METHODS OF ADMINISTERING A STING AGONIST

1 . A method of preventing or treating a tumor in a subject in need thereof, comprising administering to the subject a dose of a stimulator of interferon genes protein (STING) agonist, wherein the amount of the STING agonist in the dose is about 0.01 μg to about 100 μg.

2 . The method of claim 1 , wherein the STING agonist is delivered by an extracellular vesicle.

3 . The method of claim 1 or 2 , wherein the STING agonist is associated with an extracellular vesicle.

4 . The method of any one of claims 1 to 3 , wherein the dose comprises from about 0.1 μg to about 1 μg, about 0.1 μg to about 10 μg, about 0.1 μg to about 20 μg, about 0.1 μg to about 30 μg, from about 0.1 μg to about 40 μg, from about 0.1 μg to about 50 μg, from about 0.1 μg to about 60 μg, from about 0.1 μg to about 70 μg, from about 0.1 μg to about 80 μg, 0.1 μg to about 90 μg, from about 0.1 to about 100 μg, from about 1 μg to about 10 μg, from about 1 μg to about 20 μg, from about 1 μg to about 30 μg, from about 1 μg to about 40 μg, from about 1 μg to about 50 μg, from about 1 μg to about 60 μg, from about 1 μg to about 70 μg, from about 1 μg to about 80 μg, from about 1 μg to about 90 μg, from about 1 μg to about 100 μg, from about 10 μg to about 20 μg, from about 10 μg to about 30 μg, from about 10 μg to about 40 μg, from about 10 μg to about 50 μg, from about 10 μg to about 60 μg, from about 10 μg to about 70 μg, from about 10 μg to about 80 μg, from about 90 μg to about 10 μg; from about 20 μg to about 30 μg, from about 20 μg to about 40 μg, from about 20 μg to about 50 μg, from about 20 μg to about 60 μg, from about 20 μg to about 80 μg, from about 20 μg to about 90 μg, from about 20 μg to about 100 μg, from about 30 μg to about 40 μg, from about 30 μg to about 50 μg, from about 30 μg to about 60 μg, from about 30 μg to about 70 μg, from about 30 μg to about 80 μg, from about 30 μg to about 90 μg, from about 30 μg to about 100 μg, from about 40 μg to about 50 μg, from about 40 μg to about 60 μg, from about 40 μg to about 70 μg, from about 40 μg to about 80 μg, from about 40 μg to about 90 μg, from about 40 μg to about 100 μg, from about 50 μg to about 60 μg, from about 50 μg to about 70 μg, from about 50 μg to about 80 μg, from about 50 μg to about 90 μg, from about 50 μg to about 100 μg, from about 60 μg to about 70 μg, from about 60 μg to about 80 μg, from about 60 μg to about 90 μg, from about 60 μg to about 100 μg, from about 70 μg to about 80 μg, from about 70 μg to about 90 μg, from about 70 μg to about 100 μg, from about 80 μg to about 90 μg, from about 80 μg to about 100 μg, or from about 90 μg to about 100 μg of the STING agonist.

5 . The method of any one of claims 1 to 4 , wherein the dose comprises about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0 μg, about 1.1 μg, about 1.2 μg, about 1.3 μg, about 1.4 μg, about 1.5 μg, about 1.6 μg, about 1.7 μg, about 1.8 μg, about 1.9 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about 3.5 μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 5.5 μg, about 6.0 μg, about 6.5 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg, about 8.5 μg, about 9.0 μg, about 9.5 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, or about 20 μg of the STING agonist.

6 . The method of any one of claims 1 to 5 , wherein the dose comprises about 0.3 μg of the STING agonist.

7 . The method of any one of claims 1 to 5 , wherein the dose comprises about 1.0 μg of the STING agonist.

8 . The method of any one of claims 1 to 5 , wherein the dose comprises about 3.0 μg of the STING agonist.

