Methods of treating tumors

Abstract

Provided herein are methods of preventing or treating a tumor in a subject in need thereof, the method comprising administering to the subject a dose of an IL-12 polypeptide wherein the amount of the IL-12 polypeptide in the dose is from about 0.1 [mu] g to about 30 [mu] g.

Description

Background Art

[0001] Interleukin-12 (IL-12) is a promising anticancer agent that has been hampered by unwanted systemic exposure and unpredictable pharmacology. Surface display of functional IL-12 on engineered extracellular vesicles (EVs) provides a promising approach for more targeted delivery of IL-12. However, there remains a need in the art for improved methods of delivering IL-12. Summary of the invention

[0002] Some aspects of the present disclosure relate to a method of preventing or treating a tumor in a subject in need thereof, the method 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.

[0003] In some aspects, the IL-12 polypeptide is delivered via an extracellular vesicle. In some aspects, the IL-12 polypeptide is associated with an extracellular vesicle.

[0004] In some aspects, 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 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. In some aspects, the dosage comprises at least about 0.3 μg of the IL-12 polypeptide. In some aspects, the dosage comprises at least about 1.0 μg of the IL-12 polypeptide. In some aspects, the dosage comprises at least about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 12.0 μg of the IL-12 polypeptide.

[0005] In some aspects, the dose of the IL-12 polypeptide is administered at least twice, at least three times, at least four times, at least five times, or at least six times. In some aspects, the dose of the IL-12 polypeptide is administered about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every six weeks, or about once every eight weeks.

[0006] In some aspects, 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.

[0007] In some aspects, the extracellular vesicle comprises a scaffold portion. In some aspects, the scaffold portion comprises a prostaglandin F2 receptor negative regulator (PTGFRN) protein or a portion thereof. In some aspects, the PTGFRN protein comprises SEQ ID NO:33. In some aspects, 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. In some aspects, the PTGFRN protein comprises an amino acid sequence as shown in SEQ ID NO:1. In some aspects, the IL-12 polypeptide is connected to the scaffold portion. In some aspects, the IL-12 polypeptide is connected to the scaffold portion by a peptide bond.

[0008] In some aspects, the IL-12 is associated with the outer surface of the extracellular vesicle. In some aspects, the IL-12 polypeptide is connected to the N-terminus of the scaffold portion. In some aspects, the IL-12 polypeptide is connected to the N-terminus of the PTGFRN protein or the portion thereof. In some aspects, the IL-12 is located in the lumen of the extracellular vesicle. In some aspects, the IL-12 polypeptide is connected to the C-terminus of the scaffold portion. In some aspects, the IL-12 polypeptide is connected to the C-terminus of the PTGFRN protein or the portion thereof.

[0009] In some aspects, the amount of the IL-12 polypeptide is measured by fluorimetry, IL-12α LISA, or a combination thereof.

[0010] In some aspects, the tumor is a primary tumor, a secondary tumor, or both a primary tumor and a secondary tumor. In some aspects, the administration reduces the volume of the tumor. In some aspects, the administration reduces the volume of the tumor by at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least nine times, or at least ten times, compared to the volume of the tumor after the administration of the IL-12 polypeptide in the absence of extracellular vesicles. In some aspects, the administration reduces the volume of the primary tumor. In some aspects, the administration can reduce the volume of the primary tumor by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, or at least about 5 times, compared to the monotherapy after the administration of the 14th day. In some aspects, the administration reduces the growth of the tumor.

[0011] In some aspects, the administration 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 administration of extracellular vesicles comprising a STING agonist or the IL-12 polypeptide ("monotherapy").

[0012] In some aspects, the method further comprises administering an additional anticancer agent. In some aspects, the additional anticancer agent comprises a checkpoint inhibitor. In some aspects, 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. In some aspects, the checkpoint inhibitor is an anti-PD-1 antibody.

[0013] In some aspects, 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 re-encapsulated vesicle, or an extruded cell. In some aspects, the EV is an exosome. In some aspects, the extracellular vesicle is produced by cells that overexpress PTGFRN protein.

[0014] In some aspects, the extracellular vesicle further comprises a ligand, a cytokine or an antibody. In some aspects, the antibody comprises an antagonist antibody and / or an agonist antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a diagram of engineered exosomes with surface displayed IL-12 ("exoIL-12").

[0016] Figure 2A -2D is a graph showing tumor retention in untreated mice and mice treated with free recombinant IL-12 or exoIL-12 ( Figure 2A )、IFN-γAUC( Figure 2B ), tumor growth ( FIG. 2C ), and scatter plots of tumor growth after rechallenge, as indicated. FIG. 2E is a box plot showing the percentage of antigen-specific CD8+ T cells in control and exoIL-12 treated mice.

[0017] Figures 3A-3C is the pharmacokinetics of exoIL-12 as measured in the skin and plasma following administration of 0.3 μg, 1.0 μg or 3.0 μg of IL-12 polypeptide ( Figure 3A ) and as through the skin ( Figure 3B ) and plasma ( Figure 3C ) is a graphical representation of tissue pharmacodynamics measured by fold change of IP-10 relative to vehicle over time.

[0018] Figures 4A-4B In healthy volunteers ( Figure 4A ) and cancer patients ( Figure 4B ) is a schematic diagram of the clinical study evaluating the safety and efficacy of exoIL-12 treatment. DETAILED DESCRIPTION

[0019] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific aspects described, as such aspects may of course vary. It should also be understood that the terms used herein are only for the purpose of describing specific aspects, and are not intended to be restrictive, as the scope of the present invention will only be limited by the appended claims.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0021] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and is incorporated herein by reference to disclose and describe the methods and / or materials in connection with the cited publication.

[0022] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several aspects without departing from the scope or spirit of the invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0023] I. Definitions

[0024] It should be noted that, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein. It should be further noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for using such exclusive terms as "solely," "only," or the use of a "negative" limitation in conjunction with the recitation of claim elements.

[0025] Furthermore, the use of "and / or" herein should be considered as explicitly disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0026] It should be understood that wherever herein aspects are described with the language "comprising," otherwise similar aspects described according to "consisting of" and / or "consisting essentially of" are also provided.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the relevant field of the present disclosure. For example, the following provides a common dictionary of many terms used in the present disclosure for technical personnel: "Concise Dictionary of Biomedicine and Molecular Biology", Juo, Pei-Show, 2nd edition, 2002, CRC Press (CRC Press); "The Dictionary of Cell and Molecular Biology", 3rd edition, 1999, Academic Press (Academic Press); and "Oxford Dictionary of Biochemistry and Molecular Biology", revised edition, 2000, Oxford University Press (Oxford University Press).

