METHODS OF TREATING A TUMOR

1 . A method of preventing or treating a tumor in a subject in need thereof comprising administering to the subject a dose of an IL-12 polypeptide, wherein the amount of the IL-12 polypeptide in the dose is about 0.1 μg to about 30 μg.

2 . The method of claim 1 , wherein the IL-12 polypeptide is delivered by an extracellular vesicle.

3 . The method of claim 1 or 2 , wherein the IL-12 polypeptide is associated with an extracellular vesicle.

4 . The method of any one of claims 1 to 3 , wherein the dose comprises at least about 0.1 μg, at least about 0.2 μg, at least about 0.3 μg, at least about 0.4 μg, at least about 0.5 μg, at least about 0.6 μg, at least about 0.7 μg, at least about 0.8 μg, at least about 0.9 μg, at least about 1.0 μg, at least about 1.1 μg, at least about 1.2 μg, at least about 1.3 μg, at least about 1.4 μg, at least about 1.5 μg, at least about 1.6 μg, at least about 1.7 μg, at least about 1.8 μg, at least about 1.9 μg, at least about 2.0 μg, at least about 2.1 μg, at least about 2.2 μg, at least about 2.3 μg, at least about 2.4 μg, at least about 2.5 μg, at least about 2.6 μg, at least about 2.7 μg, at least about 2.8 μg, at least about 2.9 μg, at least about 3.0 μg, at least about 3.1 μg, at least about 3.2 μg, at least about 3.3 μg, at least about 3.4 μg, at least about 3.5 μg, at least about 3.6 μg, at least about 3.7 μg, at least about 3.8 μg, at least about 3.9 μg, at least about 4.0 μg, at least about 4.1 μg, at least about 4.2 μg, at least about 4.3 μg, at least about 4.4 μg, at least about 4.5 μg, at least about 4.6 μg, at least about 4.7 μg, at least about 4.8 μg, at least about 4.9 μg, at least about 5.0 μg, at least about 5.1 μg, at least about 5.2 μg, at least about 5.3 μg, at least about 5.4 μg, at least about 5.5 μg, at least about 5.6 μg, at least about 5.7 μg, at least about 5.8 μg, at least about 5.9 μg, at least about 6.0 μg, at least about 6.5 μg, at least about 7.0 μg, at least about 7.5 μg, at least about 8.0 μg, at least about 8.5 μg, at least about 9.0 μg, at least about 9.5 μg, at least about 10.0 μg, at least about 10.5 μg, at least about 11.0 μg, at least about 11.5 μg, at least about 12.0 μg, at least about 12.5 μg, at least about 13.0 μg, at least about 14 μg, at least about 15 μg, at least about 16 μg, at least about 17 μg, at least about 18 μg, at least about 19 μg, at least about 20 μg, at least about 25 μg, or at least about 30 μg of the IL-12 polypeptide.

5 . The method of any one of claims 1 to 3 , wherein the dose comprises at least about 0.3 μg of the IL-12 polypeptide.

6 . The method of any one of claims 1 to 3 , wherein the dose comprises at least about 1.0 μg of the IL-12 polypeptide.

7 . The method of any one of claims 1 to 3 , wherein the dose comprises at least about 3.0 μg of the IL-12 polypeptide.

8 . The method of any one of claims 1 to 3 , wherein the dose comprises at least about 6.0 μg of the IL-12 polypeptide.

9 . The method of any one of claims 1 to 3 , wherein the dose comprises at least about 12.0 μg of the IL-12 polypeptide.

10 . The method of any one of claims 1 to 9 , wherein the dose of the IL-12 polypeptide is administered at least two times, at least three times, at least four times, at least five times, or at least six times.

11 . The method of any one of claims 1 to 10 , wherein the dose of the IL-12 polypeptide is administered once about every week, once about every two weeks, once about every three weeks, once about every four weeks, once about every six weeks, or once about every eight weeks.

12 . The method of any one of claims 1 to 11 , wherein the IL-12 polypeptide comprises a single chain polypeptide having at least about 70%, at least about 75%, at least about 80%, 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% sequence identity to SEQ ID NO: 13.

