodiments, the methods further comprise administering to the subject a therapeutically effective amount of one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprises one or more analgesic agents. In some embodiments, the one or more analgesic agents comprises acetaminophen. In some embodiments, the one or more additional therapeutic agents comprises one or more antihistamines. In some embodiments, the one or more antihistamines comprises diphenhydramine. In some embodiments, the one or more additional therapeutic agents comprises one or more serotonin 5-HT3 receptor antagonists. In some embodiments, the one or more serotonin 5-HT3 receptor antagonists comprises ondansetron. In some embodiments, the one or more additional therapeutic agents, such as acetaminophen, diphenhydramine, and/or ondansetron, is administered to the subject before administering the IL-2 conjugate. In some embodiments, the one or more additional therapeutic agents, such as acetaminophen, diphenhydramine, and/or ondansetron, is administered to the subject after administering the IL-2 conjugate. In some embodiments, the one or more additional therapeutic agents, such as acetaminophen, diphenhydramine, and/or ondansetron, is administered to the subject simultaneously with the administration of the IL-2 conjugate.
Kits/ Article of Manufacture
[0315] 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. [0316] A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
[0317] 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.
[0318] 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.
EXAMPLES
[0319] 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.
[0320] An exemplary method with details for preparing IL-2 conjugates described herein is provided in this Example.
[0321] 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 /V6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) was incorporated and (ii) a tRNA derived fromM 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 fromM 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.
Scheme 1.
Figure imgf000082_0002
Figure imgf000082_0001
IL-2 Azk_PEG variant proteins
Scheme 2.
Cytokine variant protein
Figure imgf000083_0001
Figure imgf000083_0002
Cytokine Azk_L1_PEG variant proteins
Example 2. Clinical study of biomarker effects following IL-2 conjugate administration (8 μg/kg, 16 μg/kg, and 24 μg/kg [Q3W]).
Cohort Using 24 mg/kg Dose [Q3W]
[0322] A study was performed to characterize immunological effects of in vivo administration of an IL-2 conjugate described herein. The IL-2 conjugate comprised SEQ ID NO: 2, wherein position 64 is AzK_Ll_PEG30kD, where AzK_Ll_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 compound was prepared using methods wherein a protein was first prepared having SEQ ID NO: 1 in which the proline at position 64 was replaced by V6-((2-azidoetho\y)-carbonyl)-L-lysine AzK. The AzK- containing protein was then allowed to react under click chemistry conditions with DBCO comprising a methoxy, linear PEG group having an average molecular weight of 30kDa, followed by purification and formulation employing standard procedures. Here and throughout the cohorts of Example 2, drug mass per kg subject (e.g., 24 μg/kg) refers to IL-2 mass exclusive of PEG and linker mass.
[0323] The IL-2 conjugate was administered via IV infusion at a dose of 24 μg/kg for 30 minutes every 3 weeks [Q3W], Effects on the following biomarkers were analyzed as surrogate predictors of safety and/or efficacy:
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 g (IFN- g): Cytokine surrogate marker for IL-2 induced activation of CD8+ cytotoxic T lymphocytes.
[0324] 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.
[0325] 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.
[0326] 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 effective, or not accessible. Measurable disease per RECIST vl.l. Adequate laboratory parameters including: Absolute lymphocyte count > 0.5 times lower limit of normal; Platelet count > 100 x 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 x 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 x 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.
[0327] Q3W dosing. 11 individuals (4 [36.4%] male, 7 [63.6%] female, [9 [81.8%] Caucasian) having advanced or metastatic solid tumors and whose median age was 67, ranging from (37- 78), received the IL-2 conjugate at a 24 μg/kg dose Q3W for up to nine cycles (1 dose per cycle). Here and throughout discussion of the first cohort of Example 2, drug mass per kg subject (e.g., 24 μg/kg) refers to IL-2 mass exclusive of PEG and linker mass.
[0328] One subject had partial response at initial scan confirmed on second and third scan (prior PD-1 exposure) ongoing for 6+ months; five subjects had initial disease stabilization (at the 6-week assessment), three subjects had progressive disease at first assessment, and one subject came off treatment for an adverse event. All subjects had peak post-dose CD8+ Ki67 expression levels that exceeded 50 percent (50%-85%).
[0329] One 73 year old male subject with squamous cell carcinoma of unknown origin who received 7 cycles of treatment (24 μg/kg Q3W) and who had also received two lines of systemic therapy including anti-PD-1 (best response on anti-PD-1: SD) showed tumor reduction of 31% after two cycles. The maximal tumor responses in other patients with immune sensitive tumors were found to be renal cell carcinoma (RCC) (16% growth) and Melanoma (10% growth observed in two subjects; 2% reduction; and 20% reduction).
[0330] The peripheral expansion of CD8+ T effector cells averaged 4.47-fold above baseline. All subjects had elevated post-dose NK Cell Ki67 expression levels. The subjects had peak post- dose peripheral expansion of NK cells that averaged 7.67-fold above baseline.
[0331] Efficacy biomarkers. Data for efficacy biomarkers was based on data available for up to 10 subjects receiving the IL-2 conjugate at 24 μg/kg. Peripheral CD8+ Teff cell counts were measured (FIG. 1A-C). Prolonged CD8+ expansion over baseline (e.g., greater than or equal to 2-fold change) was observed at 3 weeks after the previous dose in some subjects. The percentage of CD8+ Teff cells expressing Ki67 was also measured (FIG. 2). Peripheral CD8+ memory cells counts are shown in FIG. 3A-B.
[0332] Peripheral NK cell counts are shown in FIG. 4A-D. An increase in NK cell count was observed in each subject. The percentage of NK cells expressing Ki67 was also measured (FIG. 5).
[0333] Peripheral CD4+ Treg counts are shown in FIG. 6A-B. The percentage of CD4+ Treg cells expressing Ki67 was also measured (FIG. 7).
[0334] Eosinophil counts were measured (FIG. 8A-C). The measured values did not exceed a four-fold increase and were consistently below the range of 2328-15958 eosinophils/μL in patients with IL-2 induced eosinophilia as reported in Pisani et ah, Blood 1991 Sep 15;78(6): 1538-44. Levels of IFN-g, IL-5, and IL-6 were also measured (FIG. 9A-C). The measured values show that IFN-g was induced, but low amounts of IL-5 and IL-6, cytokines associated with VLS and CRS, respectively, were induced, except for one subject in whom IL-6 levels increased to about 1100 μg/mL at 24 hours after treatment (after receiving tocilizumab) but decreased thereafter. [0335] Anti-drug Antibodies (ADAs). Samples from treated subjects were assayed after each dose cycle for anti-drug antibodies (ADAs). Anti-polyethylene glycol autoantibodies were detected by direct immunoassays (detection limit: 36 ng/mL). A bridging MesoScale Discovery ELISA was performed with a labeled form of the IL-2 conjugate, having a detection limit of 4.66 ng/mL. Additionally, a cell-based assay for neutralizing antibodies against the IL-2 conjugate was performed using the CTLL-2 cell line, with STAT5 phosphorylation as the readout (detection limit: 6.3 μg/mL).
[0336] Samples were collected and analyzed after each dose cycle from two subjects who received 5 dose cycles and one subject who received 4 dose cycles. An assay-specific cut point was determined during assay qualification as a signal to negative ratio of 1.09 or higher for the IL-2 conjugate ADA assay and 2.08 for the PEG ADA assay. Samples that gave positive or inconclusive results in the IL-2 conjugate assay were subjected to confirmatory testing in which samples and controls were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in blocking solution). Samples that gave positive or inconclusive results in the PEG assay were subjected to confirmatory testing in which samples and controls were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in 6% horse serum). Samples will be considered “confirmed” if their absorbance signal is inhibited by equal to or greater than an assay-specific cut point determined during assay qualification (14.5% for the IL-2 conjugate or 42.4% for PEG) in the detection step. No confirmed ADA against the IL-2 conjugate or PEG were detected (data not shown).
[0337] Summary of Results; Discussion. All subjects tested had post-dose CD8+ Ki67 expression levels exceeding 50% (50%-85%) at one or more time points and peripheral expansion of CD8+ T effector (Teff) cells. All subjects tested also had post-dose NK cell Ki67 expression levels exceeding 50% (50%-100%) at one or more time points with peripheral expansion of NK cells. There were no meaningful elevations in IL-5 levels and the subject whose IL-6 level was increased at day 3 showed a reduction the following day. No ADAs were induced in any of the tested subjects.
[0338] An AE was any untoward medical occurrence in a clinical investigation subject administered a pharmaceutical product, regardless of causal attribution. Dose-limiting toxicities were defined as an AE occurring within Day 1 through Day 29 (inclusive) ±1 day of a treatment cycle that was not clearly or incontrovertibly solely related to an extraneous cause and that met at least one of the following criteria:
• Grade 3 neutropenia (absolute neutrophil count < 1000/mm3 > 500/mm3) lasting > 7 days, or Grade 4 neutropenia of any duration
• Grade 3+ febrile neutropenia • Grade 4+ thrombocytopenia (platelet count < 25,000/mm3)
• Grade 3+ thrombocytopenia (platelet count < 50,000-25,000/mm3) lasting > 5 days, or associated with clinically significant bleeding or requiring platelet transfusion
• Failure to meet recovery criteria of an absolute neutrophil count of at least 1,000 cells/mm3 and a platelet count of at least 75,000 cells/mm3 within 10 days
• Any other grade 4+ hematologic toxicity lasting > 5 days
• Grade 3+ ALT or AST in combination with a bilirubin > 2 times ULN with no evidence of cholestasis or another cause such as viral infection or other drugs (i.e. Hy’s law)
• Grade 3 infusion-related reaction that occurs with premedication; Grade 4 infusion- related reaction
• Grade 3 Vascular Leak Syndrome defined as hypotension associated with fluid retention and pulmonary edema
• Grade 3+ anaphylaxis
• Grade 3+ hypotension
• Grade 3+ AE that does not resolve to grade < 2 within 7 days of starting accepted standard of care medical management
• Grade 3+ cytokine release syndrome
The following exceptions applied to non-hematologic AEs:
• 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 (e.g. systemic corticosteroid therapy)
If a subject had grade 1 or 2 ALT or AST elevation at baseline considered secondhand to liver metastases, a grade 3 elevation must also be > 3 times baseline and last > 7 days.
[0339] Serious AEs were defined as any AE that results in any of the following outcomes: Death; Life-threatening AE; Inpatient hospitalization or prolongation of an existing hospitalization; A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions; or a congenital anomaly /birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, they may jeopardize the subject and may require medical or surgical intervention to prevent one of the outcomes listed above. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.
[0340] There were no dose-limiting toxicities reported. There was no cumulative toxicity. There were two treatment-related SAEs (1 G3 acute kidney injury and 1 G4 cytokine release syndrome) which resolved with accepted standard of care. Overall, the IL-2 conjugate was considered well-tolerated.
[0341] All subjects had at least one treatment-emergent AE (TEAE). TEAEs are detailed in Table 1. No TEAEs were grade 5. Two subjects had a grade 3 event and three subjects had grade 4 events. The grade 3 events included: 1 ALT/AST elevation, 1 neutrophil count decrease, and 1 acute kidney injury. The grade 4 events included: 1 CRS, 1 lymphocyte count increase, and 2 lymphocyte count decreases.
Table 1. Treatment Emergent Adverse Events (TEAE) (n=ll)
Figure imgf000089_0001
Figure imgf000090_0001
[0342] TEAEs mostly consisted of flu-like symptoms, nausea, or vomiting. The TEAEs resolved with accepted standard of care. Treatment-related AEs were transient. AEs of fever, hypotension, and hypoxia did not correlate with IL-5/IL-6 cytokine elevation. One subject presented with IL-6 elevation at 24 hours to 1000 μg/mL (post tocilizumab treatment), which declined to below 100 μg/mL by 72 hours. There was no notable impact to vital signs, no QTc prolongation, or other cardiac toxicity.
[0343] Accordingly, the IL-2 conjugate demonstrated encouraging PD data and was generally well-tolerated. It was determined that the in vivo half-life of the IL-2 conjugate was about 10 hours. 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.
Cohorts Using 8 mg/kg and 16 mg/kg Doses [Q3W]
[0344] The IL-2 conjugate was administered via IV infusion at a dose of 8 μg/kg or 16 μg/kg for 30 minutes every 3 weeks [Q3W], Effects on the same biomarkers described above for the studies using a 24 μg/kg dose were analyzed as surrogate predictors of safety and/or efficacy. Subjects in these studies met the same criteria as the subjects receiving the 24 μg/kg dose.
Cohort 1: 8 mg/kg [Q3WJ dosing.
[0345] Cohort 1 (individuals having malignant solid tumors) received the IL-2 conjugate at an 8 μg/kg dose Q3W for five dose cycles.
[0346] Four individuals that had initial disease stabilization (at the 6-week assessment; one patient had -12% tumor regression) were treated with the IL-2 conjugate. These four subjects had post-dose CD8+ Ki67 expression levels that exceeded 60 percent (65%-80%).
[0347] Biomarkers were determined for 4 individuals in Cohort 1 as follows. The peripheral expansion of CD8+ T effector cells averaged 1.53-fold above baseline; one subject was 2.1-fold above baseline. All four subjects had post-dose NK Cell Ki67 expression levels of nearly 100 percent. All four subjects had post-dose peripheral expansion of NK cells that averaged 3.9-fold above baseline at day 3; one subject was 5.0-fold above baseline at day 3. There were no changes in the PK parameters from cycle 1 to cycle 2. There were no anti-drug antibodies detected in the first three subjects; these were measured out to cycle 5 for two subjects and out to cycle 4 for one subject.
[0348] Serum IFNy, IL-6, and IL-5 levels were measured at 1, 2, and 3 days post-dosing during cycles 1 and 2. Means and ranges are shown in Table 2. The top values of the range were observed 1 day post-dosing for all subjects.
Table 2. Safety/Toxicity Biomarkers - Cytokine Summary Cohort 1: Q3W, 8 mg/kg
Figure imgf000091_0001
[0349] Measured cytokine levels are shown graphically in FIG. 36.
[0350] It was reported in Teachey et al., Cancer Discov. 2016; 6(6); 664-79, that in acute lymphoblastic leukemia patients treated with CAR-T cells, severe cytokine release syndrome (CRS level 4 or 5) was associated with higher values of each of the three cytokines measured in the present study than nonsevere cytokine release syndrome (CRS level 0-3). Data from Teachey et al. for IFNy, IL-6, and IL-5, expressed as median (range), are reproduced in Table 3.
Table 3. IFNy, IL-6, and IL-5 levels reported as associated with CRS levels 0-3 and 4-5.
Figure imgf000091_0002
[0351] Accordingly, the results in Table 2 and FIG. 36 are consistent with absence of severe CRS.
[0352] 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.
Cohort 2: 16 mg/kg [Q3WJ dosing.
[0353] This example reports results for up to 6 individuals having malignant solid tumors who received the IL-2 conjugate at a 16 μg/kg dose Q3W for at least 2 cycles. After the first dose, one subject had a post dose peripheral expansion of CD8+ T effector cells of 4.1 -fold; the average across the three patients was 2.2-fold expansion. All three subjects had post-dose peripheral expansion of NK cells that exceeded 4-fold above baseline at day 3; one subject was 11.4-fold above baseline and the average was 7.2-fold.
[0354] Serum IFNy, IL-6, and IL-5 levels were measured at 1, 2, and 3 days post-dosing during Cycles 1 and 2. Means and ranges are shown in Table 4. The top values of the range were observed 1 day post-dosing for the indicated 3 subjects.
Table 4. Safety/Toxicity Biomarkers - Cytokine Summary Cohort 2: Q3W, 16 μg/kg
Figure imgf000092_0001
[0355] Measured cytokine levels for 4 subjects are shown graphically in FIG. 37. These results are also consistent with absence of severe CRS.