[0028] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers defining ranges. In the case of a range of narrated values, it is to be understood that each intermediate integer value and each fraction thereof between the narrated upper and lower limits of the range are also specifically disclosed, as well as each subrange between such values. The upper and lower limits of any range may be independently included in the range or excluded from the range, and each range including any limit, not including two limits or including two limits is also encompassed in the present disclosure. Therefore, the range narrated herein should be understood as a shorthand for all values ​​within the range, including the endpoints narrated. For example, a range of 1 to 10 should be understood as including any number, combination of numbers or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0029] Where a value is explicitly stated, it is understood that values ​​of a quantity or amount approximately the same as the stated value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed separately, combinations thereof are also disclosed. Where any element disclosed is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded individually or in any combination with other alternatives are also hereby disclosed; more than one element disclosed may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0030] Nucleotide is represented by its generally accepted single letter code.Unless otherwise indicated, nucleotide sequence is written from left to right with 5' to 3' direction.Nucleotide is represented by the commonly known single letter symbol recommended by IUPAC-IUB Biochemical Nomenclature Commission in this article.Therefore, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0031] Amino acid sequences are written left to right in amino to carboxyl orientation.Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0032] The terms "about" or "approximately" are used herein to mean approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the numerical values ​​set forth. The term table used herein means within 5% of the reference amount, for example, about 50% is understood to cover a range of values ​​from 47.5% to 52.5%.

[0033] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane that wraps an internal space. Extracellular vesicles include all membrane-bound vesicles (e.g., exosomes, nanovesicles) whose diameter is less than the diameter of the cell from which it is derived. Typically, the diameter of an extracellular vesicle is in the range of 20nm to 1000nm, and may include being in the internal space (i.e., lumen), displayed on the outer surface of the extracellular vesicle, and / or spanning various macromolecular payloads of the membrane. The payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, an extracellular vesicle includes a scaffold portion. For example, and not limitation, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles, vesicle organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosomes and plasma membrane fusion). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some aspects, extracellular vesicles are produced by cells expressing one or more transgenic products.

[0034] As used herein, the term "exosome" refers to a cell-derived small (diameter between 20-300nm, for example, diameter between 40-200nm) vesicle, the cell-derived vesicle comprising a membrane that encapsulates the internal space (ie, lumen), and produced from the cell by direct plasma membrane budding or by late endosome fusion with the plasma membrane. In some aspects, EV (eg, exosome) is about 20nm to about 300nm. Exosomes are extracellular vesicles of a species. Exosomes contain lipids or fatty acids and polypeptides, and optionally contain a payload (eg, a therapeutic agent), an acceptor (eg, a targeting moiety), a polynucleotide (eg, nucleic acid, RNA or DNA), a sugar (eg, a monosaccharide, a polysaccharide or a polysaccharide) or other molecules. In some aspects, exosomes include a scaffold portion. Exosomes can be derived from producer cells and can be separated from producer cells based on their size, density, biochemical parameters or a combination thereof. In some aspects, the exosomes disclosed herein are produced by cells expressing one or more transgenic products.

[0035] As used herein, the term "nanovesicle" refers to a cell-derived small (diameter between 20-250nm, more preferably between 30-150nm) vesicle, the cell-derived vesicle comprising a membrane that wraps the inner space, and produced from the cell by direct or indirect manipulation, so that the producer cell does not produce the nanovesicle in the absence of the manipulation. Suitable manipulation of producer cells includes but is not limited to continuous extrusion, treatment with alkaline solution, ultrasonic treatment or its combination. In some cases, the generation of nanovesicles can cause the destruction of producer cells. Preferably, the nanovesicle colony is substantially free of vesicles derived from producer cells by direct budding from the plasma membrane or late endosomes and plasma membrane fusion. The nanovesicle comprises lipids or fatty acids and polypeptides, and optionally comprises a payload (e.g., a therapeutic agent), an acceptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA or DNA), a sugar (e.g., a monosaccharide, a polysaccharide or a polysaccharide) or other molecules. In some aspects, the nanovesicle comprises a scaffold portion. When the nanovesicles are derived from producer cells according to the manipulations described, they can be isolated from the producer cells based on their size, density, biochemical parameters, or a combination thereof.

[0036] The term "modified" when used in the context of exosomes described herein refers to the alteration or engineering of EVs such that the modified EVs differ from naturally occurring EVs. In some aspects, the modified EVs described herein comprise a membrane that differs from the membrane of a naturally occurring EV in terms of the composition of proteins, lipids, small molecules, carbohydrates, etc. (e.g., the membrane comprises a higher density or amount of native EV proteins and / or the membrane comprises proteins that do not naturally occur in EVs. In certain aspects, such modifications to the membrane alter the outer surface of the EV. In certain aspects, such modifications to the membrane alter the lumen of the EV.

[0037] As used herein, the term "scaffold moiety" refers to a molecule that can be used to anchor an IL-12 moiety and / or any other compound of interest (e.g., a payload) to an EV, the luminal surface of an EV, or the outer surface of an EV. In certain aspects, the scaffold moiety comprises a synthetic molecule. In certain aspects, the scaffold moiety comprises a non-polypeptide moiety. In other aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that is naturally present in an EV. In certain aspects, the scaffold moiety comprises a lipid, carbohydrate, or protein that is not naturally present in an exosome. In certain aspects, the scaffold moiety is Scaffold X. In certain aspects, the scaffold moiety is Scaffold Y. In other aspects, the scaffold moiety comprises both Scaffold X and Scaffold Y. In certain aspects, the scaffold portion comprises Lamp-1, Lamp-2, CD13, CD86, Flotillin, Syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, Integrin α4, L1CAM, LFA-1, Mac-1 α and β, Vti-1A and B, CD3ε and ζ, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2 / 3, HLA-DM (MHC II), immunoglobulin, MHC-I or MHC-II component, TCRβ, a tetraspanin, or a combination thereof.

[0038] As used herein, the term "Scaffold X" refers to an exosomal protein recently identified on the surface of exosomes. See, e.g., U.S. Pat. No. 10,195,290, which is incorporated herein by reference in its entirety. Non-limiting examples of Scaffold X proteins include: prostaglandin F2 receptor negative regulator ("PTGFRN protein"); basic glycoprotein ("BSG protein"); immunoglobulin superfamily member 2 ("IGSF2 protein"); immunoglobulin superfamily member 3 ("IGSF3 protein"); immunoglobulin superfamily member 8 ("IGSF8 protein"); integrin beta-1 ("ITGB1 protein"); integrin alpha-4 ("ITGA4 protein"); 4F2 cell surface antigen heavy chain ("SLC3A2 protein"); and a class of ATP transporters ("ATP1A1 protein", "ATP1A2 protein", "ATP1A3 protein", "ATP1A4 protein", "ATP1B3 protein", "ATP2B1 protein", "ATP2B2 protein", "ATP2B3 protein", "ATP2B protein"). In some aspects, the Scaffold X protein can be a whole protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of anchoring another portion to the outer surface or lumen surface of an EV (e.g., an exosome)). In some aspects, Scaffold X can anchor a moiety (e.g., an IL-12 moiety) to the exterior surface or the luminal surface of an EV (e.g., an exosome).

[0039] As used herein, the term "scaffold Y" refers to an exosomal protein recently identified within the lumen surface of an exosome. See, for example, International Publication No. WO / 2019 / 099942, which is incorporated herein by reference in its entirety. Non-limiting examples of scaffold Y proteins include: protein kinase C substrate rich in myristoylated alanine ("MARCKS protein"); protein kinase C substrate-like 1 rich in myristoylated alanine ("MARCKSL1 protein"); and brain acid soluble protein 1 ("BASP1 protein"). In some aspects, the scaffold Y protein can be a whole protein or a fragment thereof (e.g., a functional fragment, e.g., a minimal fragment capable of anchoring a portion on the lumen surface of an EV (e.g., an exosome)). In some aspects, scaffold Y can anchor a portion (e.g., an IL-12 portion) to the lumen of an EV (e.g., an exosome).