9 . The method of any one of claims 1 to 5 , wherein the dose comprises about 6.0 μg of the STING agonist.

10 . The method of any one of claims 1 to 5 , wherein the dose comprises about 12.0 μg of the STING agonist.

11 . The method of any one of claims 1 to 10 , wherein the tumor is a primary tumor, a secondary tumor, or both a primary tumor and a secondary tumor.

12 . The method of any one of claims 1 to 11 , wherein the tumor is advanced or metastatic.

13 . The method of any one of claims 1 to 12 , wherein the tumor is recurrent, relapsed, or refractory.

14 . The method of any one of claims 1 to 13 , wherein the tumor is selected from the group consisting of a cancer selected from cancers of the lung, ovarian, cervical, endometrial, breast, brain, colon, prostate, gastrointestinal cancer, head and neck cancer, non-small cell lung cancer, cancer of the nervous system, kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic cancer, genital-urinary cancer and bladder cancer, melanoma, leukemia, brain cancer (e.g., glioma, astrocytomas, ependymomas, oligodendrogliomas, and tumors with mixtures of two or more cell types, called mixed gliomas, Acoustic Neuroma (Neurilemmoma, Schwannoma. Neurinoma), Adenoma, Astrocytoma, Low-Grade Astrocytoma, giant cell astrocytomas, Mid- and High-Grade Astrocytoma, Recurrent tumors, Brain Stem Glioma, Chordoma, Choroid Plexus Papilloma, CNS Lymphoma (Primary Malignant Lymphoma), Cysts, Dermoid cysts, Epidermoid cysts, Craniopharyngioma, Ependymoma Anaplastic ependymoma, Gangliocytoma (Ganglioneuroma), Ganglioglioma, Glioblastoma Multiforme (GBM), Malignant Astrocytoma, Glioma, Hemangioblastoma, Inoperable Brain Tumors, Lymphoma, Medulloblastoma (MDL), Meningioma, Metastatic Brain Tumors, Mixed Glioma, Neurofibromatosis, Oligodendroglioma. Optic Nerve Glioma, Pineal Region Tumors, Pituitary Adenoma, PNET (Primitive Neuroectodermal Tumor), Spinal Tumors, Subependymoma, and Tuberous Sclerosis (Bourneville's Disease), squamous cell carcinoma of the head and neck, triple negative breast cancer, anaplastic thyroid carcinoma, and cutaneous squamous cell carcinoma.

15 . The method of any one of claims 1 to 14 , wherein administering the composition prevents metastasis of the tumor, reduces the growth rate of the tumor, reduces the size of the tumor, or combinations thereof.

16 . The method of any one of claims 1 to 15 , wherein the subject has been subjected previously to at least two anti-cancer treatments.

17 . The method of any one of claims 1 to 16 , wherein the administering is parenteral, oral, intravenous, intramuscular, intratumoral, or intraperitoneal.

18 . The method any one of claims 1 to 17 , wherein the administering is intratumoral.

19 . The method of any one of claims 1 to 18 , comprising administering a second dose, a third dose, a fourth dose, or a fifth dose of the STING agonist.

20 . The method of any one of claims 1 to 19 , wherein one or more of the second dose, a third dose, a fourth dose, or a fifth dose is administered at the same dose as the initial dose.

21 . The method of any one of claims 1 to 20 , wherein one or more of the second dose, a third dose, a fourth dose, or a fifth dose is administered at a different dose than the initial dose

22 . The method of any one of claims 2 to 21 , wherein the extracellular vesicle is selected from an exosome, a nanovesicle, an apoptotic body, a microvesicle, a lysosome, an endosome, a liposome, a lipid nanoparticle, a micelle, a multilamellar structure, a revesiculated vesicle, and an extruded cell.

23 . The method of any one of claims 2 to 22 , wherein the extracellular vesicle is an exosome.

24 . The method of any one of claims 2 to 23 , wherein the STING agonist is encapsulated within the extracellular vesicle.