[0356] Eosinophil counts were measured by FACS and CBC for cohorts 1-2 (FIGs. 38A-D). The measured values were consistently below the range of 2328-15958 eosinophils/μL in patients with IL-2 induced eosinophilia as reported in Pisani et ah, Blood 1991 Sep 15;78(6): 1538-44. Peripheral lymphocyte count was also measured for Cohorts 1 and 2 (FIGs. 39A-D).
[0357] Efficacy biomarkers for Cohorts 1 and 2. Peripheral CD8+ Teff Counts were measured for Cohorts 1 and 2 (FIGs. 40A-D). Prolonged CD8+ expansion over baseline (e.g., greater than or equal to 2-fold change) was observed at 3 weeks after the previous dose in some subjects. The percentage of CD8+ Teff cells expressing Ki67 was also measured for Cohorts 1 and 2 (FIGs. 41A-B). [0358] Peripheral memory CD8+ counts are shown in FIGs. 42A-B. Peripheral NK cell counts are shown in FIGs. 43A-D. Prolonged NK cell expansion over baseline (e.g., greater than or equal to 5-fold change) was observed at 3 weeks after the previous dose in some subjects. The percentage of NK cells expressing Ki67 was also measured for Cohorts 1 and 2 (FIGs. 44A-B). [0359] Peripheral CD4+ Treg counts for Cohorts 1 and 2 are shown in FIGs. 45A-B. The percentage of CD4+ Treg cells expressing Ki67 was also measured for Cohorts 1 and 2 (FIGs. 46A-B).
[0360] Summary of Results; Discussion. The subjects discussed above receiving the 8 μg/kg dose had post-dose CD8+ Ki67 expression levels exceeding 60% (65%-80%), with peripheral expansion of CD8+ T effector (Teff) cells averaging 1.53-fold above baseline. All 4 subjects also had post-dose NK cell Ki67 expression levels of nearly 100%, with peripheral expansion of NK cells averaging 3.9-fold above baseline at day 3. Of the 3 subjects discussed above who received 16 μg/kg doses, 1 had a post-dose peripheral expansion of CD8+ Teff cells 4.1-fold above baseline at day 7; the average expansion across the 3 subjects was 2.2-fold. There was no observation of anti-drug antibodies (IL-2 or PEG), and no meaningful elevations in IL-5 and IL- 6 levels. Also, the PK data does not indicate a decrease in AUC from cycle 1 to cycle 2 (data not shown).
[0361] There were no dose-limiting toxicities reported at either dose and there were no treatment-related adverse events (TRAE) leading to discontinuation or treatment-related serious AEs reported.
[0362] TEAEs for 10 subjects receiving Q3W 8 or 16 μg/kg doses are detailed in Table 5. No TEAEs were Grade 5. Two subjects had a Grade 4 event (one AST elevation and one lymphocyte count decrease).
Table 5. Treatment Emergent Adverse Events (TEAE)
Figure imgf000093_0001
Figure imgf000094_0001
[0363] TEAEs mostly consisted of flu-like symptoms, nausea, or vomiting. The TEAEs resolved with accepted standard of care. Treatment-related AEs were transient. AEs of fever, hypotension, and hypoxia did not correlate with IL-5/IL-6 cytokine elevation. There was no notable impact to vital signs, no QTc prolongation, or other cardiac toxicity. Accordingly, the IL-2 conjugate demonstrated encouraging PD data and was generally well-tolerated. It was determined that the in vivo half-life of the IL-2 conjugate was about 10 hours. 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. [0364] Selected individual results. One subject having prostate adenocarcinoma received 10 cycles of Q3W 16 μg/kg doses and showed stable disease (24% decrease after two cycles). This subject came off treatment after the 10th cycle due to rising PSA.
[0365] One subject having non-small cell lung cancer received at least 6 cycles of Q3W 16 μg/kg doses and showed stable disease (17.9% decrease after 5 cycles).
[0366] Anti-drug Antibodies (ADAs). Samples from treated subjects were assayed after each dose cycle for anti-drug antibodies (ADAs). Anti-polyethylene glycol autoantibodies were detected by direct immunoassays (detection limit: 36 ng/mL). A bridging MesoScale Discovery ELISA was performed with a labeled form of the IL-2 conjugate, having a detection limit of 4.66 ng/mL. Additionally, a cell-based assay for neutralizing antibodies against the IL-2 conjugate was performed using the CTLL-2 cell line, with STAT5 phosphorylation as the readout (detection limit: 6.3 μg/mL).
[0367] Samples were collected and analyzed after each dose cycle from two subjects who received 5 dose cycles and one subject who received 4 dose cycles. An assay-specific cut point was determined during assay qualification as a signal to negative ratio of 1.09 or higher for the IL-2 conjugate ADA assay and 2.08 for the PEG ADA assay. Samples that gave positive or inconclusive results in the IL-2 conjugate assay were subjected to confirmatory testing in which samples and controls were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in blocking solution). Samples that gave positive or inconclusive results in the PEG assay were subjected to confirmatory testing in which samples and controls were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in 6% horse serum). Samples will be considered “confirmed” if their absorbance signal is inhibited by equal to or greater than an assay-specific cut point determined during assay qualification (14.5% for the IL-2 conjugate or 42.4% for PEG) in the detection step. No confirmed ADA against the IL-2 conjugate or PEG were detected (data not shown).
Example 3. Clinical Study of Biomarker Effects Following IL-2 Conjugate and Anti-PD-1 Antibody Administration.
[0368] A study was performed to characterize immunological effects of in vivo administration of an IL-2 conjugate described herein in combination with an anti-PD-1 antibody (in this study, pembrolizumab). The same IL-2 conjugate used in Example 2 was used in this study. The IL-2 conjugate and pembrolizumab were administered via IV infusion for 30 minutes every 3 weeks [Q3W], Effects on the following biomarkers were analyzed as surrogate predictors of safety and/or efficacy: 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 g (IFN- g): Cytokine surrogate marker for IL-2 induced activation of CD8+ cytotoxic T lymphocytes and NK cells.
[0369] Effects on the cell counts of 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.
[0370] 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 included cervical cancer, head and neck squamous cell carcinoma, basal cell carcinoma, melanoma and non-small cell lung cancer.
[0371] 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 effective, or not accessible. Measurable disease per RECIST vl.l. Adequate laboratory parameters including: Absolute lymphocyte count > 0.5 times lower limit of normal; Platelet count > 100 x 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 x 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 x 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.
Cohorts treated with 8 mg/kg and 16 mg/kg doses
[0372] Q3W dosing. 14 adults (9 [64.3%] male, 5 [35.7%] female, 9 [64.2%] Caucasian) having advanced or metastatic solid tumors and whose age ranged from 29-74 years received a) the IL-2 conjugate at an 8 μg/kg dose IV Q3Wor 16 μg/kg dose IV Q3W and b) pembrolizumab at a dose of 200 mg IV Q3W sequentially for at least one cycle. Here and throughout Example 3, drug mass per kg subject (e.g., 8 μg/kg) refers to IL-2 mass exclusive of PEG and linker mass. The results below are for subjects receiving an 8 μg/kg dose IV Q3W and pembrolizumab (4 subjects) or 16 μg/kg dose IV Q3W and pembrolizumab (6 subjects), who received treatment for 2-19 cycles.
[0373] Two subjects who received 8 μg/kg IL-2 conjugate and pembrolizumab had confirmed partial responses (PRs; 1 PD- 1 -naive basal cell carcinoma, 1 head and neck squamous cell carcinoma, who had received prior anti-PD-1) ongoing for 22+ months. One subject (non-small cell lung cancer) who received 16 μg/kg IL-2 conjugate and pembrolizumab had disease stabilization for about 6 months. Six subjects had disease progression (at the 6-week assessment); one subject had initial disease stabilization (at the 6 week assessment; followed by progressive disease). The four subjects receiving 8 μg/kg IL-2 conjugate and pembrolizumab had increased post-dose CD8+ Ki67 expression levels (15%-70%).
[0374] One 59 year old male with head and neck squamous cell carcinoma receiving 8 μg/kg IL-2 conjugate and pembrolizumab received 30+ cycles and had a confirmed partial response (39% decrease after 8 cycles; 47% decrease after 11 cycles). This subject had previously received 4 lines of systemic therapy including 2 anti-PDl treatments; the best response to an anti-PDl treatment had been stable disease. [0375] One 50 year old male with basal cell carcinoma receiving 8 μg/kg IL-2 conjugate and pembrolizumab received 30 cycles and had a confirmed partial response (50% decrease after 2 cycles, and 80% decrease after 8 cycles). This subject had previously undergone surgeries and radiation therapy.
[0376] The maximal tumor responses in other patients with immune sensitive tumors were found to be melanoma (23% and 11% growth), basal cell carcinoma (4% growth), and non-small cell lung cancer (29% reduction).
[0377] The peak peripheral expansion of CD8+ T effector cells averaged 2.06-fold above baseline in subjects receiving 8 μg/kg IL-2 conjugate and pembrolizumab. All four subjects had post-dose NK Cell Ki67 expression levels of nearly 100 percent. The subjects had post-dose peak peripheral expansion of NK cells that averaged 6.73-fold above baseline at day 3. The peak peripheral expansion of CD8+ T effector cells averaged 3.71-fold above baseline in subjects receiving 16 μg/kg IL-2 conjugate and pembrolizumab.
[0378] Efficacy biomarkers. Data relating to efficacy biomarkers was based on data available for 10 subjects (4 subjects receiving the IL-2 conjugate at 8 μg/kg; 6 subjects receiving the IL-2 conjugate at 16 μg/kg). Peripheral CD8+ Teff cell counts were measured (FIGS. 10A-C). Prolonged CD8+ expansion over baseline (e.g., greater than or equal to 1.5-fold change) was observed at 3 weeks after the previous dose in some subjects. The percentage of CD8+ Teff cells expressing Ki67 was also measured (FIG. 11).
[0379] Peripheral NK cell counts are shown in FIGS. 12A-C. Prolonged NK cell expansion over baseline (e.g., greater than or equal to 2-fold change) was observed at 3 weeks after the previous dose in some subjects. The percentage of NK cells expressing Ki67 was also measured
(FIG. 13)
[0380] Peripheral CD4+ Treg counts are shown in FIGS. 14A-C. The percentage of CD4+ Treg cells expressing Ki67 was also measured (FIG. 15).
[0381] Eosinophil counts were measured (FIGS. 16A-C). The measured values were consistently below the range of 2328-15958 eosinophils/μL in patients with IL-2 induced eosinophilia as reported in Pisani et al, Blood 1991 Sep 15;78(6): 1538-44. Levels of IFN-g, IL- 5, and IL-6 were also measured (FIGS. 17A-D). The measured values show that IFN-g was induced, but low amounts of IL-5 and IL-6, cytokines associated with VLS and CRS, respectively, were induced.
[0382] Mean concentrations of the IL-2 conjugate, administered at a dose of 8 μg/kg, after 1 and 2 cycles are shown in FIG. 18A and FIG. 18B, respectively. Mean concentrations of the IL-2 conjugate, administered at a dose of 16 μg/kg, after 1 and 2 cycles are shown in FIG. 18C and FIG. 18D, respectively. [0383] Anti-drug Antibodies (ADAs). Samples from treated subjects were assayed after each dose cycle for anti-drug antibodies (ADAs). Anti-polyethylene glycol autoantibodies were detected by direct immunoassays (detection limit: 36 ng/mL). A bridging MesoScale Discovery ELISA was performed with a labeled form of the IL-2 conjugate, having a detection limit of 4.66 ng/mL. Additionally, a cell-based assay for neutralizing antibodies against the IL-2 conjugate was performed using the CTLL-2 cell line, with STAT5 phosphorylation as the readout (detection limit: 6.3 μg/mL).
[0384] Samples were collected and analyzed after each dose cycle from four subjects where 2 patients received 2 cycles and the other two patients received 10 or 11 cycles. An assay-specific cut point was determined during assay qualification as a signal to negative ratio of 1.09 or higher for the IL-2 conjugate ADA assay and 2.08 for the PEG ADA assay. Samples that gave positive or inconclusive results in the IL-2 conjugate assay were subjected to confirmatory testing in which samples and controls were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in blocking solution). Samples that gave positive or inconclusive results in the PEG assay were subjected to confirmatory testing in which samples and controls were assayed in the presence and absence of confirmatory buffer (10 μg/mL IL-2 conjugate in 6% horse serum). Samples will be considered “confirmed” if their absorbance signal is inhibited by equal to or greater than an assay-specific cut point determined during assay qualification (14.5% for the IL-2 conjugate or 42.4% for PEG) in the detection step. No confirmed ADA against the IL-2 conjugate or PEG were detected (data not shown).
[0385] Summary of Results; Discussion. All subjects had elevated post-dose CD8+ Ki67 expression levels (FIG. 11), with peripheral expansion of CD8+ T effector (Teff) cells averaging 1.95-fold above baseline. All 4 subjects also had elevated post-dose NK cell Ki67 expression levels (FIG. 13), with peripheral expansion of NK cells averaging 6.73-fold above baseline at day 3. There were no meaningful elevations in IL-5 and IL-6 levels.
[0386] An AE was any untoward medical occurrence in a clinical investigation subject administered a pharmaceutical product, regardless of causal attribution. Dose-limiting toxicities were defined as an AE occurring within Day 1 through Day 29 (inclusive) ±1 day of a treatment cycle that was not clearly or incontrovertibly solely related to an extraneous cause and that met at least one of the following criteria:
• Grade 3 neutropenia (absolute neutrophil count < 1000/mm3 > 500/mm3) lasting > 7 days, or Grade 4 neutropenia of any duration
• Grade 3+ febrile neutropenia
• Grade 4+ thrombocytopenia (platelet count < 25,000/mm3) • Grade 3+ thrombocytopenia (platelet count < 50,000-25,000/mm3) lasting > 5 days, or associated with clinically significant bleeding or requiring platelet transfusion
• Failure to meet recovery criteria of an absolute neutrophil count of at least 1,000 cells/mm3 and a platelet count of at least 75,000 cells/mm3 within 10 days
• Any other grade 4+ hematologic toxicity lasting > 5 days
• Grade 3+ ALT or AST in combination with a bilirubin > 2 times ULN with no evidence of cholestasis or another cause such as viral infection or other drugs (i.e. Hy’s law)
• Grade 3 infusion-related reaction that occurs with premedication; Grade 4 infusion- related reaction
• Grade 3 Vascular Leak Syndrome defined as hypotension associated with fluid retention and pulmonary edema
• Grade 3+ anaphylaxis
• Grade 3+ hypotension
• Grade 3+ AE that does not resolve to grade < 2 within 7 days of starting accepted standard of care medical management
• Grade 3+ cytokine release syndrome
The following exceptions applied to non-hematologic AEs:
• 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 (e.g. systemic corticosteroid therapy)
If a subject had grade 1 or 2 ALT or AST elevation at baseline considered secondhand to liver metastases, a grade 3 elevation must also be > 3 times baseline and last > 7 days.
[0387] Serious AEs were defined as any AE that results in any of the following outcomes: Death; Life-threatening AE; Inpatient hospitalization or prolongation of an existing hospitalization; A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions; or a congenital anomaly /birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, they may jeopardize the subject and may require medical or surgical intervention to prevent one of the outcomes listed above. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.
[0388] There were no dose-limiting toxicities reported at either dose and there were no treatment-related adverse events (TRAE) leading to discontinuation. One TRAE led to dosage reduction. There were 5 treatment-related serious AEs reported in three of the patients treated at 16 μg/kg dose IV Q3W.
[0389] The most common TRAEs (> 2 patients) of all grades by SOC included general disorders and administration conditions, investigations, metabolism and nutrition, nervous system disorders, respiratory, thoracic and mediastinal disorders, vascular disorders, skin and subcutaneous disorders, blood and lymphatic disorders, cardiac disorders, gastrointestinal disorders, immune sysmte disorders, infections and infestations, and musculoskeletal. TEAEs by preferred terms are detailed in Table 6.
Table 6.
Figure imgf000101_0001
Figure imgf000102_0001