[0040] As used herein, the term "fragment" of a protein (e.g., a therapeutic protein, scaffold X, or scaffold Y) refers to an amino acid sequence of a protein that is shorter than the naturally occurring sequence of the protein, the portion of the N-terminus and / or the C-terminus that is missing, or any portion thereof, as compared to the naturally occurring protein. As used herein, the term "functional fragment" refers to a protein fragment that retains the function of the protein. Thus, in some aspects, a functional fragment of a scaffold X protein retains the ability to anchor a moiety to the luminal surface and / or the outer surface of an EV. Similarly, in some aspects, a functional fragment of a scaffold Y protein retains the ability to anchor a moiety to the luminal surface of an EV. Whether a fragment is a functional fragment can be assessed by any known method for determining the protein content of an EV, including Western blot, FACS analysis, and fusion of a fragment with an autofluorescent protein, such as GFP. In some aspects, a functional fragment of a scaffold X protein retains the ability of a naturally occurring scaffold X protein, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability to anchor a moiety. In some aspects, a functional fragment of a Scaffold Y protein retains the ability of a naturally occurring Scaffold Y protein, e.g., at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the ability to anchor another molecule.

[0041] As used herein, the term "variant" of a molecule (e.g., a functional molecule, an antigen, a scaffold X and / or a scaffold Y) refers to a molecule that shares certain structural and functional identities with another molecule after comparison by methods known in the art. For example, a variant of a protein may include a substitution, insertion, deletion, frameshift or rearrangement in another protein.

[0042] In some aspects, variants of Scaffold X comprise variants having at least about 70% identity to full-length, mature PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2 or ATP transporter or fragments (e.g., functional fragments) of PTGFRN, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, SLC3A2 or ATP transporter. In some aspects, variants of PTGFRN or variants of fragments share at least about 70%, 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%, or at least about 99% sequence identity with PTGFRN according to SEQ ID NO: 1 or with a functional fragment thereof.

[0043] In some aspects, variants of Scaffold Y comprise variants having at least 70% identity with MARCKS, MARCKSL1, BASP1 or fragments of MARCKS, MARCKSL1 or BASP1. In some aspects, variants of MARCKS or variants of fragments share at least about 70%, 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% or at least about 99% sequence identity with MARCKS according to SEQ ID NO: 401 or a functional fragment thereof. In some aspects, variants of MARCKSL1 or variants of fragments share at least about 70%, 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% or at least about 99% sequence identity with MARCKSL1 according to SEQ ID NO: 402 or a functional fragment thereof. In some aspects, the variants of BASP1 or variants of fragments share at least about 70%, 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%, or at least about 99% sequence identity with BASP1 according to SEQ ID NO: 403 or a functional fragment thereof. In some aspects, the variants of Scaffold Y protein or variants of fragments retain the ability to specifically target to the lumen of EVs. In some aspects, Scaffold Y comprises one or more mutations, e.g., conservative amino acid substitutions.

[0044] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered to be conservative. On the other hand, a string of amino acids can be conservatively replaced by a structurally similar string that differs in the order and / or composition of the side chain family members.

[0045] The term "percentage of sequence identity" or "percentage of identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are not nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted because the target sequence nucleotides or amino acids are counted, not the nucleotides or amino acids from the reference sequence.

[0046] The percentage of sequence identity is calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in the two sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The comparison of the sequence and the determination of the percentage of sequence identity between the two sequences can be completed using readily available software that can be used and downloaded online. Suitable software programs can be obtained from various sources, and can be used for the comparison of protein sequences and nucleotide sequences. A suitable program for determining the percentage of sequence identity is bl2seq, which is a part of the BLAST program suite that can be obtained from the National Center for Biotechnology Information (National Center for Biotechnology Information) BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs are for example Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0047] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It should be noted that percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that length values ​​will always be integers.

[0048] Those skilled in the art will appreciate that the generation of sequence alignments for calculating sequence identity percentages is not limited to binary sequence-sequence comparisons driven completely by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. A suitable program for generating multiple sequence alignments is ClustalW2, which can be obtained from www.clustal.org. Another suitable program is MUSCLE, which can be obtained from www.drive5.com / muscle / . ClustalW2 and MUSCLE can alternatively be obtained from, for example, EBI.

[0049] It should also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystal protein structures), functional data (e.g., the location of mutations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, which is available at www.tcoffee.org and alternatively available from, for example, EBI. It should also be understood that the final alignment for calculating percent sequence identity can be automatically or manually curated.

[0050] Polynucleotide variants can contain changes in coding region, noncoding region or both.On the one hand, polynucleotide variants contain silent substitutions, additions or deletions that do not change the characteristics or active changes of the encoded polypeptide.On the other hand, due to the degeneracy of the genetic code, nucleotide variants are produced by silent substitutions.In other aspects, wherein 5-10, 1-5 or 1-2 amino acids are substituted, lacked or added variants in any combination.Polynucleotide variants can be produced for a variety of reasons, for example, for optimizing the codon expression of a specific host (codons in human mRNA are changed to other codons, for example bacterial hosts, such as Escherichia coli (E.coli)).

[0051] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on a chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants may vary at the polynucleotide and / or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or direct synthesis.

[0052] Using known methods of protein engineering and recombinant DNA technology, variants can be generated to improve or change the properties of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without significant loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993), which is incorporated herein by reference in its entirety, reported variant KGF proteins that had heparin binding activity even after the deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma exhibits up to ten times higher activity after the deletion of 8-10 amino acid residues from the carboxyl terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988), which is incorporated herein by reference in its entirety.)

[0053] In addition, there is sufficient evidence to show that variants generally retain biological activities similar to those of naturally occurring proteins. For example, Gayle and colleagues (Journal of Biological Chemistry 268:22105-22111 (1993), the document is incorporated herein by reference in its entirety) have been subjected to extensive mutation analysis of human cytokine IL-1a. It uses random mutagenesis to produce more than 3,500 individual IL-1a mutants with an average of 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations are examined at each possible amino acid position. The researchers found that "[most of] the molecule can be changed, with little effect on [combination or biological activity]. "(See Abstract.) In fact, in the more than 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins significantly different in activity from the wild type.

[0054] As described above, polypeptide variants include, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. In some aspects, Scaffold X and / or Scaffold Y are modified at any convenient location.

[0055] As used herein, the term "producer cell" refers to a cell used to produce EVs. Producer cells can be cells cultured in vitro or cells cultured in vivo. Producer cells include, but are not limited to, cells known to be effective in producing EVs (e.g., exosomes), for example, HEK293 cells, Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, s9f cells, fHDF fibroblasts, Neuronal precursor cells, In some aspects, the producer cell is an antigen presenting cell. In some aspects, the producer cell is a bacterial cell. In some aspects, the producer cell is a dendritic cell, a B cell, a mast cell, a macrophage, a neutrophil, a Kupffer-Browicz cell or a cell derived from any of these cells or any combination thereof. In some aspects, the producer cell is not a bacterial cell. In other aspects, the producer cell is not an antigen presenting cell.