13 . The method of any one of claims 1 to 12 , wherein the extracellular vesicle comprises a scaffold moiety.

14 . The method of claim 13 , wherein the scaffold moiety comprises a Prostaglandin F2 receptor negative regulator (PTGFRN) protein or a portion thereof.

15 . The method of claim 14 , wherein the PTGFRN protein comprises SEQ ID NO: 33.

16 . The method of claim 14 , 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.

17 . The method of claim 16 , wherein the PTGFRN protein comprises the amino acid sequence as set forth in SEQ ID NO: 1.

18 . The method of any one of claims 13 to 17 , wherein the IL-12 polypeptide is linked to the scaffold moiety.

19 . The method of claim 18 , wherein the IL-12 polypeptide is linked to the scaffold moiety by a peptide bond.

20 . The method of any one of claims 1 to 19 , wherein the IL-12 is associated with the exterior surface of the extracellular vesicle.

21 . The method of any one of claims 13 to 20 , wherein the IL-12 polypeptide is linked to the N-terminus of the scaffold moiety.

22 . The method of any one of claims 13 to 21 , wherein the IL-12 polypeptide is linked to the N-terminus of the PTGFRN protein of the portion thereof.

23 . The method of any one of claims 1 to 22 , wherein the IL-12 is within the lumen of the extracellular vesicle.

24 . The method of any one of claims 13 to 24 , wherein the IL-12 polypeptide is linked to the C-terminus of the scaffold moiety.

25 . The method of any one of claims 13 to 24 , wherein the IL-12 polypeptide is linked to the C-terminus of the PTGFRN protein of the portion thereof.

26 . The method of any one of claims 1 to 25 , wherein the amount of IL-12 polypeptide is measured by fluorescence assay, IL-12 AlphaLISA, or a combination thereof.

27 . The method of any one of claims 1 to 26 , wherein the tumor is a primary tumor, a secondary tumor, or both a primary tumor and a secondary tumor.

28 . The method of any one of claims 1 to 27 , wherein the administering reduces the volume of the tumor.

29 . The method of any one of claims 1 to 28 , wherein the administering reduces the volume of the tumor by at least two fold, at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least nine fold, or at least ten fold compared to the tumor volume after administering the IL-12 polypeptide in the absence of an extracellular vesicle.

30 . The method of claim 28 or 29 , wherein the administering reduces the volume of the primary tumor.

31 . The method of claim 30 , wherein the administering is capable of reducing the volume of the primary tumor by at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, or at least about 5 fold compared to the monotherapy after day 14 of the administering.

32 . The method of any one of claims 1 to 31 , wherein the administering reduces the growth of the tumor.

33 . The method of any one of claims 1 to 32 , wherein the administering reduces the growth of the tumor by at least two fold, at least three fold, at least four fold, at least five fold, at least six fold, at least seven fold, at least nine fold, or at least ten fold compared to the tumor volume after administering either an extracellular vesicle comprising the STING agonist or the IL-12 polypeptide (“monotherapy”).

34 . The method of any one of claims 1 to 33 , further comprising administering an additional anti-cancer agent.

35 . The method of claim 34 , wherein the additional anti-cancer agent comprises a checkpoint inhibitor.

36 . The method of claim 35 , wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, or any combination thereof.

37 . The method of claim 36 , wherein the checkpoint inhibitor is an anti-PD-1 antibody.

38 . The method of any one of claims 2 to 37 , wherein the extracellular vesicle is 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, or an extruded cell.

39 . The method of any one of claims 2 to 38 , wherein the EV is an exosome.

40 . The method of any one of claims 2 to 39 , wherein the extracellular vesicle is produced by a cell that overexpresses a PTGFRN protein.

41 . The method of any one of claims 2 to 40 , wherein the extracellular vesicle further comprises a ligand, a cytokine, or an antibody.

42 . The method of claim 41 , wherein the antibody comprises an antagonistic antibody and/or an agonistic antibody.