[0390] Treatment-related AEs were transient and resolved with accepted standard of care. AEs of fever, hypotension, and hypoxia did not correlate with IL-5/IL-6 cytokine elevation. No cumulative toxicity, end organ toxicity, vascular leak syndrome, or eosinophilia was observed. IL-5 levels remained at or below the lowest level of detection. One subject had G2 hypotension which resolved with hydration. One subject had G3 cytokine release syndrome (fever + hypotension requiring pressors; subject had baseline orthostatic hypotension). One subject developed recurrent G2 cytokine release syndrome with fever and hypoxia (patient had underlying COPD managed with supportive care including one dose of tociluzimab with resolution). The subject’s dose was reduced to 8 μg/kg; the subject then developed G2 pneumonitis, and was rechallenged following improvement to Gl. Subsequently, the subject developed recurrent G3 pneumonitis and did not receive further therapy. There was no notable impact to vital signs, no QTc prolongation, or other cardiac toxicity. Accordingly, the IL-2 conjugate in combination with pembrolizumab demonstrated encouraging PD data and was generally well-tolerated with no discontinuations due to TRAE. It was determined that the in vivo half-life of the IL-2 conjugate was about 10 hours. Overall, the results are considered to support non-alpha preferential activity of the IL-2 conjugate, with a tolerable safety profile in combination with pembrolizumab as well as encouraging PD and preliminary evidence of activity in patients with immune-sensitive tumors.
Cohort treated with 24 mg/kg dose [0391] Ten individuals (male [100%], 6 [60.0%] Caucasian) with a median age of 61 years, ranging from 46-68 years of age, having advanced or metastatic solid tumors received the IL-2 conjugate at a 24 μg/kg dose Q3W. Tumor types included lung cancer, basal cell carcinoma, and colon cancer.
[0392] Each subject was treated with a) the IL-2 conjugate administered via IV infusion at a dose of 24 μg/kg for 30 minutes, and b) pembrolizumab administered at a dose of 200 mg IV sequentially. Treatment was given every 3 weeks [Q3W], Effects on the same biomarkers described above for the 8 μg/kg and 16 μg/kg doses of the IL-2 conjugate were analyzed as surrogate predictors of safety and/or efficacy. Subjects in these studies met the same criteria as the subjects treated 8 μg/kg and 16 μg/kg doses.
[0393] All 10 subjects experienced at least one TEAE, and 7 (70.0%) of 10 subjects experienced at least 1 Grade 3-4 related TEAEs (1 Grade 3 and 6 Grade 4). There was one Grade 3 ALT/AST elevation (also with Grade 3 hypophosphatemia), G2 hyperbilirubinemia in the setting of G2 CRS, and 5 Grade 4 lymphocyte count decrease (one in a subject with Grade 3 AST/ALT elevation, Grade 2 hyperbilirubinemia-DLT along with Grade 2 CRS) and 1 G4 lymphopenia. The lymphocyte count recovered to at least Grade 3 in 48 hours.
[0394] Six subjects experienced related SAEs: one Grade 1 fever in a subject with adrenal insufficiency requiring steroid adjustment, and one Grade 2 cytokine release syndrome (fever and hypotension requiring fluids and dexamethasone) associated with Grade 3 AST/ALT elevation and G2 hyperbilirubinemia. Additionally, one subject developed G2 hypotension managed with supportive care, one subject developed an infusion related reaction during C1D1 followedy by G2 CRS with fever, chills and hypotension managed with supportive care following cycle 2, an additional subject developed a G3 infusion related reaction during Cl followed by a cytokine release syndrome G1 during cycle 2, and one subject developed G2 CRS during cycle 2 managed with supportive care. There was one instance of a DLT: a subject with Grade 3 AST/ALT elevation along with Grade 2 hyperbilirubinemia associated with Grade 2 CRS (fever and hypotension requiring hydration and dexamethasone). For this subject, the dose was reduced for C2D1. No drug discontinuations resulted from TEAEs. TEAEs are detailed in Table 7.
Table 7. Treatment Emergent Adverse Events (TEAE) (n=10)
Figure imgf000104_0001
[0395] The following related events were reported: one Grade 3 AST/ALT and Grade 2 bilirubin (DLT) in the seting of Grade 2 CRS (fever, hypotension [BP97/56 mm Hg] and hypoxemia [Sp0292%]) managed with fluid bolus, supplemental oxygen and dexamethasone with resolution required a dose reduction for C2D1; one patient fever, chills, rigors and hypoxemia (92%) requiring supportive care and oxygen (C2D1); one Grade 3 AST/ALT (C2D8) presumed related to IL-2 conjugate and pembrolizumab without other symptoms in the seting of alcoholism; and three Grade 4 lymphocyte count decrease.
[0396] Efficacy biomarkers. Data relating to efficacy biomarkers was based on data available for 6 subjects receiving the IL-2 conjugate at 24 μg/kg. Peripheral CD8+ Teff cell counts were measured (FIG. 19), and peripheral NK cell counts are shown in FIG. 20. Peripheral CD4+ Treg cell counts are shown in FIG. 21, and peripheral eosinophil cell counts are shown in FIG. 22. [0397] Mean concentrations of the IL-2 conjugate after 1 and 2 cycles are shown in FIG. 23A and FIG. 23B, respectively.
[0398] Cytokine levels (IFN-g, IL-6, and IL-5) are shown in FIG. 24.
[0399] Accordingly, the IL-2 conjugate in combination with pembrolizumab demonstrated encouraging PD data and was generally well-tolerated with no discontinuations due to TRAE. Overall, the results are considered to support non-alpha preferential activity of the IL-2 conjugate, with a tolerable safety profile in combination with pembrolizumab as well as encouraging PD and preliminary evidence of activity in patients with immune-sensitive tumors.
Example 4. Administration of IL-2 conjugate at varying doses to subjects having skin cancer.
[0400] A total of 6 individuals with skin cancer received the IL-2 conjugate used in the study described for Example 2. 3 individuals having melanoma received the IL-2 conjugate at a 8 μg/kg dose Q3W, 1 individual having melanoma received the IL-2 conjugate at a 24 μg/kg dose Q3W, 1 individual having basal cell carcinoma (BCC) received the IL-2 conjugate at a 8 μg/kg dose (together with pembrolizumab) Q3W, 1 individual having basal cell carcinoma (BCC) received the IL-2 conjugate at a 16 μg/kg dose (together with pembrolizumab) Q3W, and 1 individual having basal cell carcinoma (BCC) received the IL-2 conjugate at a 24 μg/kg dose (together with pembrolizumab), each for up to 13 cycles (1 dose per cycle). Here and throughout Example 4, drug mass per kg subject (e.g., 24 μg/kg) refers to IL-2 mass exclusive of PEG and linker mass.
[0401] The clinical outcome for the subjects having basal cell carcinoma was a partial response for the individuals receiving the 8 μg/kg and 24 μg/kg doses and stable disease for the individual receiving the 16 μg/kg dose. The individual receiving the 8 μg/kg dose showed a 50% decrease after two treatment cycles, which became a 66.7% decrease after five treatment cycles and an 80% decrease after eight treatment cycles. The individual receiving the 24 μg/kg dose had a confirmed partial response with 85% decrease in target lesion size after 8 cycles. The reported decreases are in the sum of the products of maximal diameters in target lesions.
Efficacy biomarkers
[0402] CD8+ Teff cells. Peripheral CD8+ Teff cell counts were measured for each of the 6 individuals. Table 8 summarizes the fold change of CD8+ Teff cell counts normalized to the cell count prior to treatment. The data demonstrate that administration of the IL-2 conjugate promotes CD8+ expansion over baseline (e.g., up to approximately 2-fold change) in subjects having melanoma or BCC.
[0403] NK cells. Peripheral NK cell counts were measured for each of the 6 individuals. Table 9 summarizes the fold change of peripheral NK cell counts normalized to the cell count prior to treatment. An increase in NK cell count was observed in each subject.
[0404] CD4+ Treg cells. Peripheral CD4+ Treg cell counts were measured for each of the 6 individuals. Table 10 summarizes the fold change of peripheral CD4+ Treg cell counts normalized to the cell count prior to treatment. The data demonstrate that administration of the IL-2 conjugate does not significantly promote CD4+ expansion over baseline.
[0405] Lymphocytes. Lymphocyte cell counts were measured for each of the 6 individuals. Table 11 summarizes the fold change of lymphocyte cell counts normalized to the cell count prior to treatment. The data show that administration of the IL-2 conjugate promotes lymphocyte expansion over baseline (e.g., up to approximately 2-fold change).
[0406] Eosinophils. Eosinophil cell counts were measured for each of the 6 individuals. Table 12 summarizes the fold change of eosinophil cell counts normalized to the cell count prior to treatment. The measured values did not exceed a 5 -fold increase.
[0407] IFN-y, IL-5, andIL-6. Levels of IFN-g, IL-5, and IL-6 were measured for each of the 6 individuals. The cytokine levels of the patients having melanoma are shown in FIG. 25A. The measured values indicate that IFN-g was induced, but low amounts of IL-5 and IL-6, cytokines associated with VLS and CRS, respectively, were induced, except for one subject in whom IL-6 levels increased to about 200 μg/mL at 4 hours after treatment but decreased thereafter. The cytokine levels of the patients having BCC are shown in FIG. 25B. For these patients, the measured values show that IFN-g was induced, but low amounts of IL-5 and IL-6 were induced.
Attorney Docket No. 01183-0107-00PCT
Table 8. Normalized CD8+ Teff cell counts.
Figure imgf000107_0001
Attorney Docket No. 01183-0107-00PCT
Table 8 (Cont’d).
Figure imgf000108_0001
Attorney Docket No. 01183-0107-00PCT
Table 9. Normalized peripheral NK cell counts.
Figure imgf000109_0001
Attorney Docket No. 01183-0107-00PCT
Table 9 (Cont’d).
Figure imgf000110_0001
Attorney Docket No. 01183-0107-00PCT
Table 10. Normalized peripheral CD4+ Treg cell counts.
Figure imgf000111_0001
Attorney Docket No. 01183-0107-00PCT
Table 10 (Cont’d).
Figure imgf000112_0001
Attorney Docket No. 01183-0107-00PCT
Table 11. Normalized lymphocyte cell counts.
Figure imgf000113_0001
Attorney Docket No. 01183-0107-00PCT
Table 11 (Cont’d).
Figure imgf000114_0001
Attorney Docket No. 01183-0107-00PCT
Table 12. Normalized eosinophil cell counts.
Figure imgf000115_0001
Attorney Docket No. 01183-0107-00PCT
Table 12 (Cont’d).
Figure imgf000116_0001
Example 5. Use of an IL-2 conjugate plus a checkpoint inhibitor in the treatment of CT-26 tumor-bearing Balb/c mice.
[0408] An IL-2 conjugate “IL-2_P65[AzK_PEG30kD]” (also referred to herein and in the Figures as “Compound A”) comprising SEQ ID NO: 3 was used in this study:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKlAzK PEG30kDlLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 3) wherein [AzK_ PEG30kD] is N6-((2-azidoethoxy)-carbonyl)-L-lysine stably -conjugated to PEG via DBCO-mediated click chemistry to form compounds comprising a structure of Formula (I), supra, in which Z is CH2, Y is , q is 3, and W is a methoxy,
Figure imgf000117_0001
linear PEG group having an average molecular weight of 30kDa and/or compounds comprising a structure of Formula (I) whereinY is CH2 and Z is , q is 3, and
Figure imgf000117_0002
W is a methoxy, linear PEG group having an average molecular weight of 30kDa.
The compound was prepared using methods wherein a protein was first prepared having SEQ ID NO: 4 in which the proline at position 65 was replaced by /V6-((2-azidoethoxy)-carbonyl)-L- lysine AzK.
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFSQSIISTLT (SEQ ID NO: 4)
The AzK-containing protein was then allowed to react under click chemistry conditions with DBCO comprising a methoxy, linear PEG group having an average molecular weight of 30kDa, followed by purification and formulation employing standard procedures.
[0409] Studies of Compound A as monotherapy and in combination with an anti-PD-1 antibody were undertaken in Balb/c female mice. Balb/c female mice, 6-8 weeks of age, with an average weight of 16 g to 21 g were purchased from Jackson Laboratories (Sacramento, CA) for studies 1 and 2. Balb/c female mice, 7-8 weeks of age, with an average weight of 18 to 22 g were purchased from Taconic Biosciences by HD Biosciences for study 3. Cryogenically preserved vials of CT-26 colon cancer cells were purchased from American Tissue Type
Collection (ATCC, Manassas, VA). Cells were thawed and cultured according to the manufacturer’s protocol. On the day of tumor cell inoculation, cells were washed in serum-free media, counted, and resuspended in cold serum-free media at a concentration of 250,000 (studies 1 and 2) or 300,000 (study 3) viable cells per 0.1 mL. The CT-26 cells (0.1 mL) were injected subcutaneously into the flanks of individual mice and tumors were allowed to grow.
[0410] For studies 1 and 2 in which a combination of Compound A and an anti-PD-1 antibody were used, the antibody used was anti-mouse PD-1 (BioXcell; RMPl-14) and the control antibody was IgGl isotype antibody (BioXcell; catalog #BP0089, lot #2A3). For study 3 in which an anti-PD-1 antibody was used, the antibody used was anti-mouse PD-1 (BioXcell; catalog #BP0146, RMPl-14, lot #695318A1) and the control antibody was IgGl isotype antibody (BioXcell; catalog #BP0089, lot #2A3).
[0411] Lyophilized Compound A was reconstituted into 10 mg/mL stock with 0.1 M acetic acid. It was then further diluted into working concentration with lx phosphate buffered saline (PBS). The compound was reconstituted and diluted within an hour of dosing of animals and kept on ice until dosing. The lyophilized compound was stored at -80°C before use. Vehicle was stored at 4°C.
[0412] Three separate efficacy studies were performed using CT-26 tumor-bearing Balb/c mice. The design for study 1, which evaluated Compound A for dose-dependent efficacy as a single agent, is outlined in Table 13. The designs for studies 2 and 3, which evaluated the efficacy of Compound A in combination with an anti-PD-1 antibody, are outlined in Table 14 and Table 15, respectively. The route of administration for Compound A was intravenous (IV). IV dosing in the mice was done via the tail vein. The antibody was administered intraperitoneally (IP). All agents were administered based on the individual body weight of each animal obtained immediately prior to each dosing. Details on the dosing regimen are described below.
Table 13. Study #1: Control and Test Treatment Groups in CT-26 Tumor-Bearing Mice.
Figure imgf000118_0001
IV = intravenous; QWx3 = once a week for a total of 3 doses; Q2Wx2 = once every 2 weeks for a total of 2 doses.
Table 14. Study #2: Control and Test Treatment Groups in CT-26 Tumor-Bearing Mice.
Figure imgf000119_0001
intravenous; QWx3 = once a week for a total of 3 doses.
Table 15. Study #3: Control and Test Treatment Groups in CT-26 Tumor-Bearing Mice.
Figure imgf000119_0002
intravenous; QWx3 = once a week for a total of 3 doses; Q2Wx2 = once every 2 weeks for a total of 2 doses.
[0413] In Study 1, CT-26 tumor-bearing mice were treated with vehicle IV once a week for a total of 3 doses (QWx3) or Compound A at 0.3, 1, or 3 mg/kg IV, either once a week for a total of three doses (QWx3), or once every 2 weeks for a total of 2 doses (Q2Wx2), starting on Day 4 following tumor cell inoculation when the average tumor volume was ~80 mm3. [0414] In Study 2, CT-26 tumor-bearing mice were treated on Day 5 following tumor cell inoculation when the average tumor volume was ~80 mm3. Dosing was with vehicle IV QWx3 + IgG isotype control IP or Compound A at 3 or 6 mg/kg IV, on a QWx3 dosing schedule, or anti- PD-1 antibody at 10 mg/kg IP, or the combination of Compound A at 6 mg/kg IV QWx3 + anti- PD-1 antibody at 10 mg/kg IP. The IP dosing of the antibody in all cases was twice a week for 3 weeks with a total of 6 doses (BIWx3).
[0415] In Study 3, CT-26 tumor-bearing mice were treated on Day 7 following tumor cell inoculation when the average tumor volume was ~70 mm3. Dosing was with vehicle IV QWx3 + IgG isotype control IP BIWx3; or Compound A at 1, 3, 6, or 9 mg/kg IV on a QWx3 dosing schedule, or anti-PD-1 antibody at 10 mg/kg IP BIWx3; or the combination of Compound A at 1, 3, or 6 mg/kg IV QWx3 + anti-PD-1 antibody at 10 mg/kg IP BIWx3.
[0416] A summary of all three studies is shown in Table 16. Animals were observed daily for clinical signs. In accordance with IACUC guidelines, animals were humanely euthanized when tumors grew over 2000 mm3 in volume or they were observed to have a continuing deteriorating condition or showing obvious signs of severe distress and/or pain.
[0417] The survival of each mouse was monitored for over 100 days, at which time surviving tumor-free animals in Studies 2 and 3 were included in a re-challenge continuation of the study for two cycles, 2 months apart. Specifically, tumor-free animals were re-challenged via inoculation of the same type of tumor cells (CT-26) in the opposite lower flank. Control animals were age-matched naive mice that were concurrently inoculated with the same number of CT-26 tumor cells in the opposite lower flank.
[0418] Tumor growth was monitored using digital caliper measurements every 3 to 4 days until the end of the study. Tumor volume was calculated as Width2 x Length/2, where width is the smallest dimension and length is the largest. Raw tumor volume data are presented in the study reports.