[0056] As used herein, the term "associated with" refers to encapsulating a first portion (e.g., an IL-12 portion) into a second portion (e.g., an extracellular vesicle), or refers to a covalent bond or non-covalent bond formed between a first portion (e.g., an IL-12 portion) and a second portion (e.g., an extracellular vesicle). For example, in some aspects, a scaffold portion, e.g., a scaffold X (e.g., a PTGFRN protein) is expressed in or on an extracellular vesicle, and the IL-12 portion is loaded on the outer surface of the extracellular vesicle. On the one hand, the term "associated with" means a covalent bond, a non-peptide bond, or a non-covalent bond. For example, the amino acid cysteine ​​comprises a thiol group that can form a disulfide bond or a bridge with a sulfhydryl group on a second cysteine ​​residue. Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, σ bonds, π bonds, δ bonds, glycosidic bonds, π hydrogen bonds, bent bonds, dipole bonds, π reverse bonds, double bonds, triple bonds, quadruple bonds, pentapole bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, Haptor number, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Walls forces, London dispersion forces, mechanical bonds, halogen bonds, gold affinity, intercalation, stacking, entropy forces, or chemical polarity. In other aspects, the term "associated with..." means that the first part (e.g., an extracellular vesicle) encapsulates the second part (e.g., an IL-12 part). In some aspects, the first part and the second part may be connected to each other. In other aspects, the first part and the second part are not physically and / or chemically connected to each other.

[0057] As used herein, the terms "linked to" or "conjugated to" are used interchangeably and refer to a covalent bond or non-covalent bond formed between a first portion and a second portion (e.g., an IL-12 portion and an extracellular vesicle, respectively). In some aspects, the scaffold portion is expressed in or on an extracellular vesicle, e.g., Scaffold X (e.g., PTGFRN protein), and the IL-12 portion is linked or conjugated to a portion of the Scaffold X protein (e.g., PTGFRN protein) that is exposed on the surface of the extracellular vesicle (e.g., "surface display of IL-12"). In some aspects, the scaffold portion is expressed in or on an extracellular vesicle, e.g., Scaffold X (e.g., PTGFRN protein), and the IL-12 portion is linked or conjugated to a portion of the Scaffold X protein (e.g., PTGFRN protein) that is exposed to the lumen of the extracellular vesicle.

[0058] As used herein, the term "loading" or grammatically different forms of the term (e.g., load or loaded) refers to the state or process of associating a first portion (e.g., IL-12 portion) with a second portion (e.g., EV, e.g., and exosome). In some aspects, the first portion is chemically or physically linked to the second portion. In some aspects, the first portion is not chemically or physically linked to the second portion. In some aspects, the first portion is present within the second portion, e.g., within the lumen of the EV (e.g., exosome), e.g., "encapsulated". In some aspects, the first portion is associated with the outer surface of the second portion, e.g., linked or conjugated to the surface of the EV (e.g., exosome), e.g., "surface display" of the second portion.

[0059] The term "encapsulated" or grammatically different forms of the term (e.g., encapsulation or encapsulating) refers to the state or process of having a first portion (e.g., an IL-12 portion) located inside a second portion (e.g., an EV, e.g., an exosome) without chemically or physically connecting the two portions. In some aspects, the term "encapsulated" can be used interchangeably with "in the lumen of." Non-limiting examples of encapsulating a first portion (e.g., an IL-12 portion) into a second portion (e.g., an EV, e.g., an exosome) are disclosed elsewhere herein.

[0060] As used herein, the terms "isolate," "isolated," and "isolating," or "purify," "purified," and "purifying," and "extracted," and "extracting," are used interchangeably and refer to the state (e.g., multiple known or unknown amounts and / or concentrations) of a desired preparation of EVs that have been subjected to one or more purification processes, e.g., selection or enrichment of a desired EV preparation. In some aspects, as used herein, isolation or purification is the process of removing, partially removing (e.g., fractionating) EVs from a sample containing producer cells. In some aspects, the isolated EV composition has no detectable undesirable activity, or alternatively, the level or amount of the undesirable activity is at or below an acceptable level or amount. In other aspects, the isolated EV composition has an amount and / or concentration of the desired EVs that is at or above an acceptable amount and / or concentration. In other aspects, the isolated EV composition is enriched compared to the starting material (e.g., producer cell preparation) from which the composition is obtained. Compared to the starting material, this enrichment can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999% or greater than 99.9999%. In some aspects, the isolated EV preparation is substantially free of residual biological products. In some aspects, the isolated EV preparation is 100% free, 99% free, 98% free, 97% free, 96% free, 95% free, 94% free, 93% free, 92% free, 91% free or 90% free of any contaminating biological substances. Residual biological products may include non-biological materials (including chemicals) or undesirable nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products may also mean that the EV composition contains no detectable producer cells, and only EVs are detectable.

[0061] As used herein, the term "agonist" refers to a molecule that binds to a receptor and activates the receptor to produce a biological response. Receptors can be activated by endogenous or exogenous agonists. Non-limiting examples of endogenous agonists include hormones, neurotransmitters, and cyclic dinucleotides. Non-limiting examples of exogenous agonists include drugs, small molecules, and cyclic dinucleotides. Agonists can be complete agonists, partial agonists, or inverse agonists.

[0062] As used herein, the term "antagonist" refers to a molecule that blocks or retards an agonist-mediated response rather than itself stimulating a biological response when bound to a receptor. Many antagonists achieve their effectiveness by competing with endogenous ligands or substrates at a structurally defined binding site on a receptor. Non-limiting examples of antagonists include alpha blockers, beta blockers, and calcium channel blockers. Antagonists can be competitive, noncompetitive, or noncompetitive antagonists.

[0063] As used herein, the term "free IL-12 portion" means an IL-12 portion that is not associated with an extracellular vesicle, but is otherwise identical to the IL-12 portion associated with an extracellular vesicle. In particular, the free IL-12 portion is the same IL-12 portion associated with an extracellular vesicle when compared to an extracellular vesicle associated with the IL-12 portion. In some aspects, when the free IL-12 portion is compared to an extracellular vesicle comprising an IL-12 portion in terms of its efficacy, toxicity, and / or any other properties, the amount of the free IL-12 portion is the same as the amount of the IL-12 portion associated with an EV when compared to the IL-12 portion associated with an extracellular vesicle.

[0064] As used herein, the term "exoIL-12" refers to an exosome loaded with an IL-12 portion, for example, an IL-12 polypeptide or a fragment thereof. In some aspects, the IL-12 portion is associated with the outer surface of the exosome (e.g., the surface display of the IL-12 portion). Non-limiting examples of exosomes comprising IL-12 portions can be found in, for example, U.S. Patent No. 10,723,782 and International Publication No. WO 2019 / 133934 A2, each of which is incorporated herein by reference in its entirety. In some aspects, the IL-12 portion is connected or conjugated to the outer surface of the exosome. In some aspects, the IL-12 portion is connected or conjugated to a surface-exposed scaffold protein, for example, a scaffold X protein (e.g., PTGFRN protein). In some aspects, the IL-12 portion is connected or conjugated to the lipid bilayer of the exosome. In some aspects, the exosome comprises an IL-12 portion located in the lumen of the exosome. In some aspects, the IL-12 portion is associated with the luminal surface of the exosome, e.g., with a scaffold protein, e.g., Scaffold X (e.g., PTGFRN). In some aspects, the IL-12 portion is encapsulated within the lumen of the exosome and is not associated with a scaffold protein. In some aspects, the IL-12 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 13.