25 . The method of any one of claims 2 to 24 , wherein the STING agonist is linked to a lipid bilayer of the extracellular vesicle.

26 . The method of claim 25 , wherein the STING agonist is linked to the lipid bilayer of the extracellular vesicle by a linker.

27 . The method of claim 26 , wherein the linker is a cleavable linker.

28 . The method of any one of claims 2 to 27 , wherein the extracellular vesicle overexpresses a PTGFRN protein or a portion thereof.

29 . The method of claim 28 , wherein the STING agonist is linked to the PTGFRN protein or the portion thereof.

30 . The method of claim 27 or 28 , wherein the STING agonist is linked to the PTGFRN protein or the portion thereof by a linker.

31 . The method of any one of claims 2 to 30 , wherein the extracellular vesicle is produced by a cell that overexpresses a PTGFRN protein.

32 . The method of any one of claims 1 to 31 , wherein the STING agonist is a cyclic dinucleotide.

33 . The method of any one of claims 1 to 32 , wherein the STING agonist is a non-cyclic dinucleotide.

34 . The method of any one of claims 1 to 33 , wherein the STING agonist comprises a lipid-binding tag.

35 . The method of any one of claims 1 to 34 , wherein the STING agonist is physically and/or chemically modified.

36 . The method, EV or composition of claim 35 , wherein the modified STING agonist has a polarity and/or a charge different from the corresponding unmodified STING agonist.

37 . The method of any one of claims 1 to 36 , wherein the STING agonist comprises:

wherein:

X 1 is H, OH, or F;

X 2 is H, OH, or F;

Z is OH, OR 1 , SH or SR 1 , wherein:

i) R 1 is Na or NH 4 , or

ii) R 1 is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl;

B1 and B2 are bases chosen from:

With the proviso that:

in Formula (I): X 1 and X 2 are not OH,

in Formula (II): when X 1 and X 2 are OH, B1 is not Adenine and B2 is not Guanine, and

in Formula (III): when X 1 and X 2 are OH, B1 is not Adenine, B2 is not Guanine and Z is not OH, or a pharmaceutically acceptable salt thereof.

38 . The method of any one of claims 1 to 36 , wherein the STING agonist is selected from the group consisting of:

and a pharmaceutically acceptable salt thereof.

39 . The method of any one of claims 1 to 38 , wherein the STING agonist is

or a pharmaceutically acceptable salt thereof.

40 . The method of any one of claims 1 to 39 further comprising administering a second anticancer agent to the subject.

41 . The method of claim 40 , wherein the second anticancer agent comprises a chemotherapy, an antibody or an antigen-binding portion thereof, an additional extracellular vesicle, or any combination thereof.

42 . The method of any one of claims 29 to 41 , wherein the PTGFRN protein comprises SEQ ID NO: 33.

43 . The method of any one of claims 29 to 42 , wherein the PTGFRN protein comprises at least about 70%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 1.

44 . The method any one of claims 29 to 43 , wherein the PTGFRN protein comprises the amino acid sequence as set forth in SEQ ID NO: 1.

45 . A method of treating a tumor in a subject comprising:

a. a first administering of a first composition of extracellular vesicles comprising a first STING agonist to the patient, the first composition comprising about 0.3 micrograms of the first STING agonist;

b. a second administering of a second composition of extracellular vesicles comprising a second STING agonist to the patient, the second composition comprising about 1.0 microgram of the second STING agonist; and

c. a third administering of a third composition of extracellular vesicles comprising a third STING agonist to the patient, the third composition comprising about 2.0 micrograms of the third STING agonist;

wherein the first, second, and third STING agonists are the same;

wherein the compositions are administered intratumorally; and

wherein said tumor is selected from the group consisting of squamous cell carcinoma of the head and neck, triple negative breast cancer, anaplastic thyroid carcinoma, and cutaneous squamous cell carcinoma

wherein the patient has been subjected previously to at least two anti-cancer treatments.