[0419] Mean tumor volume data for each group was plotted over time with standard error of the mean (SEM) bars. Additionally, individual tumor volume data for the last day before animal sacrifice was plotted along with mean and SEM bars to examine the distribution of the data. [0420] A statistical analysis of the tumor volume data for the last day before animal sacrifice was performed using the using GraphPad Prism v.7.0. Data was analyzed for significance using a one-way ANOVA. Pairwise comparisons were made using Tukey’s test procedures (2-sided). The p-value for each individual comparison was reported.
[0421] The percent tumor growth inhibition (%TGI) in each treated group vs. a control group was calculated as: [(Control - Control baseline) - (Treated - Treated baseline)] / (Control - Control baseline) x 100%.
[0422] The survival of each mouse was recorded, and a Kaplan-Meir plot was generated to show survival by treatments group and the significance was assessed by log-rank (Mantel-Cox) test. Survival was monitored for over 100 days following treatment initiation in Studies #1, #2, and #3, and over the two re-challenge cycles in surviving tumor-free mice in Studies #2 and #3. Analyses were performed using GraphPad Prism version 7.0.
Table 16. Tumor Growth Inhibition in Mice with Compound A as Monotherapy and in Combination with Anti-Mouse PD-1 Antibody.
Figure imgf000121_0001
[0423] In Study 1, Compound A was evaluated for dose-dependent efficacy as a single agent in female Balb/c mice bearing subcutaneously established CT-26 colon tumors. The study formally ended on Day 15 after treatment initiation according to the humane endpoint set forth by the IACUC when several tumors in the control group reached over 2000 mm3 in volume. FIG. 26 shows mean tumor volume over time for groups treated QWx3 dosing with Compound A. FIG. 27 shows tumor volumes on Day 15 post treatment for each animal treated QWx3 dosing with Compound A. FIG. 28 shows mean tumor volume over time for groups treated Q2Wx2 dosing with Compound A. FIG. 29 shows tumor volumes on Day 15 post treatment for each animal with Q2Wx2 dosing with Compound A.
[0424] On the QWx3 dosing schedule, Compound A demonstrated dose-dependent single agent anti -tumor activity resulting in %TGI compared to the vehicle control of 31%, 19%, and 52% for the 0.3, 1, and 3 mg/kg dose groups, respectively. Similarly, on the Q2Wx2 dosing schedule, Compound A demonstrated dose-dependent single agent anti-tumor activity resulting %TGI compared to the vehicle control of 20%, 27%, and 45% for the 0.3, 1, and 3 mg/kg dose groups, respectively. However, on both dosing schedules, only the 3 mg/kg dose was statistically significant (p <0.05) compared to the vehicle control. Both dosing schedules demonstrated comparable anti-tumor activity. Therefore, for the subsequent studies in this mouse model, the QWx3 dosing schedule was chosen.
[0425] In FIGS. 26, 28, 30, and 33, black arrows denote days of Compound A dosing. Data in FIGS. 26 and 28 are mean tumor growth curves with QWx3 dosing and Q2Wx2 dosing with Compound A; black arrows denote days of Compound A dosing. Data in FIGS. 27 and 29 represent individual tumor volume and mean tumor volume ± standard error of the mean (SEM) (10 mice/group) on day 15 post-treatment with QWx3 and Q2Wx2 dosing with Compound A. Data represent individual tumor volumes; the mean ± SEM and %TGI compared to the vehicle control are also displayed.
[0426] Data in FIG. 28 represents mean tumor volume ± standard error of the mean (SEM)
(10 mice/group) in animals with Q2Wx2 dosing with Compound A. Data in FIG. 29 represents individual and mean tumor volume data on Day 15 post treatment with Q2Wx2 dosing with Compound A. * p<0.05 vs. vehicle control on Day 15.
[0427] There were two separate studies (Studies 2 and 3) conducted in CT-26 colon tumor- bearing mice to assess Compound A as a single agent and in combination with a murine anti- PD-1 checkpoint inhibitor antibody. The dose ranges for Compound A between the studies overlapped, with Study 3 having a wider dose range. In both studies, Compound A was administered QWx3 and the same dose level of antibody was administered BIWx3.
[0428] In Study 2, anti-tumor activity of Compound A was evaluated as a single agent at 3 and 6 mg/kg (QWx3) in female Balb/c mice bearing subcutaneously established CT-26 colon tumors. Additionally, the combination anti-tumor activity was evaluated with IV dosing of Compound A at 6 mg/kg (QWx3) and anti-PD-1 antibody at 10 mg/kg IP (BIWx3). The %TGI was calculated on Day 15 after treatment initiation because several tumors in the vehicle control group reached over 2000 mm3 in volume. However, the animals in treatment groups that demonstrated complete tumor regression were followed with tumor measurements at a frequency of once or twice a week. [0429] Compound A demonstrated single agent anti-tumor activity resulting in %TGI compared to the vehicle control of 56.3% and 35.6% for the 3 and 6 mg/kg dose groups, respectively. In the combination study, CT-26 tumor-bearing mice were treated IV with Compound A at 6 mg/kg QWx3, or IP with anti-PD-1 antibody BIWx3, or the combination with the same dosing schedules, starting 5 days following tumor cell inoculation when the average tumor volume was ~80 mm3. Mean tumor growth curves are shown in FIG. 30 for treatment of mice with vehicle, 6 mg/kg Compound A as a single agent, anti-PD-1 antibody as a single agent, and the combination of 6 mg/kg Compound A and anti-PD-1 antibody. Data in FIG. 30 represent mean tumor volume ± SEM (14 mice/group). Upper arrows denote days of Compound A dosing and lower arrows denote days of anti-PD-1 antibody dosing. The combination anti- tumor activity was significantly enhanced compared to Compound A or anti-PD-1 antibody alone (p<0.05). The %TGI data is shown in FIG. 31 and shows significant anti -tumor effects on Day 15 post treatment in the group treated with the combination of Compound A and anti-PD-1 antibody, compared to the groups treated with vehicle, Compound A alone or the anti-PD-1 antibody alone (35.6% for the Compound A alone group; 44.1% for the anti-PD-1 antibody alone group; and 74.6% for the group administered the combination of Compound A and anti- PD-1 antibody). Data represent individual tumor volumes; the mean ± SEM and %TGI compared to the vehicle control are also displayed. *p<0.05, **p<0.01, and ***p<0.01; vs. vehicle control. "Lp<0.05 vs. anti-PD-1 antibody. #p<0.05 vs. Compound A. The median survival times of the groups are shown in FIG. 32 and were 17, 27, 27.5, and 38 days for the control, Compound A, anti-PD-1 antibody, and Compound A + anti-PD-1 antibody groups, respectively. The median survival time of the combination group was significantly longer than both the Compound A (p<0.05) and anti-PD-1 antibody (p<0.05) single agent treatment groups. At 98 days post treatment, only 1 out of 14 animals (7%) in each of Compound A and anti-PD-1 antibody dose groups survived tumor-free, while 4 of 14 animals (29%) in the combination group survived tumor-free. Data in FIG. 32 represent Kaplan-Meier survival curves for treatment groups. *p<0.05 vs. vehicle control. "Lp<0.05 vs. anti-PD-1 antibody. #p<0.05 vs. Compound A.
[0430] In Study 3, the single agent anti -tumor activity of Compound A was evaluated in female Balb/c mice bearing SC CT-26 colon tumors at a wider dose range (1, 3, 6, and 9 mg/kg) as compared to Study 2 on the same IV QWx3 dosing schedule. Data in FIG. 33 represent mean tumor growth curves when Compound A was dosed a single agent at 1 mg/kg, 3 mg/kg, 6 mg/kg, and 9 mg/kg. Data represent mean tumor volume ± SEM (14 mice/group; except 12 mice/group for 9 mg/kg Compound A). Black arrows denote days of Compound A dosing. Compound A dosed alone at 1 mg/kg, 3 mg/kg, 6 mg/kg, and 9 mg/kg also demonstrated dose- dependent anti -tumor activity resulting in %TGI compared to the vehicle control of 29.8%, 58.8%, 86.2%, and 84.8% for 1, 3, 6, and 9 mg/kg dose groups, respectively (FIG. 34). The %TGI was calculated on Day 15 after treatment initiation because several tumors in the vehicle control group reached over 2000 mm3. However, the animals in treatment groups that demonstrated complete tumor regression were followed with tumor measurements at a frequency of once or twice a week. Data in FIG. 34 represent individual tumor volumes on Day 15 post treatment. Data represent individual tumor volumes; the mean ± SEM and %TGI compared to the vehicle control are also displayed. ***p<0.01 vs. vehicle control. The lowest dose (1 mg/kg) did not show statistically significant anti -tumor activity while the other 3 dose groups were statistically significant (p<0.001) compared to the vehicle treated group. The data also showed that %TGI for the two high dose groups (6 mg/kg and 9 mg/kg) were similar indicating maximal anti-tumor activity was reached at the 6 mg/kg dose. In the 9 mg/kg dose group, 2 of the 14 animals were found dead following >15% body weight loss due to treatment. [0431] In the combination phase of the study, Compound A at 1, 3, or 6 mg/kg (QWx3) was dosed with anti-PD-1 antibody at 10 mg/kg IP (BIWx3). CT-26 tumor-bearing mice were treated IV with Compound A at 1, 3, 6, or 9 mg/kg QWx3, or IP anti-PD-1 antibody BIWx3, or the combination with the same dosing schedules, starting 7 days following tumor cell inoculation when the average tumor volume was ~70 mm3. Note that for the 9 mg/kg Compound A single agent group, two animals were found dead after >15% body weight loss and are not included in the analysis. With the combination of 1 mg/kg Compound A + anti-PD-1 antibody, no additive anti-tumor activity was observed based on survival data. At Compound A days post treatment, 1 of 14 animals (7%) in the anti-PD-1 antibody group survived, while 0 of the animals in the 3 mg/kg single agent group survived. However, in the 3 mg/kg Compound A + anti-PD-1 antibody group, 2 of 14 animals (14%) survived up to Compound A days. As shown in FIG. 35, the combination of 6 mg/kg Compound A + anti-PD-1 antibody resulted in prolonged survival compared to each single agent alone. The median survival times were 21, 35, 24.5, and 49 days for the vehicle control, Compound A (6 mg/kg), anti-PD-1 antibody (10 mg/kg), and Compound A + anti-PD-1 antibody groups (6 mg/kg Compound A and 10 mg/kg anti-PD-1 antibody), respectively. The median survival time of the combination group was significantly longer than the Compound A and anti-PD-1 antibody (p<0.05) single agent treatment groups. Specifically, at Compound A days post treatment, 0 of the animals in the 6 mg/kg Compound A group survived while only 1 of 14 animals (7%) in the anti-PD-1 antibody group survived tumor-free. However, in the combination group, 5 of 14 (36%) animals survived tumor-free (p<0.05). Data in FIG. 35 represent Kaplan-Meier survival curves for treatment groups. *p<0.05 vs. vehicle control. -½><0.05 vs. anti-PD-1 antibody. #p<0.05 vs. Compound A. Example 6. PK/PD Studies in Naive (E3826-U1704) and B16-F10 tumor-tearing (E3826- U1803) C57BL/6 mice.
[0432] This study describes the effect of certain IL-2 conjugates as described herein in a murine melanoma model. The study designs are summarized in Table 17 and Table 18, wherein the dose was calculated by reference to the mass of the protein component not including the mass of the PEG moiety. Terminal blood samples were collected via cardiac puncture at the points indicated. Study E3826-U1704 included 13 time points (0.13, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120 h) sacrificing 3 mice per each time point, and study E3826-U1803 included 9 time points (2, 8, 12, 24, 48, 72, 120, 168, and 240 h) sacrificing 4-7 mice per each time point. Plasma and blood cells (in both studies) and tumors in study E3826-U1803 were collected for PK and PD analyses.
[0433] The IL-2 conjugates P65_30kD and E62_30kD were used in this study. The IL-2 conjugate P65_30kD refers to the conjugate “IL-2_P65[AzK_PEG30kD]” described in Example 5. The IL-2 conjugate E62_30kD comprises SEQ ID NO: 5:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEfAzK PEG30kDlLKPLEEVLNLAOSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 5) wherein [AzK_ 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 (I) in which Z, Y, W, and q were as described in Example 5 for “IL-2_P65[AzK_PEG30kD]”. The IL-2 conjugate E62_30kD was prepared using methods wherein a protein was first prepared having SEQ ID NO: 4 in which the glutamic acid at position 62 was replaced by N6-((2- azidoethoxy)-carbonyl)-L-lysine AzK. The AzK-containing protein was then allowed to react under click chemistry conditions with DBCO comprising a methoxy, linear PEG group having an average molecular weight of 30kDa, followed by purification and formulation employing standard procedures.
[0434] Bioanalysis of plasma samples was performed using a qualified human IL-2 ELISA assay (Abeam, Cambridge, UK). Concentrations of Aldesleukin, E62_30kD and P65_30kD and the internal standard in samples derived from plasma were determined using an ELISA assay.
PK data analysis was performed at NW Solutions (Seattle, WA). The PK data were imported into Phoenix WinNonlin v6.4 (Certara/Pharsight, Princeton, NJ) for analysis. The group mean plasma concentration versus time data were analyzed with noncompartmental methods using an IV bolus administration model. Table 17. PK/PD Study No. E3826-U1704 - Control and Test Treatment groups in Naive C57/BL6 Mice.
Figure imgf000126_0002
Table 18. PK/PD Study No. E3826-U1803 - Control and Test Treatment groups in B16F-10 Melanoma Tumor-Bearing Mice.
Figure imgf000126_0003
[0435] In study E3826-U1704, both P65_30kD and E62_30kD exhibit a superior PK profile relative to aldesleukin as summarized in Table 19. Following a single IV bolus dose of aldesleukin, the Tmax was observed at 0.03 h post-dose (the first measured time point after dosing) and mean plasma concentrations were measurable out to 4 h post-dose. After single IV bolus dosing of P65_30kD and E62_30kD, the Tmax was observed at 0.03 h post-dose and mean plasma concentrations were measurable out to 120 h post-dose (the last measured time point). In a separate study, after IV dosing of E62_5kD (an IL-2 conjugate analogous to E62_30kD but having a PEG group with an average molecular weight of 5kDa), the Tmax was observed at 0.133 hr post-dose and mean plasma concentrations were measurable out to 12 hr post-dose.
[0436] Exposure based on Cmax and
Figure imgf000126_0001
was as follows: P65_30kD >E62_30kD » E62_5kD> aldesleukin. E62_5kD with a smaller PEG had a PK profile closer to rIL-2 (Table 15). P65_30kD exposure was 5.5 and 200 times higher than aldesleukin based on Cmax and AUCO-t, respectively. In addition, P65_30kD demonstrated 23-fold extended tl/2 (13.3 h vs. 0.57 h) and about 198-fold reduced CL (6.58 vs 1300 mL/h/Kg) compared to the aldesleukin. For both P65_30kD and E62_30kD, the distribution volume (82.4 and 92.3 mL/Kg respectively) was about 4.2 to 4.7-fold reduced relative to aldesleukin, and similar to the blood volume in a mouse (85 mL/Kg; [Boersen 2013]). This suggests that P65_30kD and E62_30kD are mostly distributed within systemic circulation.
Table 19. P65 30kD PK Parameters in C57BL/6 Female Mice.
Figure imgf000127_0001
Example 7. Pharmacodynamics Studies of an IL-2 conjugate.
Pharmacodynamics observations in peripheral blood compartment.
[0437] STAT5 phosphorylation and induction of cell proliferation (the early molecular marker Ki-67 and cell counts) were used as pharmacodynamics readouts to assess the pharmacological profile of P65_30kD, described in Example 4, relative to its pharmacokinetics. The pSTAT5 PD marker showed good correlation with PK for both P65_30kD and aldesleukin in CD8+ effector T cells (Table 13). Persistent elevation of pSTAT5 was observed in both NK and CD8+ T cells up to 72 h, and up to 24 h in Tregs. pSTAT5 induction returned to baseline after only 2 h in mice dosed with aldesleukin. STAT5 phosphorylation translated into proliferative responses (72 - 120 hrs) of CD8+ effector T cells and NK cells but not with T regs, Phenotypic analysis of CD8+ effector T cells revealed substantial expansion of CD44+ memory cells within this population.
Pharmacodynamics observations in Tumor comyartment in B16-F10 Tumor -Bearing ( Έ3826- U1803) C57BL/6 mice.
[0438] Table 20 shows the plasma and tumor drug concentration following a single dose of P65_30kD at 3 mg/kg in B16-F10 tumor-bearing mice, wherein the dose was calculated by reference to the mass of the protein component not including the mass of the PEG moiety. The tumor half-life was twice the plasma half-life (24.4 vs 12.6), indicating that the P65_30kD penetrates the tumor and is retained in the tumor. The tail end of the curves cross showing the plasma eliminates faster than the tumor (data not shown). The tumorplasma AUC ratio was 9.7% and 8.4% for the 1 and 3 mg/kg doses respectively.
Table 20. P65_30kD Plasma and Tumor PK Parameters B16-F10 tumor-bearing C57BL/6 Female Mice.
Figure imgf000128_0001
MTD study in Balb/c mice E3826-U1802
[0439] A dose ranging study of P65_30kD was conducted in naive female Balb/c mice at Crown Biosciences, Inc. (San Diego, CA). The study design is shown in Table 21, wherein the dose was calculated by reference to the mass of the protein component not including the mass of the PEG moiety. Blood samples were drawn via sub mandibular vein at 8 time points (0.25, 1, 4, 12, 24, 34, 48 & 72 h). Both plasma and blood cells were collected for PK and PD analyses. [0440] All plasma samples were analyzed for human IL-2 as well as mouse IL-2, TNF-a, IFNy, IL-5, and IL-6 cytokines, employing commercially-available ELISA kits.