[0065] As used herein, the term "ligand" refers to a molecule that binds to a receptor and modulates the receptor to produce a biological response. Modulation can be activation, inactivation, blocking or retardation of a receptor-mediated biological response. Receptors can be modulated by endogenous ligands or exogenous ligands. Non-limiting examples of endogenous ligands include antibodies and peptides. Non-limiting examples of exogenous agonists include drugs, small molecules and cyclic dinucleotides. A ligand can be a complete ligand, a partial ligand or a reverse ligand.

[0066] As used herein, the term "antibody" encompasses immunoglobulins and fragments thereof that are produced naturally or partially or completely synthetically. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. "Antibodies" further include polypeptides that specifically bind to and recognize an antigen and comprise framework regions from an immunoglobulin gene or a fragment thereof. The use of the term antibody is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and further include single-chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, such as scFv, (scFv) 2 , Fab, Fab' and F(ab') 2 、F(ab1) 2 , Fv, dAb and Fd fragments, bifunctional antibodies and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0067] As used herein, the term "therapeutically effective amount" is an amount of an agent or pharmaceutical compound sufficient to produce a desired therapeutic, pharmacological and / or physiological effect on a subject in need thereof. A therapeutically effective amount can be a "prophylactically effective amount" because prevention and treatment can be considered therapy.

[0068] As used herein, the term "pharmaceutical composition" refers to one or more compounds, e.g., EVs, of the compounds described herein that are mixed or doped with one or more other chemical components, such as pharmaceutically acceptable carriers and excipients, or suspended in the one or more other chemical components. One purpose of a pharmaceutical composition is to facilitate the preparation of EVs for administration to a subject. The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" and its grammatical variants cover any agent approved by a regulatory agency of the U.S. federal government or listed in the U.S. Pharmacopoeia for use in animals, including humans, and any carrier or diluent that does not produce an undesirable physiological effect to the extent that it inhibits the administration of the composition to a subject and does not eliminate the biological activity and properties of the administered compound. Excipients and carriers that can be used to prepare pharmaceutical compositions and are generally safe, non-toxic and desirable are included.

[0069] As used herein, the term "payload" refers to a therapeutic agent that acts on a target (e.g., a target cell) that contacts an EV. Payloads that can be introduced into EVs and / or producer cells include therapeutic agents such as nucleotides (e.g., nucleotides that contain a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g., DNA or mRNA molecules that encode polypeptides such as enzymes, or RNA molecules with regulatory functions such as miRNA, dsDNA, lncRNA, and siRNA), amino acids (e.g., amino acids that contain a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g., enzymes), lipids, carbohydrates, and small molecules (e.g., small molecule drugs and toxins).

[0070] The terms "administration," "administering," and variations thereof refer to the introduction of a composition (such as an EV) or an agent into a subject and include simultaneous and sequential introduction of a composition or agent. The composition or agent is introduced into a subject by any suitable route, including intratumorally, orally, pulmonary, intranasally, parenterally (intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly, or topically. In some aspects, the composition is delivered to the subject by intravesical administration. Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0071] As used herein, the terms "treat," "treatment," or "treating" refer to, for example, reducing the severity of a disease or condition; reducing the duration of a disease process; ameliorating or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject with a disease or condition, but not necessarily curing the disease or condition. The terms also include preventing or preventing a disease or condition, or a symptom thereof. In one aspect, the terms "treating" or "treatment" means inducing an immune response to an antigen in a subject.

[0072] As used herein, the term "prevent" or "preventing" refers to reducing or decreasing the incidence or severity of a particular outcome. In some aspects, an outcome is prevented by prophylactic treatment.

[0073] As used herein, the terms "modulate", "modulate", "modify" and / or "modulator" generally refer to the ability to change by increasing or decreasing, for example, directly or indirectly promoting / stimulating / upregulating or interfering / inhibiting / downregulating a specific concentration, level, expression, function or behavior, such as acting as an antagonist or agonist. In some cases, a modulator can increase and / or decrease a certain concentration, level, activity or function relative to a control or relative to a generally expected average activity level or relative to a control activity level.

[0074] As used herein, "mammalian subjects" include all mammals, including but not limited to humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.).

[0075] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapy and veterinary applications. In some aspects, the subject is a mammal, and in other aspects, the subject is a human.

[0076] As used herein, the term "substantially free" means that the sample containing EVs contains less than 10% macromolecules by mass / volume (m / v) percentage concentration. Some fractions may contain less than 0.001%, less than 0.01%, less than 0.05%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9% or less than 10% (m / v) macromolecules.

[0077] As used herein, the term "macromolecule" means nucleic acid, exogenous protein, lipid, carbohydrate, metabolite or a combination thereof.

[0078] As used herein, "primary tumor" refers to the original or first tumor in a subject's body where the tumor begins to grow. Primary tumors are used in contrast to "secondary tumors," which refer to tumors that arise at a location other than the location of the primary tumor after the primary tumor begins to grow, such as due to metastasis of cells in the primary tumor.

[0079] The ranges recited herein are to be understood as shorthand for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 50 is to be understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0080] Unless otherwise stated, reference to a compound having one or more stereocenters is intended to refer to each and every stereoisomer thereof and to all combinations of stereoisomers thereof.

[0081] II. Treatment Methods

[0082] Certain aspects of the present disclosure relate to a method of preventing or treating a tumor in a subject in need thereof, the method 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. In some aspects, the IL-12 polypeptide is delivered via extracellular vesicles. In some aspects, the IL-12 polypeptide is associated with an EV.

[0083] In some aspects, the tumor that can be treated by the method of the present invention is a primary tumor, a secondary tumor, or both a primary tumor and a secondary tumor. In some aspects, administration reduces the volume of the primary tumor. In some aspects, administration can reduce the volume of the primary tumor by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to the monotherapy after administration of the 14th day.

[0084] In some aspects, use reduces the tumor growth rate of the primary tumor. In some aspects, administration can reduce the growth rate of the primary tumor by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times, compared to monotherapy after administration of the 14th day. In some aspects, administration can ablate or prevent primary tumor growth.

[0085] The method of any one of claims 4 to 8, wherein the administration reduces the volume of secondary tumors. In some aspects, the administration can reduce the volume of secondary tumors by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to monotherapy after day 14 of administration.

[0086] In some aspects, use reduces the tumor growth rate of secondary tumors. In some aspects, administration can reduce the growth rate of secondary tumors by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times compared to monotherapy after administration of day 14. In some aspects, administration can ablate or prevent secondary tumor growth.

[0087] Other aspects of the present disclosure relate to methods of treating a disease or disorder (e.g., a tumor) in a subject in need thereof, the method comprising administering an extracellular vesicle (EV) comprising an interleukin 12 (IL-12) portion, wherein the method does not comprise administering an EV comprising a STING agonist.

[0088] In some aspects, the method further comprises an anticancer agent. In some aspects, the anticancer agent comprises a checkpoint inhibitor. In some aspects, 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. In some aspects, the checkpoint inhibitor is an anti-PD-1 antibody.

[0089] In some aspects, the administration of the IL-12 portion causes an objective response rate (ORR) of about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, or about 80% to about 100%. In some aspects, the administration of the IL-12 portion causes an ORR of about 20% to about 90%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%. In some aspects, the administration of the IL-12 portion causes an ORR of about 25% to about 56%. In some aspects, administration of the IL-12 moiety results in at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% ORR. In some aspects, for example, for CTCL, the response rate is measured using the mSWAT analysis.