Table 21. PK/PD and MTD Study No. E3826-U1802 - Control and Test Treatment groups in Naive Balb/C Mice.
Figure imgf000128_0002
Figure imgf000129_0001
Toxicology observations in the MTD study using Balb/c mice
[0441] A major of toxicity associated with high-dose aldesleukin is vascular leak syndrome and associated Cytokine Release Syndrome (CRS). To evaluate the potential for this effect in mice, a single dose IV administration of P65_30kD at doses ranging from 0.01-5.0 mg/kg dose was performed (Table 21), wherein the dose was calculated by reference to the mass of the protein component not including the mass of the PEG moiety. The analysis performed was hematology, histopathology, organ weight, and cytokine analyses. Abnormalities were not observed with hematology, histopathology or body weights relative to the vehicle control mice with both P65_30kD or aldesleukin. With respect to the cytokine analysis, it was observed that aldesleukin elevated plasma IL-5 levels starting at 1 mg/kg to 5 mg/kg. With P65_30kD, a moderate increase in IL-5 (but less compared to aldesleukin) was seen only at 5 mg/kg dose. A transient elevation in the systemic levels of IKNg was observed with both aldesleukin and P65_30kD.
Example 8. Clinical study of combination therapy using an IL-2 conjugate and cemiplimab.
[0442] A Phase 1/2 non-randomized, open-label, multi-cohort, multi-center study assessing the clinical benefit of the IL-2 conjugate described in Example 1 in combination with cemiplimab for the treatment of participants with advanced unresectable or metastatic skin cancers was undertaken. Cohort A participants were patients with immune checkpoint inhibitor (ICI)-naive locally advanced, unresectable or metastatic melanoma who have not received prior treatment (i.e., the IL-2 conjugate treatment is 1L or first-line therapy; the subject is treatment- naive). Cohort B participants were patients with immune checkpoint inhibitor (ICI)-naive metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation, and who have received no more than 2 prior lines of systemic therapy (i.e., the IL-2 conjugate treatment is 1-3L, or first- to third-line therapy).
[0443] Participants of both Cohort A and Cohort B received the IL-2 conjugate (16 or 24 μg/kg dose) and cemiplimab (350 mg) once every 3 weeks (on Day 1 of each cycle). Prior to treatment for the first 4 cycles, participants were administered acetaminophen, diphenhydramine, and ondansetron (or equivalents). For subsequent cycles, prior to treatment, the supervising physician may have decided to administer acetaminophen, diphenhydramine, and/or ondansetron if determined to be medically necessary or suitable. Treatment is being repeated for up to a total of 35 cycles (1 cycle is 21 days).
[0444] Key Inclusion Criteria. All participants were males or females 18 years of age or older. Participants in Cohort A had histologically-confirmed diagnosis of unresectable locally advanced or metastatic melanoma not amenable to local therapy. Participants in Cohort B had histologically-confirmed diagnosis of locally advanced or metastatic cutaneous squamous cell carcinoma (CSCC), with no more than 2 prior lines of systemic therapy, and who were not candidates for curative surgery or radiation. Participants in Cohort A had at least one measurable lesion per RECIST 1.1 criteria. Participants in Cohort B had at least one measurable lesion per RECIST 1.1 or at least a lesion that could be followed by serial digital medical photographs using modified WHO criteria. All participants had adequate cardiovascular, hematological, liver, renal function, and laboratory parameters.
[0445] Key Exclusion Criteria. Participants did not have a history of allogenic tissue or solid organ transplant. Participants did not have immune-mediated/related toxicity from prior immuno-oncology therapy of Grade 4 or leading to discontinuation. Participants did not have ongoing AEs caused by any prior anti-cancer therapy >Grade 2. Participants did not have baseline oxygen saturation (Sp02) <92% (without oxygen therapy). Participants may have temporarily (for at least 36 hours) withheld antihypertensive medications prior to each IL-2 conjugate dosing. Participants did not have any medical or clinical condition, laboratory abnormality, or any specific situation as judged by the supervising physician that would preclude protocol therapy or would make the subject inappropriate for the study. Participants in Cohort A did not have uveal or ocular or desmoplastic melanoma. Participants in Cohort B did not have dry red lip (vermillion) or anogenital area as the primary site of CSCC and mixed CSCC histologies (e.g., sarcomatoid, adenosquamous). All participants had an ECOG performance status of less than 2.
[0446] The progression of disease is being monitored in patients according to various criteria. The objective response rate (ORR) is being evaluated in patients following administration of the first dose of the IL-2 conjugate and cemiplimab combination treatment per RECIST 1.1, using modified WHO criteria, or composite, optionally up to the date of the first documented progression. The incidence of treatment emergent adverse events (TEAEs), dose-limiting toxicities (DLTs), serious adverse events (SAEs), and laboratory abnormalities is being evaluated from the signing of the informed consent form (ICF) until 90 days following last administration of study treatment or until the participant initiates another anticancer therapy, whichever is earlier, according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v 5.0 and the American Society for Transplantation and Cellular Therapy (ASTCT) consensus gradings. The complete response rate (CRR) and the time to CR per RECIST 1.1 is being evaluated for Cohort A participants and, when applicable, for Cohort B participants, up to approximately 6 months following administration of the first dose of the IL-2 conjugate and cemiplimab combination treatment. The times at which the foregoing parameters are evaluated is being adjusted as necessary, based on the supervising physician’s assessment. For example, any of the variables described above may be evaluated at up to 36 months following administration of the first treatment dose.
[0447] The following parameters are also being evaluated in patients up to approximately 36 months following administration of the first dose of the IL-2 conjugate and cemiplimab combination treatment: (1) time to response (TTR), per RECIST 1.1, modified WHO criteria, or composite criteria; (2) duration of response (DoR), defined as the time from the first documented evidence of CR or PR until progressive disease (PD) determined per RECIST 1.1, modified WHO Criteria for medical photographs, or composite criteria, or death from any cause, whichever occurs first; (3) clinical benefit rate (CBR), including confirmed CR or PR at any time plus stable disease (SD) of at least 6 months per RECIST 1.1, modified WHO criteria for medical photographs, or composite criteria; and (4) progression free survival (PFS), defined as the time from the date of first administration of IL-2 conjugate and cemiplimab combination treatment to the date of the first documented disease progression, as per RECIST 1.1 or modified WHO Criteria for medical photographs, or death due to any cause, whichever occurs first. Pharmacokinetic parameters, such as concentration of IL-2 conjugate, incidence of anti-drug antibodies (AD As) against the IL-2 conjugate, Ctrough of cemiplimab, and Cend_ofjnfusion of cemiplimab, are also being evaluated in patients at various time points throughout the study. [0448] The number (%) of participants with treatment emergent adverse events (TEAE) by primary system organ class (SOC) and worst grade by participant (PT) for Cohort A (5 patients) and Cohort B (4 patients) is detailed in Table 22. Table 22. Treatment Emergent Adverse Events (TEAE)
Figure imgf000132_0001
Figure imgf000133_0001
[0449] Four individuals having CSCC (Cohort B) received the IL-2 conjugate at a dose of 16 μg/kg Q3W in combination with cemiplimab (350 mg Q3W). For 3 of the 4 individuals, investigators reported an unconfirmed response (i.e., an apparent decrease in the size of target lesions) after the first tumor assessment; for these 3 individuals, the first tumor assessment occurred following 3 cycles. For the remaining individual, the investigator reported a confirmed pseudo-progression during the first cycle, then later reported an unconfirmed response (i.e., an apparent decrease in the size of target lesion(s)) after 7 weeks).
[0450] Five individuals having melanoma received the IL-2 conjugate at a dose of 16 μg/kg Q3W in combination with cemiplimab (350 mg Q3W). In some embodiments, an individual shows a decrease in the size of target lesion(s) after one cycle of treatment. In some embodiments, an individual shows a decrease in the size of target lesion(s) after the first tumor assessment. In some embodiments, an individual shows a response (i.e., a decrease in the size of target lesions) after the second, third, or fourth tumor assessment. In some embodiments, the individual shows a response (i.e., a decrease in the size of target lesions) after 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 cycles of treatment. In some embodiments, the individual shows a response (i.e., a decrease in the size of target lesions) after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 weeks following the first treatment.
[0451] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein in their entirety by reference.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cemiplimab, 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 imgf000135_0001
wherein:
Figure imgf000135_0002
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000135_0003
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
2. A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate, and (b) cemiplimab, wherein: the skin cancer is unresectable skin cancer, locally advanced cutaneous squamous cell carcinoma, or metastatic skin cancer; and 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 imgf000136_0002
wherein:
Figure imgf000136_0003
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000136_0001
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
3. A method of treating skin cancer in a subject in need thereof, comprising: selecting a subject having skin cancer, wherein the subject is selected on the basis of one or more attributes comprising (i) the skin cancer being unresectable skin cancer; (ii) the skin cancer being locally advanced cutaneous squamous cell carcinoma; or (iii) the skin cancer being metastatic skin cancer; and administering to the subject (a) an IL-2 conjugate, and (b) cemiplimab, 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 imgf000137_0001
wherein:
Figure imgf000137_0002
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000138_0001
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
4. A method of treating skin cancer in a subject in need thereof, comprising administering to the subject (a) about 8 μg/kg, 16 μg/kg, or 24 μg/kg of an IL-2 conjugate and (b) cemiplimab, 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 imgf000138_0002
wherein:
Figure imgf000138_0003
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3;
X is an L-amino acid having the structure:
Figure imgf000139_0001
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
5. The method of any one of claims 1-4, wherein the skin cancer is melanoma.
6. The method of any one of claims 1-4, wherein the skin cancer is cutaneous squamous cell carcinoma.
7. The method of any one of claims 1-4, wherein the skin cancer is locally advanced cutaneous squamous cell carcinoma.
8. The method of any one of claims 1-7, wherein the skin cancer is unresectable.
9. The method of any one of claims 1-8, wherein the skin cancer is metastatic.
10. The method of any one of claims 1-9, wherein the skin cancer is not amenable to local therapy.
11. The method of any one of claims 1-10, wherein the skin cancer is advanced.
12. The method of any one of claims 1-11, wherein the skin cancer is immune checkpoint inhibitor-naive.
13. The method of any one of claims 1-12, comprising administering to the subject about 8 μg/kg of the IL-2 conjugate.
14. The method of any one of claims 1-12, comprising administering to the subject about 16 μg/kg of the IL-2 conjugate.
15. The method of any one of claims 1-12, comprising administering to the subject about 24 μg/kg of the IL-2 conjugate.
16. The method of any one of claims 1-15, wherein in the IL-2 conjugate the PEG group has an average molecular weight of about 30 kDa.
17. The method of any one of claims 1-16, wherein in the IL-2 conjugate Z is CH2 and Y is
Figure imgf000140_0002
18. The method of any one of claims 1-16, wherein in the IL-2 conjugate Y is CH2 and Z is
Figure imgf000140_0003
19. The method of any one of claims 1-16, wherein in the IL-2 conjugate Z is CH2 and Y is
Figure imgf000140_0004
20. The method of any one of claims 1-16, wherein in the IL-2 conjugate Y is CH2 and Z is
Figure imgf000140_0005
21. The method of any one of claims 1-16, 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 imgf000140_0001
wherein:
W is a PEG group having an average molecular weight of about 25 kDa - 35 kDa; q is 1, 2, or 3; X is an L-amino acid having the structure:
Figure imgf000141_0001
X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the following amino acid residue.
22. The method of any one of claims 1-16, 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 imgf000141_0002
wherein: n is an integer such that has a molecular weight of about 30 kDa;
Figure imgf000141_0003
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.
23. The method of any one of claims 1-22, wherein q is 1.
24. The method of any one of claims 1-22, wherein q is 2.
25. The method of any one of claims 1-22, wherein q is 3.
26. The method of any one of claims 1-25, wherein the IL-2 conjugate is administered to the subject about once every two weeks, about once every three weeks, or about once every 4 weeks.
27. The method of any one of claims 1-26, wherein the IL-2 conjugate and cemiplimab are administered to the subject about once every two weeks, about once every three weeks, or about once every 4 weeks.
28. The method of any one of claims 1-27, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.
29. The method of any one of claims 1-28, wherein cemiplimab is administered at a dose of about 350 mg every 3 weeks.
30. The method of any one of claims 1-29, wherein the IL-2 conjugate and cemiplimab are administered separately.
31. The method of claim 30, wherein the IL-2 conjugate and cemiplimab are administered sequentially.
32. The method of claim 30 or 31, wherein the IL-2 conjugate is administered before cemiplimab.
33. The method of claim 30 or 31, wherein the IL-2 conjugate is administered after cemiplimab.
34. The method of any one of claims 1-33, wherein the IL-2 conjugate is administered to the subject by intravenous administration.
35. The method of any one of claims 1-34, wherein the IL-2 conjugate and cemiplimab are administered to the subject by intravenous administration.
36. The method of any one of claims 1-35, further comprising administering acetaminophen to the subject.
37. The method of any one of claims 1-36, further comprising administering diphenhydramine to the subject.
38. The method of any one of claims 1-37, further comprising administering ondansetron to the subject.
39. The method of any one of claims 36-38, wherein the acetaminophen, diphenhydramine, and/or ondansetron is administered to the subject before administering the IL-2 conjugate.
40. The method of any one of claims 1-39, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being unresectable skin cancer.
41. The method of any one of claims 1-40, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being the skin cancer being locally advanced cutaneous squamous cell carcinoma.
42. The method of any one of claims 1-41, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being metastatic skin cancer.
43. The method of any one of claims 1-42, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being not amenable to local therapy.
44. The method of any one of claims 1-43, further comprising selecting the subject to whom the IL-2 conjugate and cemiplimab are administered at least in part on the basis of the skin cancer being immune checkpoint inhibitor-naive.
45. An IL-2 conjugate for use in the method of any one of claims 1-44.
46. Use of an IL-2 conjugate for the manufacture of a medicament for the method of any one of claims 1-45.
PCT/US2022/016216 2021-02-12 2022-02-11 Skin cancer combination therapy with il-2 conjugates and cemiplimab WO2022174101A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202163149081P 2021-02-12 2021-02-12
US63/149,081 2021-02-12
US202163276955P 2021-11-08 2021-11-08
US63/276,955 2021-11-08