[0090] In some aspects, the administration of IL-12 moieties causes a complete response in a subject. In some aspects, the administration of IL-12 moieties causes at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the subject to experience a complete response (CR). In some aspects, the administration of IL-12 moieties causes at least about 20% of the subject to experience CR. In some aspects, the administration of IL-12 moieties causes at least about 22% of the subject to experience CR.

[0091] Without being bound by any particular mechanism, in some aspects, administration of an IL-12 portion disclosed herein activates immune cells that target and kill tumor cells. In some aspects, an IL-12 portion disclosed herein (e.g., an EV loaded with an IL-12 portion) activates T cells. In some aspects, an IL-12 portion disclosed herein (e.g., an EV loaded with an IL-12 portion) activates CD8 + T cells. In some aspects, an IL-12 portion disclosed herein (e.g., an EV loaded with an IL-12 portion) activates NK cells.

[0092] The IL-12 portion can be administered to a subject by any route known in the art. In some aspects, the IL-12 portion is administered parenterally, orally, intravenously (IV), intramuscularly (IM), intratumorally (IT), intranasally, subcutaneously, or intraperitoneally (IP). In certain aspects, the IL-12 portion is administered directly to the tumor microenvironment, for example, by IT delivery. In some aspects, IT delivery of the IL-12 portion disclosed herein reduces systemic exposure, enhances selective immune cell activation, increases tissue retention (provides a prolonged response), or any combination thereof.

[0093] II.A. Interleukin-12 (IL-12)

[0094] Interleukin 12 (IL-12) is a heterodimeric cytokine produced by dendritic cells, macrophages, and neutrophils. See, e.g., Interleukin-12 Signaling, Reactom, available at reactome.org / content / detail / R-HSA-9020591; and UniProtKB–P29459 (IL-12A subunit) P29460 (IL-12B subunit). It is encoded by the genes interleukin-12 subunit alpha (IL12A) and interleukin-12 subunit beta (IL12B), which encode a 35-kDa light chain (p35) and a 40-kDa heavy chain (p40), respectively. The active IL-12 heterodimer is sometimes referred to as p70. The p35 component is homologous to single-chain cytokines, while p40 is homologous to the extracellular domain of members of the hematopoietic cytokine receptor family. Therefore, IL-12 heterodimers are similar to cytokines connected to soluble receptors. IL-12 participates in the differentiation of naive T cells to Th1 cells and is sometimes referred to as a T cell stimulator. IL-12 enhances the cytotoxic activity of natural killer cells and CD8+ cytotoxic T lymphocytes. IL-12 also has anti-angiogenic activity, which is mediated by increasing the production of CXCL10 by interferon gamma. Non-limiting examples IL-12 that can be used for the present disclosure can be found in, for example, U.S. Patent No. 10,723,782, International Publication No. WO 2019 / 133934A2, and International Application No. PCT / US2020 / 028778, each of which is incorporated herein by reference in its entirety.

[0095] The IL-12 receptor is a heterodimer formed by the interleukin-12 receptor subunit β-1 (IL12RB1) and the interleukin-12 receptor subunit β-2 (IL12RB2), both of which have extensive homology with IL6ST (gp130), which is a signal transduction receptor subunit of the IL6-like cytokine superfamily. IL-12RB2 is believed to play a key role in IL-12 function, in part because its expression on activated T cells is stimulated by cytokines that promote the development of Th1 cells and inhibited by cytokines that promote the development of Th2 cells. In addition, IL-12 binding causes IL12RB2 tyrosine phosphorylation, which provides binding sites for the kinase non-receptor tyrosine protein kinase TYK2 and the tyrosine protein kinase JAK2. These activate transcription factor proteins in the signal transducer and activator of transcription (STAT) family, particularly STAT4, which is a cytokine produced by bone marrow cells and other cell types.

[0096] The amino acid sequences of IL-12 A and B subunits are shown in Table 1A.

[0097] Table 1A: Human IL-12A and IL-12B subunit amino acid sequences.

[0098]

[0099] In some aspects, the IL-12 portion comprises an IL-12 polypeptide. In some aspects, the IL-12 portion comprises an IL-12 protein. In some aspects, the IL-12 polypeptide comprises a full-length human IL-12, such as an IL-12 heterodimer. In some aspects, the IL-12 heterodimer comprises a fusion protein in which an IL-12 α subunit is covalently linked to an IL-12 β subunit (SEQ ID NO: 13; Table 1B). In some aspects, the IL-12 portion comprises an IL-12 α subunit. In some aspects, the IL-12 portion comprises an IL-12 β subunit.

[0100] Table 1B: Human IL-12A-IL-12B fusion amino acid sequence.

[0101]

[0102] In some aspects, the IL-12 heterodimer comprises an IL-12 alpha subunit covalently linked to an IL-12 beta subunit via a linker. In some aspects, the linker comprises one or more amino acids. In some aspects, the linker is a linker disclosed herein. In some aspects, the linker comprises a Gly / Ser linker. In some aspects, the linker is a cleavable linker. In some aspects, the linker comprises a disulfide bond.

[0103] In some aspects, the IL-12 portion comprises a molecule with IL-12 activity. In some aspects, the molecule with IL-12 activity is an IL-12 analog. In some aspects, the molecule with IL-12 activity comprises a molecule that activates an IL-12 receptor.

[0104] In some aspects, the IL-12 portion comprises a nucleic acid molecule encoding an IL-12 protein (e.g., an IL-12 α subunit, an IL-12 β subunit, and / or an IL-12 heterodimer). In some aspects, the nucleic acid molecule encodes an IL-12 α subunit. In some aspects, the nucleic acid molecule encodes an IL-12 β subunit. In some aspects, the nucleic acid molecule encodes an IL-12 α subunit and an IL-12 β subunit. In some aspects, the nucleic acid molecule encodes an IL-12 α subunit covalently attached to an IL-12 β subunit. In some aspects, the nucleic acid molecule encodes an IL-12 heterodimer.

[0105] In some aspects, the IL-12 portion comprises a nucleic acid molecule, wherein the nucleic acid molecule is packaged in a vector. In some aspects, the vector is a viral vector. In some aspects, the vector is based on a DNA virus, such as adenovirus, adeno-associated virus (AAV) and herpes virus, and a retroviral vector. In some aspects, the vector is a slow virus. In some aspects, the virus is AAV.

[0106] In some aspects, the IL-12 portion comprises a p35 polypeptide or a fragment thereof. In some aspects, IL-12 comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, 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: 11. In some aspects, IL-12 comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 11. In some aspects, IL-12 comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 11. In some aspects, IL-12 comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 11. In some aspects, IL-12 comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 11. In some aspects, the IL-12 comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 11. In some aspects, the IL-12 comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 11. In some aspects, the IL-12 comprises the amino acid sequence shown in SEQ ID NO: 11. In some aspects, the IL-12 portion lacks a signal peptide (see Table 1A).

[0107] In some aspects, the IL-12 portion comprises a p40 polypeptide or a fragment thereof. In some aspects, the IL-12 comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, 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: 12. In some aspects, the IL-12 comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 12. In some aspects, the IL-12 comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 12. In some aspects, the IL-12 comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 12. In some aspects, the IL-12 comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 12. In some aspects, the IL-12 comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 12. In some aspects, the IL-12 comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 12. In some aspects, the IL-12 comprises the amino acid sequence shown in SEQ ID NO: 12. In some aspects, the IL-12 portion lacks a signal peptide (see Table 1A).

[0108] In some aspects, the IL-12 portion comprises a p35 polypeptide or a fragment thereof and a p40 polypeptide or a fragment thereof. In some aspects, the IL-12 portion comprises a single polypeptide, wherein the p35 polypeptide or a fragment thereof is linked to the p40 polypeptide or a fragment thereof. In some aspects, the p35 polypeptide or a fragment thereof is linked to the p40 polypeptide or a fragment thereof via a linker. In some aspects, the linker is a peptide linker. In some aspects, the linker comprises one or more amino acids. In some aspects, the linker comprises a Gly-Ser (GS) linker. In some aspects, the GS linker comprises (G 4 S) n , wherein n is an integer between 1 and 10. In some aspects, the GS linker comprises (G 3 S) n , where n is an integer between 1 and 10.

[0109] In certain aspects, the IL-12 portion comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, 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. In some aspects, IL-12 comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 13. In some aspects, IL-12 comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 13. In some aspects, IL-12 comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 13. In some aspects, IL-12 comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 13. In some aspects, IL-12 comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 13. In some aspects, IL-12 comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 13. In some aspects, IL-12 comprises the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, IL-12 consists of the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, IL-12 consists essentially of the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, the IL-12 portion lacks a signal peptide (see Table 1B).

[0110] II.B. Administration

[0111] In some aspects, the method comprises administering to a 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. In some aspects, the amount of the IL-12 polypeptide is measured using a fluorescence assay. In some aspects, the amount of the IL-12 polypeptide is measured using an IL-12α LISA. In some aspects, the dose of the IL-12 polypeptide administered is based on the weight of the IL-12 polypeptide, excluding the weight of an anchoring portion, a scaffold portion, or other linkers that can be used to associate the IL-12 polypeptide with an exosome.

[0112] In some aspects, 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 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. 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.

[0113] In some aspects, the dose comprises at least about 0.3 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 0.4 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 0.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 0.6 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 0.7 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 0.8 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 0.9 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 1.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 1.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 2.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 2.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 3.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 4.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 4.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 5.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 5.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 6.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 7.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 7.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 8.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 8.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 9.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 9.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 10.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 10.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 11.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 11.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 12.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 13.0 μg of the IL-12 polypeptide.In some aspects, the dose comprises at least about 13.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 14.0 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 14.5 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 15 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 16 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 17 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 18 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 19 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 20 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 21 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 22 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 23 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 24 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 25 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 26 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 27 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 28 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 29 μg of the IL-12 polypeptide. In some aspects, the dose comprises at least about 30 μg of the IL-12 polypeptide.

[0114] In some aspects, the dose of the IL-12 polypeptide is administered at least twice, at least three times, at least four times, at least five times, or at least six times. In some aspects, the dose of the IL-12 polypeptide is administered about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every six weeks, or about once every eight weeks. In some aspects, the IL-12 polypeptide is administered about once a week. In some aspects, the IL-12 polypeptide is administered about once every two weeks. In some aspects, the IL-12 polypeptide is administered about once every three weeks. In some aspects, the IL-12 polypeptide is administered about once every four weeks.

[0115] In some aspects, the dosage is used once a week, once every other week, once every three weeks, or once every four weeks. In some aspects, the dosage is used once every other week. In some aspects, the dosage is used once every 7 to 21 days, once every 7 to 18 days, once every 7 to 14 days, once every 10 to 21 days, once every 10 to 18 days, once every 10 to 14 days, once every 10 to 16 days, once every 12 to about 20 days, once every 12 to about 18 days, once every 12 to about 16 days, once every 12 to about 14 days, once every 14 to about 21 days, once every 14 to about 16 days, once every 14 to about 18 days, or once every 14 to about 21 days. In some aspects, the dosage is used once every 7 days. In some aspects, the dosage is used once every 10 days. In some aspects, the dose is administered about once every 12 days. In some aspects, the dose is administered about once every 14 days. In some aspects, the dose is administered about once every 16 days. In some aspects, the dose is administered about once every 18 days. In some aspects, the dose is administered about once every 20 days. In some aspects, the dose is administered about once every 21 days.

[0116] In some aspects, the dose of the IL-12 polypeptide is administered at a therapeutically effective amount of between about 5 μg and about 7 μg of the IL-12 polypeptide, such as about 6 μg of the IL-12 polypeptide, about once a week, about once every other week, about once every three weeks, or about once every four weeks. In some aspects, the dose of the IL-12 polypeptide is administered at a therapeutically effective amount of between about 5 μg and about 7 μg of the IL-12 polypeptide, such as about 6 μg of the IL-12 polypeptide, about once every other week. In some aspects, the dose of the IL-12 polypeptide is administered at a therapeutically effective amount of between about 5 μg and about 7 μg of the IL-12 polypeptide, such as about 6 μg of the IL-12 polypeptide, about once every 10 to 18 days, about once every 12 to about 16 days, about once every 14 to about 21 days, about once every 10 to about 14 days, or about once every 14 to about 18 days. In some aspects, the dose of the IL-12 polypeptide is administered at a therapeutically effective amount of between about 5 μg and about 7 μg of the IL-12 polypeptide, such as about 6 μg of the IL-12 polypeptide, about once every 14 days.

[0117] In some aspects, the dose of the IL-12 polypeptide is administered about once a week at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 7 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 8 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 9 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 10 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 11 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 12 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 13 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 14 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 17 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 18 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 19 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 20 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 21 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 3 weeks at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once per week at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide.In some aspects, the dose of the IL-12 polypeptide is administered about once every 7 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 8 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 9 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 10 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 11 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 12 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 13 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 14 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 17 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 18 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 19 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 20 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 21 days at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 3 weeks at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide.

[0118] In some aspects, the dose of the IL-12 polypeptide is administered about once a week at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 7 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 8 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 9 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 10 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 11 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 12 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 13 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 14 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 17 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 18 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 19 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 20 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 21 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 3 weeks at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once per week at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide.In some aspects, the dose of the IL-12 polypeptide is administered about once every 7 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 8 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 9 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 10 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 11 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 12 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 13 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 14 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 17 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 18 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 19 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 20 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 21 days at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 3 weeks at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide.

[0119] In some aspects, the dose of the IL-12 polypeptide is administered about once a week at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 7 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 8 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 9 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 10 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 11 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 12 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 13 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 14 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 15 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 17 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 18 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 19 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 20 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 21 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 3 weeks at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide.In some aspects, the dose of the IL-12 polypeptide is administered about once every 22 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 23 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 24 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 25 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 26 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 27 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 28 days at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once every 4 weeks at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide. In some aspects, the dose of the IL-12 polypeptide is administered about once a month at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide.