Publications (1)

Publication Number Publication Date
WO2022174101A1 true WO2022174101A1 (en) 2022-08-18

Family

ID=80685433

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/016216 WO2022174101A1 (en) 2021-02-12 2022-02-11 Skin cancer combination therapy with il-2 conjugates and cemiplimab

Country Status (2)

Country Link
TW (1) TW202245843A (en)
WO (1) WO2022174101A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11701407B2 (en) 2017-08-03 2023-07-18 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases

Citations (173)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4667025A (en) 1982-08-09 1987-05-19 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
US4835263A (en) 1983-01-27 1989-05-30 Centre National De La Recherche Scientifique Novel compounds containing an oligonucleotide sequence bonded to an intercalating agent, a process for their synthesis and their use
US4845205A (en) 1985-01-08 1989-07-04 Institut Pasteur 2,N6 -disubstituted and 2,N6 -trisubstituted adenosine-3'-phosphoramidites
US4849513A (en) 1983-12-20 1989-07-18 California Institute Of Technology Deoxyribonucleoside phosphoramidites in which an aliphatic amino group is attached to the sugar ring and their use for the preparation of oligonucleotides containing aliphatic amino groups
US4876335A (en) 1986-06-30 1989-10-24 Wakunaga Seiyaku Kabushiki Kaisha Poly-labelled oligonucleotide derivative
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
US4910300A (en) 1985-12-11 1990-03-20 Chiron Corporation Method for making nucleic acid probes
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US4981957A (en) 1984-07-19 1991-01-01 Centre National De La Recherche Scientifique Oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini
US5015733A (en) 1983-12-20 1991-05-14 California Institute Of Technology Nucleosides possessing blocked aliphatic amino groups
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
US5093232A (en) 1985-12-11 1992-03-03 Chiron Corporation Nucleic acid probes
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5112963A (en) 1987-11-12 1992-05-12 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Modified oligonucleotides
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US5130302A (en) 1989-12-20 1992-07-14 Boron Bilogicals, Inc. Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
US5319080A (en) 1991-10-17 1994-06-07 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
WO1994014226A1 (en) 1992-12-14 1994-06-23 Honeywell Inc. Motor system with individually controlled redundant windings
EP0614907A1 (en) 1993-03-06 1994-09-14 Ciba-Geigy Ag Dinucleotide and oligonucleotide analogues
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
EP0629633A2 (en) 1993-06-05 1994-12-21 Ciba-Geigy Ag Dinucleotide analogues, intermediates therefor and oligonucleotides derived therefrom
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5414077A (en) 1990-02-20 1995-05-09 Gilead Sciences Non-nucleoside linkers for convenient attachment of labels to oligonucleotides using standard synthetic methods
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US5446137A (en) 1993-12-09 1995-08-29 Syntex (U.S.A.) Inc. Oligonucleotides containing 4'-substituted nucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5466786A (en) 1989-10-24 1995-11-14 Gilead Sciences 2'modified nucleoside and nucleotide compounds
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5510475A (en) 1990-11-08 1996-04-23 Hybridon, Inc. Oligonucleotide multiple reporter precursors
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5514785A (en) 1990-05-11 1996-05-07 Becton Dickinson And Company Solid supports for nucleic acid hybridization assays
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5545730A (en) 1984-10-16 1996-08-13 Chiron Corporation Multifunctional nucleic acid monomer
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5552540A (en) 1987-06-24 1996-09-03 Howard Florey Institute Of Experimental Physiology And Medicine Nucleoside derivatives
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5567811A (en) 1990-05-03 1996-10-22 Amersham International Plc Phosphoramidite derivatives, their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5576427A (en) 1993-03-30 1996-11-19 Sterling Winthrop, Inc. Acyclic nucleoside analogs and oligonucleotide sequences containing them
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5587371A (en) 1992-01-21 1996-12-24 Pharmacyclics, Inc. Texaphyrin-oligonucleotide conjugates
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5596086A (en) 1990-09-20 1997-01-21 Gilead Sciences, Inc. Modified internucleoside linkages having one nitrogen and two carbon atoms
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5599923A (en) 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5610300A (en) 1992-07-01 1997-03-11 Ciba-Geigy Corporation Carbocyclic nucleosides containing bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5614617A (en) 1990-07-27 1997-03-25 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5639873A (en) 1992-02-05 1997-06-17 Centre National De La Recherche Scientifique (Cnrs) Oligothionucleotides
US5645985A (en) 1991-11-26 1997-07-08 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5658873A (en) 1993-04-10 1997-08-19 Degussa Aktiengesellschaft Coated sodium percarbonate particles, a process for their production and detergent, cleaning and bleaching compositions containing them
US5663312A (en) 1993-03-31 1997-09-02 Sanofi Oligonucleotide dimers with amide linkages replacing phosphodiester linkages
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
WO1997035869A1 (en) 1996-03-23 1997-10-02 Novartis Ag Dinucleotide and oligonucleotide analogues
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5677439A (en) 1990-08-03 1997-10-14 Sanofi Oligonucleotide analogues containing phosphate diester linkage substitutes, compositions thereof, and precursor dinucleotide analogues
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5750692A (en) 1990-01-11 1998-05-12 Isis Pharmaceuticals, Inc. Synthesis of 3-deazapurines
US5830653A (en) 1991-11-26 1998-11-03 Gilead Sciences, Inc. Methods of using oligomers containing modified pyrimidines
WO1999062923A2 (en) 1998-06-05 1999-12-09 Dynavax Technologies Corporation Immunostimulatory oligonucleotides with modified bases and methods of use thereof
US6268490B1 (en) 1997-03-07 2001-07-31 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogues
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
US6670461B1 (en) 1997-09-12 2003-12-30 Exiqon A/S Oligonucleotide analogues
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
US20040171570A1 (en) 2002-11-05 2004-09-02 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
WO2004106356A1 (en) 2003-05-27 2004-12-09 Syddansk Universitet Functionalized nucleotide derivatives
WO2005021570A1 (en) 2003-08-28 2005-03-10 Gene Design, Inc. Novel artificial nucleic acids of n-o bond crosslinkage type
US20050130923A1 (en) 2003-09-18 2005-06-16 Balkrishen Bhat 4'-thionucleosides and oligomeric compounds
US20060074035A1 (en) 2002-04-17 2006-04-06 Zhi Hong Dinucleotide inhibitors of de novo RNA polymerases for treatment or prevention of viral infections
US7053207B2 (en) 1999-05-04 2006-05-30 Exiqon A/S L-ribo-LNA analogues
WO2007090071A2 (en) 2006-01-27 2007-08-09 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
WO2007134181A2 (en) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. 5'-modified bicyclic nucleic acid analogs
US20080039618A1 (en) 2002-11-05 2008-02-14 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
WO2008101157A1 (en) 2007-02-15 2008-08-21 Isis Pharmaceuticals, Inc. 5'-substituted-2'-f modified nucleosides and oligomeric compounds prepared therefrom
WO2008150729A2 (en) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2008154401A2 (en) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
WO2009006478A2 (en) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
US8778631B2 (en) 2009-01-12 2014-07-15 Sutro Biopharma, Inc. Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system
US20140315245A1 (en) 2013-04-19 2014-10-23 Sutro Biopharma, Inc. Expression of biologically active proteins in a bacterial cell-free synthesis system using bacterial cells transformed to exhibit elevated levels of chaperone expression
WO2015021432A1 (en) 2013-08-08 2015-02-12 The Scripps Research Institute A method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
WO2015157555A2 (en) 2014-04-09 2015-10-15 The Scripps Research Institute Import of unnatural or modified nucleoside triphosphates into cells via nucleic acid triphosphate transporters
US9201020B2 (en) 2011-10-25 2015-12-01 Apogee Enterprises, Inc. Specimen viewing device
WO2016115168A1 (en) 2015-01-12 2016-07-21 Synthorx, Inc. Incorporation of unnatural nucleotides and methods thereof
US9402993B2 (en) 2011-04-11 2016-08-02 Boston Scientific Neuromodulation Corporation Systems and methods for enhancing paddle lead placement
WO2017106767A1 (en) 2015-12-18 2017-06-22 The Scripps Research Institute Production of unnatural nucleotides using a crispr/cas9 system
US20170283469A1 (en) 2012-10-12 2017-10-05 Sutro Biopharma, Inc. Proteolytic inactivation of select proteins in bacterial extracts for improved expression
WO2017223528A1 (en) 2016-06-24 2017-12-28 The Scripps Research Institute Novel nucleoside triphosphate transporter and uses thereof
US20180051065A1 (en) 2014-12-19 2018-02-22 Sutro Biopharma, Inc. Codon optimization for titer and fidelity improvement
US9938516B2 (en) 2013-10-11 2018-04-10 Sutro Biopharma, Inc. Non-natural amino acid tRNA synthetases for para-methylazido-L-phenylalanine
US9988619B2 (en) 2013-10-11 2018-06-05 Sutro Biopharma, Inc. Non-natural amino acid tRNA synthetases for pyridyl tetrazine
WO2018234862A1 (en) * 2017-06-19 2018-12-27 Medicenna Therapeutics Inc. Uses and methods for il-2 superagonists, agonists, and fusions thereof
WO2019014262A1 (en) 2017-07-11 2019-01-17 The Scripps Research Institute Incorporation of unnatural nucleotides and methods of use in vivo thereof
WO2019014267A1 (en) 2017-07-11 2019-01-17 Synthorx, Inc. Incorporation of unnatural nucleotides and methods thereof
WO2019028419A1 (en) 2017-08-03 2019-02-07 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
WO2020163532A1 (en) * 2019-02-06 2020-08-13 Synthorx, Inc. Il-2 conjugates and methods of use thereof
WO2021030706A1 (en) * 2019-08-15 2021-02-18 Synthorx, Inc. Immuno oncology combination therapies with il-2 conjugates

Patent Citations (200)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4667025A (en) 1982-08-09 1987-05-19 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4789737A (en) 1982-08-09 1988-12-06 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives and production thereof
US4835263A (en) 1983-01-27 1989-05-30 Centre National De La Recherche Scientifique Novel compounds containing an oligonucleotide sequence bonded to an intercalating agent, a process for their synthesis and their use
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US5541313A (en) 1983-02-22 1996-07-30 Molecular Biosystems, Inc. Single-stranded labelled oligonucleotides of preselected sequence
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US4849513A (en) 1983-12-20 1989-07-18 California Institute Of Technology Deoxyribonucleoside phosphoramidites in which an aliphatic amino group is attached to the sugar ring and their use for the preparation of oligonucleotides containing aliphatic amino groups
US5015733A (en) 1983-12-20 1991-05-14 California Institute Of Technology Nucleosides possessing blocked aliphatic amino groups
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US4981957A (en) 1984-07-19 1991-01-01 Centre National De La Recherche Scientifique Oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini
US5545730A (en) 1984-10-16 1996-08-13 Chiron Corporation Multifunctional nucleic acid monomer
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5578717A (en) 1984-10-16 1996-11-26 Chiron Corporation Nucleotides for introducing selectably cleavable and/or abasic sites into oligonucleotides
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US5552538A (en) 1984-10-16 1996-09-03 Chiron Corporation Oligonucleotides with cleavable sites
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
US4845205A (en) 1985-01-08 1989-07-04 Institut Pasteur 2,N6 -disubstituted and 2,N6 -trisubstituted adenosine-3'-phosphoramidites
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US5093232A (en) 1985-12-11 1992-03-03 Chiron Corporation Nucleic acid probes
US4910300A (en) 1985-12-11 1990-03-20 Chiron Corporation Method for making nucleic acid probes
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
US4876335A (en) 1986-06-30 1989-10-24 Wakunaga Seiyaku Kabushiki Kaisha Poly-labelled oligonucleotide derivative
US5286717A (en) 1987-03-25 1994-02-15 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
US5552540A (en) 1987-06-24 1996-09-03 Howard Florey Institute Of Experimental Physiology And Medicine Nucleoside derivatives
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
US5112963A (en) 1987-11-12 1992-05-12 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Modified oligonucleotides
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5453496A (en) 1988-05-26 1995-09-26 University Patents, Inc. Polynucleotide phosphorodithioate
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
US5599923A (en) 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5416203A (en) 1989-06-06 1995-05-16 Northwestern University Steroid modified oligonucleotides
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5466786B1 (en) 1989-10-24 1998-04-07 Gilead Sciences 2' Modified nucleoside and nucleotide compounds
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5466786A (en) 1989-10-24 1995-11-14 Gilead Sciences 2'modified nucleoside and nucleotide compounds
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5130302A (en) 1989-12-20 1992-07-14 Boron Bilogicals, Inc. Boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using same
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5750692A (en) 1990-01-11 1998-05-12 Isis Pharmaceuticals, Inc. Synthesis of 3-deazapurines
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5587469A (en) 1990-01-11 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides containing N-2 substituted purines
US5414077A (en) 1990-02-20 1995-05-09 Gilead Sciences Non-nucleoside linkers for convenient attachment of labels to oligonucleotides using standard synthetic methods
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
US5541306A (en) 1990-03-08 1996-07-30 Worcester Foundation For Biomedical Research Aminoalkylphosphotriester oligonucleotide derivatives
US5563253A (en) 1990-03-08 1996-10-08 Worcester Foundation For Biomedical Research Linear aminoalkylphosphoramidate oligonucleotide derivatives
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5536821A (en) 1990-03-08 1996-07-16 Worcester Foundation For Biomedical Research Aminoalkylphosphorothioamidate oligonucleotide deratives
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
US5567811A (en) 1990-05-03 1996-10-22 Amersham International Plc Phosphoramidite derivatives, their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
US5514785A (en) 1990-05-11 1996-05-07 Becton Dickinson And Company Solid supports for nucleic acid hybridization assays
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5614617A (en) 1990-07-27 1997-03-25 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5567810A (en) 1990-08-03 1996-10-22 Sterling Drug, Inc. Nuclease resistant compounds
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
US5677439A (en) 1990-08-03 1997-10-14 Sanofi Oligonucleotide analogues containing phosphate diester linkage substitutes, compositions thereof, and precursor dinucleotide analogues
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
US5596086A (en) 1990-09-20 1997-01-21 Gilead Sciences, Inc. Modified internucleoside linkages having one nitrogen and two carbon atoms
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
US5510475A (en) 1990-11-08 1996-04-23 Hybridon, Inc. Oligonucleotide multiple reporter precursors
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5319080A (en) 1991-10-17 1994-06-07 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
US5393878A (en) 1991-10-17 1995-02-28 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5830653A (en) 1991-11-26 1998-11-03 Gilead Sciences, Inc. Methods of using oligomers containing modified pyrimidines
US5645985A (en) 1991-11-26 1997-07-08 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
US5587371A (en) 1992-01-21 1996-12-24 Pharmacyclics, Inc. Texaphyrin-oligonucleotide conjugates
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5639873A (en) 1992-02-05 1997-06-17 Centre National De La Recherche Scientifique (Cnrs) Oligothionucleotides
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US5700920A (en) 1992-07-01 1997-12-23 Novartis Corporation Carbocyclic nucleosides containing bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5610300A (en) 1992-07-01 1997-03-11 Ciba-Geigy Corporation Carbocyclic nucleosides containing bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
WO1994014226A1 (en) 1992-12-14 1994-06-23 Honeywell Inc. Motor system with individually controlled redundant windings
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5466677A (en) 1993-03-06 1995-11-14 Ciba-Geigy Corporation Dinucleoside phosphinates and their pharmaceutical compositions
EP0614907A1 (en) 1993-03-06 1994-09-14 Ciba-Geigy Ag Dinucleotide and oligonucleotide analogues
US5576427A (en) 1993-03-30 1996-11-19 Sterling Winthrop, Inc. Acyclic nucleoside analogs and oligonucleotide sequences containing them
US5663312A (en) 1993-03-31 1997-09-02 Sanofi Oligonucleotide dimers with amide linkages replacing phosphodiester linkages
US5658873A (en) 1993-04-10 1997-08-19 Degussa Aktiengesellschaft Coated sodium percarbonate particles, a process for their production and detergent, cleaning and bleaching compositions containing them
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
EP0629633A2 (en) 1993-06-05 1994-12-21 Ciba-Geigy Ag Dinucleotide analogues, intermediates therefor and oligonucleotides derived therefrom
US5763588A (en) 1993-09-17 1998-06-09 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6005096A (en) 1993-09-17 1999-12-21 Gilead Sciences, Inc. Pyrimidine derivatives
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5446137B1 (en) 1993-12-09 1998-10-06 Behringwerke Ag Oligonucleotides containing 4'-substituted nucleotides
US5446137A (en) 1993-12-09 1995-08-29 Syntex (U.S.A.) Inc. Oligonucleotides containing 4'-substituted nucleotides
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5599928A (en) 1994-02-15 1997-02-04 Pharmacyclics, Inc. Texaphyrin compounds having improved functionalization
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5591584A (en) 1994-08-25 1997-01-07 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
WO1997035869A1 (en) 1996-03-23 1997-10-02 Novartis Ag Dinucleotide and oligonucleotide analogues
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
US6268490B1 (en) 1997-03-07 2001-07-31 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogues
US6670461B1 (en) 1997-09-12 2003-12-30 Exiqon A/S Oligonucleotide analogues
US6794499B2 (en) 1997-09-12 2004-09-21 Exiqon A/S Oligonucleotide analogues
US7034133B2 (en) 1997-09-12 2006-04-25 Exiqon A/S Oligonucleotide analogues
WO1999062923A2 (en) 1998-06-05 1999-12-09 Dynavax Technologies Corporation Immunostimulatory oligonucleotides with modified bases and methods of use thereof
US7053207B2 (en) 1999-05-04 2006-05-30 Exiqon A/S L-ribo-LNA analogues
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
US20060074035A1 (en) 2002-04-17 2006-04-06 Zhi Hong Dinucleotide inhibitors of de novo RNA polymerases for treatment or prevention of viral infections
US20080039618A1 (en) 2002-11-05 2008-02-14 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US20040171570A1 (en) 2002-11-05 2004-09-02 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
WO2004106356A1 (en) 2003-05-27 2004-12-09 Syddansk Universitet Functionalized nucleotide derivatives
WO2005021570A1 (en) 2003-08-28 2005-03-10 Gene Design, Inc. Novel artificial nucleic acids of n-o bond crosslinkage type
US7427672B2 (en) 2003-08-28 2008-09-23 Takeshi Imanishi Artificial nucleic acids of n-o bond crosslinkage type
US20050130923A1 (en) 2003-09-18 2005-06-16 Balkrishen Bhat 4'-thionucleosides and oligomeric compounds
WO2007090071A2 (en) 2006-01-27 2007-08-09 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
US7399845B2 (en) 2006-01-27 2008-07-15 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
US20070287831A1 (en) 2006-05-11 2007-12-13 Isis Pharmaceuticals, Inc 5'-modified bicyclic nucleic acid analogs
WO2007134181A2 (en) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. 5'-modified bicyclic nucleic acid analogs
WO2008101157A1 (en) 2007-02-15 2008-08-21 Isis Pharmaceuticals, Inc. 5'-substituted-2'-f modified nucleosides and oligomeric compounds prepared therefrom
WO2008150729A2 (en) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2008154401A2 (en) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
WO2009006478A2 (en) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
US8778631B2 (en) 2009-01-12 2014-07-15 Sutro Biopharma, Inc. Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system
US9402993B2 (en) 2011-04-11 2016-08-02 Boston Scientific Neuromodulation Corporation Systems and methods for enhancing paddle lead placement
US9201020B2 (en) 2011-10-25 2015-12-01 Apogee Enterprises, Inc. Specimen viewing device
US20170283469A1 (en) 2012-10-12 2017-10-05 Sutro Biopharma, Inc. Proteolytic inactivation of select proteins in bacterial extracts for improved expression
US20140315245A1 (en) 2013-04-19 2014-10-23 Sutro Biopharma, Inc. Expression of biologically active proteins in a bacterial cell-free synthesis system using bacterial cells transformed to exhibit elevated levels of chaperone expression
WO2015021432A1 (en) 2013-08-08 2015-02-12 The Scripps Research Institute A method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
US9988619B2 (en) 2013-10-11 2018-06-05 Sutro Biopharma, Inc. Non-natural amino acid tRNA synthetases for pyridyl tetrazine
US9938516B2 (en) 2013-10-11 2018-04-10 Sutro Biopharma, Inc. Non-natural amino acid tRNA synthetases for para-methylazido-L-phenylalanine
WO2015157555A2 (en) 2014-04-09 2015-10-15 The Scripps Research Institute Import of unnatural or modified nucleoside triphosphates into cells via nucleic acid triphosphate transporters
US20180051065A1 (en) 2014-12-19 2018-02-22 Sutro Biopharma, Inc. Codon optimization for titer and fidelity improvement
WO2016115168A1 (en) 2015-01-12 2016-07-21 Synthorx, Inc. Incorporation of unnatural nucleotides and methods thereof
WO2017106767A1 (en) 2015-12-18 2017-06-22 The Scripps Research Institute Production of unnatural nucleotides using a crispr/cas9 system
WO2017223528A1 (en) 2016-06-24 2017-12-28 The Scripps Research Institute Novel nucleoside triphosphate transporter and uses thereof
WO2018234862A1 (en) * 2017-06-19 2018-12-27 Medicenna Therapeutics Inc. Uses and methods for il-2 superagonists, agonists, and fusions thereof
WO2019014262A1 (en) 2017-07-11 2019-01-17 The Scripps Research Institute Incorporation of unnatural nucleotides and methods of use in vivo thereof
WO2019014267A1 (en) 2017-07-11 2019-01-17 Synthorx, Inc. Incorporation of unnatural nucleotides and methods thereof
WO2019028419A1 (en) 2017-08-03 2019-02-07 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases
WO2019028425A1 (en) 2017-08-03 2019-02-07 Synthorx, Inc. Cytokine conjugates for the treatment of autoimmune diseases
WO2020163532A1 (en) * 2019-02-06 2020-08-13 Synthorx, Inc. Il-2 conjugates and methods of use thereof
WO2021030706A1 (en) * 2019-08-15 2021-02-18 Synthorx, Inc. Immuno oncology combination therapies with il-2 conjugates

Non-Patent Citations (78)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Synthorx, Inc - Annual report - Form 10-K", 12 March 2019 (2019-03-12), XP055927347, Retrieved from the Internet <URL:https://www.sec.gov/Archives/edgar/data/1609727/000156459019007374/thor-10k_20181231.htm> [retrieved on 20220602] *
BOHRINGER ET AL., TET. LETT., vol. 34, 1993, pages 2723 - 2726
BRAASCH ET AL., CHEM. BIOL, vol. 8, 2001, pages 1 - 7
CHATTOPADHYAYA ET AL., J. ORG. CHEM., vol. 209, no. 74, 1998, pages 10035 - 10039
CHATURVEDI ET AL., NUCLEIC ACIDS RES., vol. 24, 1996, pages 2318 - 2323
CHEN ET AL., PHOSPHORUS, SULFUR AND SILICON, vol. 777, 2002, pages 1783 - 1786
COLLINGWOOD ET AL., SYNLETT, vol. 7, 1995, pages 703 - 705
CROOKE ET AL., J. PHARMACOL. EXP. THER., vol. 277, 1996, pages 2966 - 2973
DHAMI ET AL., NUCLEIC ACIDS RES., vol. 42, 2014, pages 10235 - 10244
DIAB ADI ET AL: "Clinical activity of BEMPEG plus NIVO in previously untreated patients with metastatic melanoma: updated results from the phase 1/2 PIVOT-02 study", JOURNAL FOR IMMUNOTHERAPY OF CANCER, vol. 7, no. S1, 1 November 2019 (2019-11-01), pages 205/237, XP055926961, Retrieved from the Internet <URL:http://link.springer.com/article/10.1186/s40425-019-0764-0/fulltext.html> DOI: 10.1186/s40425-019-0764-0 *
DUMAS ET AL.: "Designing logical codon reassignment - Expanding the chemistry in biology", CHEMICAL SCIENCE, vol. 6, 2015, pages 50 - 69, XP055490374, DOI: 10.1039/C4SC01534G
ELAYADI ET AL., CURR. OPINION INVENS. DRUGS, vol. 2, 2001, pages 558 - 561
EPPACHER ET AL., HELVETICA CHIMICA ACTA, vol. 87, 2004, pages 3004 - 3020
FAIRHURST ET AL., SYNLETT, vol. 4, 2001, pages 467 - 472
FELDMAN ET AL., J AM CHEM SOC, vol. 139, 2017, pages 11427 - 11433
GALLIER ET AL., EUR. J. ORG. CHEM., 2007, pages 925 - 933
GEZE ET AL., J. AM. CHEM. SOC, vol. 105, no. 26, 1983, pages 7638 - 7640
HAMPTON ET AL., J. AM. CHEM. SOC, vol. 95, no. 13, 1973, pages 4404 - 4414
HAMPTON ET AL., J. MED. CHEM., vol. 19, no. 8, 1976, pages 1371 - 1377
HUTTER ET AL., HELVETICA CHIMICA ACTA, vol. 85, 2002, pages 2777 - 2806
JAGER ET AL., BIOCHEM., vol. 27, 1988, pages 7247 - 7246
JOSEPH I B ET AL: "Abstract 3258: THOR-707, a novel not-alpha IL-2, elicits durable pharmacodynamic responses in non-human primates and efficacy as single agent and in combination with anti PD-1 in multiple syngeneic mouse models", 1 July 2019 (2019-07-01), XP055926896, Retrieved from the Internet <URL:https://aacrjournals.org/cancerres/article/79/13_Supplement/3258/635477/Abstract-3258-THOR-707-a-novel-not-alpha-IL-2> [retrieved on 20220601], DOI: 10.1158/1538-7445.AM2019-3258 *
JUNG ET AL., BIOORG. MED. CHEM., vol. 8, 2000, pages 2501 - 2509
KABANOV ET AL., FEBS LETT., vol. 259, 1990, pages 327 - 330
KANDIMALLA ET AL., BIOORG. MED. CHEM., vol. 9, 2001, pages 807 - 813
KAPPLER ET AL., J. MED. CHEM., vol. 25, 1982, pages 1179 - 1184
KAPPLER ET AL., J. MED. CHEM., vol. 29, 1986, pages 1030 - 1038
KHUSHALANI NIKHIL I ET AL: "Bempegaldesleukin plus nivolumab in untreated, unresectable or metastatic melanoma: Phase III PIVOT IO 001 study design", FUTURE ONCOLOGY, vol. 16, no. 28, 1 October 2020 (2020-10-01), GB, pages 2165 - 2175, XP055927116, ISSN: 1479-6694, DOI: 10.2217/fon-2020-0351 *
KOSHKIN ET AL., TETRAHEDRON, vol. 54, 1998, pages 3607 - 3630
KUMAR ET AL., BIOORG. MED. CHEM. LETT., vol. 8, 1998, pages 2219 - 2222
LAVERGNE ET AL., J AM CHEM SOC., vol. 135, 2013, pages 18637 - 18643
LEDBETTER ET AL., J AM CHEM SOC., vol. 140, 2018, pages 16115 - 16123
LEE ANN Y ET AL: "The Landmark Series: Non-melanoma Skin Cancers", ANNALS OF SURGICAL ONCOLOGY, SPRINGER INTERNATIONAL PUBLISHING, CHAM, vol. 27, no. 1, 23 September 2019 (2019-09-23), pages 22 - 27, XP036975410, ISSN: 1068-9265, [retrieved on 20190923], DOI: 10.1245/S10434-019-07831-9 *
LETSINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 6553 - 6556
MALYSHEV ET AL., NATURE, vol. 509, 2014, pages 385 - 388
MALYSHEV ET AL., NATURE, vol. 509, no. 7500, pages 385 - 388
MALYSHEV ET AL., PROC NATL ACAD SCI USA, vol. 109, 2012, pages 12005 - 12010
MANOHARAN ET AL., ANN. KY. ACAD. SCI., vol. 660, 1992, pages 306 - 309
MANOHARAN ET AL., BIOORG. MED. CHEM. LET., vol. 3, 1993, pages 2765 - 2770
MANOHARAN ET AL., BIOORG. MED. CHEM. LET., vol. 4, 1994, pages 1053 - 1060
MANOHARAN ET AL., NUCLEOSIDES & NUCLEOTIDES, vol. 14, 1995, pages 969 - 973
MANOHARAN ET AL., TETRAHEDRON LETT., vol. 36, 1995, pages 3651 - 3654
MATTEUCCI: "Oligonucleotides as Therapeutic Agents", 1997, JOHN WILEY AND SONS, article "Oligonucleotide Analogs: an Overview"
MESMAEKER ET AL., SYNLETT, 1997, pages 1287 - 1290
MICHAEL R. MIGDEN ET AL: "PD-1 Blockade with Cemiplimab in Advanced Cutaneous Squamous-Cell Carcinoma", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 379, no. 4, 26 July 2018 (2018-07-26), US, pages 341 - 351, XP055694330, ISSN: 0028-4793, DOI: 10.1056/NEJMoa1805131 *
MICKLEFIELD, CURR. MED. CHEM., vol. 8, 2001, pages 1157 - 1179
MICKLEFIELD, CURRENT MEDICINAL CHEMISTRY, vol. 8, 2001, pages 1157 - 1179
MIKHAILOV ET AL., NUCLEOSIDES & NUCLEOTIDES, vol. 10, no. 1-3, 1991, pages 339 - 343
MILLA M E ET AL: "THOR-707: Using synthetic biology to reprogram the therapeutic activity of interleukin-2 (IL-2).", 1 January 2019 (2019-01-01), XP055927108, Retrieved from the Internet <URL:https://ascopubs.org/doi/abs/10.1200/JCO.2019.37.15_suppl.2603> [retrieved on 20220601] *
MILLER ET AL., JACS, vol. 93, 1971, pages 6657 - 6665
MISHRA ET AL., BIOCHEM. BIOPHYS. ACTA, vol. 1264, 1995, pages 229 - 237
NAPOLITANO ET AL.: "Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli", PNAS, vol. 113, no. 38, 2016, pages E5588 - 5597, XP055409571, DOI: 10.1073/pnas.1605856113
NAWROT ET AL., OLIGONUCLEOTIDES, vol. 16, no. 1, 2006, pages 68 - 82
NELSON ET AL., JOC, vol. 62, 1997, pages 7278 - 7287
NEUMANN ET AL.: "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome", NATURE, vol. 464, no. 7287, 2010, pages 441 - 444
NIELSEN ET AL., SCIENCE, vol. 254, 1991, pages 1497 - 1500
OBERHAUSER ET AL., NUCL. ACIDS RES., vol. 20, 1992, pages 533 - 538
ORAM ET AL., CURR. OPINION MOL. THER., vol. 3, 2001, pages 239 - 243
OSTROV ET AL.: "Design, synthesis, and testing toward a 57-codon genome", SCIENCE, vol. 353, no. 6301, 2016, pages 819 - 822, XP055409587, DOI: 10.1126/science.aaf3639
PISANI ET AL., BLOOD, vol. 78, no. 6, 15 September 1991 (1991-09-15), pages 1538 - 44
SAHA ET AL., J. ORG CHEM., vol. 60, 1995, pages 788 - 789
SAISON-BEHMOARAS ET AL., EM50J, vol. 10, 1991, pages 1111 - 1118
SAKAGUCHI ET AL.: "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25", J. IMMUNOL., vol. 155, no. 3, 1995, pages 1151 - 1164
SHEA ET AL., NUCL. ACIDS RES., vol. 18, 1990, pages 3777 - 3783
SINGH ET AL., CHEM. COMMUN., vol. 4, no. 4, 1998, pages 55 - 456
SRIVASTAVA ET AL., J. AM. CHEM. SOC., vol. 129, no. 26, 2007, pages 8362 - 8379
SVINARCHUK ET AL., BIOCHIMIE, vol. 75, 1993, pages 49 - 54
TEACHEY ET AL., CANCER DISCOV., vol. 6, no. 6, 2016, pages 664 - 79
VRUDHULA ET AL., J. MED. CHEM., vol. 30, 1987, pages 888 - 894
WAHLESTEDT ET AL., PROC. NATL. ACAD. SCI. U. S. A., vol. 97, 2000, pages 5633 - 5638
WANG ET AL., BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 9, 1999, pages 885 - 890
WANG ET AL., NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS, vol. 23, no. 1 & 2, 2004, pages 317 - 337
WU ET AL., BIOCONJUGATE CHEM., vol. 10, 1999, pages 921 - 924
WU ET AL., HELVETICA CHIMICA ACTA, vol. 83, 2000, pages 1127 - 1143
YOUNG ET AL.: "Beyond the canonical 20 amino acids: expanding the genetic lexicon", J. OF BIOLOGICAL CHEMISTRY, vol. 285, no. 15, 2010, pages 11039 - 11044, XP055157080, DOI: 10.1074/jbc.R109.091306
ZHANG ET AL., NATURE, vol. 551, no. 7682, 2017, pages 644 - 647
ZHANG ET AL., PROC NATL ACAD SCI USA, vol. 114, 2017, pages 1317 - 1322
ZON: "Protocols for Oligonucleotides and Analogs, Synthesis and Properties", 1993, HUMANA PRESS, article "Oligonucleoside Phosphorothioates", pages: 165 - 190

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11701407B2 (en) 2017-08-03 2023-07-18 Synthorx, Inc. Cytokine conjugates for the treatment of proliferative and infectious diseases

Also Published As

Publication number Publication date
TW202245843A (en) 2022-12-01

Similar Documents

Publication Publication Date Title
JP2022544280A (en) Immuno-oncology combination therapy using IL-2 conjugates
US20230277627A1 (en) Immuno oncology combination therapy with il-2 conjugates and pembrolizumab
US20230416327A1 (en) Immuno oncology therapies with il-2 conjugates
EP4171648A1 (en) Immuno oncology combination therapy with il-2 conjugates and anti-egfr antibodies
EP4346904A1 (en) Head and neck cancer combination therapy comprising an il-2 conjugate and cetuximab
US20220016252A1 (en) Immuno oncology combination therapy with il-2 conjugates and anti-egfr antibodies
WO2022174101A1 (en) Skin cancer combination therapy with il-2 conjugates and cemiplimab
CA3156405A1 (en) Interleukin 10 conjugates and uses thereof
US20230381335A1 (en) Lung cancer combination therapy with il-2 conjugates and an anti-pd-1 antibody or antigen-binding fragment thereof
WO2023122573A1 (en) Head and neck cancer combination therapy comprising an il-2 conjugate and pembrolizumab
WO2023122750A1 (en) Cancer combination therapy with il-2 conjugates and cetuximab
CN116635061A (en) Immunooncology therapies with IL-2 conjugates
CN116615247A (en) Combination therapy of immunooncology with IL-2 conjugates and pembrolizumab
TW201936632A (en) IL-15 conjugates and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22709451

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22709451

Country of ref document: EP

Kind code of ref document: A1