[0120] In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide, at least about 1 dose, at least about 2 doses, at least about 3 doses, at least about 4 doses, at least about 5 doses, at least about 6 doses, at least about 7 doses, at least about 8 doses, at least about 9 doses, at least about 10 doses, at least about 11 doses, at least about 12 doses, at least about 13 doses, at least about 14 doses, at least about 15 doses, at least about 16 doses, at least about 17 doses, at least about 18 doses, at least about 19 doses, at least about 20 doses, at least about 25 doses, at least about 30 doses, at least about 35 doses, at least about 40 doses, at least about 45 doses, or at least about 50 doses. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide for at least about 2 weeks, at least about 4 weeks, at least about 6 weeks, at least about 8 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, at least about 30 weeks, at least about 32 weeks, at least about 34 weeks, at least about 36 weeks, at least about 38 weeks, at least about 40 weeks, at least about 42 weeks, at least about 44 weeks, at least about 46 weeks, at least about 48 weeks, at least about 50 weeks, or for at least about 52 weeks. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide for at least about 6 weeks. In some aspects, the dosage of the IL-12 polypeptide is administered at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide about once every 2 weeks for at least about 12 weeks. In some aspects, the dosage of the IL-12 polypeptide is administered at a therapeutically effective amount of about 3.0 μg of the IL-12 polypeptide about once every 2 weeks for as long as treatment is effective.

[0121] In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide, at least about 1 dose, at least about 2 doses, at least about 3 doses, at least about 4 doses, at least about 5 doses, at least about 6 doses, at least about 7 doses, at least about 8 doses, at least about 9 doses, at least about 10 doses, at least about 11 doses, at least about 12 doses, at least about 13 doses, at least about 14 doses, at least about 15 doses, at least about 16 doses, at least about 17 doses, at least about 18 doses, at least about 19 doses, at least about 20 doses, at least about 25 doses, at least about 30 doses, at least about 35 doses, at least about 40 doses, at least about 45 doses, or at least about 50 doses. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks for at least about 2 weeks, at least about 4 weeks, at least about 6 weeks, at least about 8 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, at least about 30 weeks, at least about 32 weeks, at least about 34 weeks, at least about 36 weeks, at least about 38 weeks, at least about 40 weeks, at least about 42 weeks, at least about 44 weeks, at least about 46 weeks, at least about 48 weeks, at least about 50 weeks, or for at least about 52 weeks. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks for at least about 6 weeks. In some aspects, the dose of the IL-12 polypeptide is administered at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide about once every 2 weeks for at least about 12 weeks. In some aspects, the dose of the IL-12 polypeptide is administered at a therapeutically effective amount of about 6.0 μg of the IL-12 polypeptide about once every 2 weeks for as long as treatment is effective.

[0122] In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide, at least about 1 dose, at least about 2 doses, at least about 3 doses, at least about 4 doses, at least about 5 doses, at least about 6 doses, at least about 7 doses, at least about 8 doses, at least about 9 doses, at least about 10 doses, at least about 11 doses, at least about 12 doses, at least about 13 doses, at least about 14 doses, at least about 15 doses, at least about 16 doses, at least about 17 doses, at least about 18 doses, at least about 19 doses, at least about 20 doses, at least about 25 doses, at least about 30 doses, at least about 35 doses, at least about 40 doses, at least about 45 doses, or at least about 50 doses. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks for at least about 2 weeks, at least about 4 weeks, at least about 6 weeks, at least about 8 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, at least about 30 weeks, at least about 32 weeks, at least about 34 weeks, at least about 36 weeks, at least about 38 weeks, at least about 40 weeks, at least about 42 weeks, at least about 44 weeks, at least about 46 weeks, at least about 48 weeks, at least about 50 weeks, or for at least about 52 weeks. In some aspects, the dose of the IL-12 polypeptide is administered about once every 2 weeks for at least about 6 weeks. In some aspects, the dosage of the IL-12 polypeptide is administered at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide about once every 2 weeks for at least about 12 weeks. In some aspects, the dosage of the IL-12 polypeptide is administered at a therapeutically effective amount of about 12.0 μg of the IL-12 polypeptide about once every 2 weeks for as long as treatment is effective.

[0123] In some aspects, the dose is administered on about day 1 and day 15 of the first 28-day cycle. In some aspects, the dose is administered on about day 1 and day 15 of the second 28-day cycle. In some aspects, the dose is administered on about day 1 of the third 28-day cycle. In some aspects, the dose is administered on day 1 of each subsequent 28-day cycle. In some aspects, the dose is administered (i) on about day 1 and day 15 of the first 28-day cycle, and (ii) on about day 1 and day 15 of the second 28-day cycle. In some aspects, the dose is administered (i) on about day 1 and day 15 of the first 28-day cycle, (ii) on about day 1 and day 15 of the second 28-day cycle, and (iii) on about day 1 of the third 28-day cycle. In certain aspects, the dose is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0124] In some aspects, a dosage of about 0.3 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 0.3 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 0.3 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0125] In some aspects, a dosage of about 1.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 1.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 1.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0126] In some aspects, a dosage of about 3.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 3.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 3.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0127] In some aspects, a dosage of about 6.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 6.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 6.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0128] In some aspects, a dosage of about 12.0 μg is applied on about the 1st and 15th day of the first 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied on about the 1st day of the second 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied on about the 1st day of the third 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied on the 1st day of each subsequent 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, and (ii) on about the 1st and 15th day of the second 28-day cycle. In some aspects, a dosage of about 12.0 μg is applied (i) on about the 1st and 15th day of the first 28-day cycle, (ii) on about the 1st and 15th day of the second 28-day cycle, and (iii) on about the 1st day of the third 28-day cycle. In certain aspects, a dose of about 12.0 μg is administered (i) on about days 1 and 15 of a first 28-day cycle, (ii) on about days 1 and 15 of a second 28-day cycle, (iii) on about day 1 of a third 28-day cycle, and (iii) about every 28 days thereafter in the third 28-day cycle.

[0129] In some aspects, administration of the IL-12 polypeptide continues until a clinical benefit is achieved.

[0130] II.C. Scaffold-engineered EVs (e.g., exosomes)

[0131] In some aspects, the EVs of the present disclosure comprise a membrane that is modified in its composition. For example, its membrane composition can be modified by changing the protein, lipid, or glycan content of the membrane.

[0132] In some aspects, surface engineered EVs are produced by chemical and / or physical methods (such as PEG-induced fusion and / or ultrasonic fusion). In other aspects, surface engineered EVs (e.g., exosomes) are produced by genetic engineering. EVs produced by genetically modified producer cells or progeny of genetically modified cells may contain modified membrane compositions. In some aspects, surface engineered EVs (e.g., exosomes) have a higher or lower density (e.g., a higher number) of scaffold portions (e.g., exosomal proteins, e.g., Scaffold X), or include variants or fragments of scaffold portions.

[0133] For example, surface engineered EVs (e.g., Scaffold X engineered or Scaffold Y engineered EVs) can be produced by cells (e.g., HEK293 cells) transformed with exogenous sequences encoding scaffold portions (e.g., exosomal proteins, e.g., Scaffold X and / or Scaffold Y) or variants or fragments thereof. EVs comprising scaffold portions expressed by exogenous sequences can include modified membrane compositions.

[0134] Various modifications or fragments of the scaffold portion can be used in various aspects of the present disclosure. For example, a scaffold portion modified to have an enhanced affinity for a binding agent can be used to produce a surface engineered EV that can be purified using a binding agent. Scaffold portions modified to more effectively target EVs (e.g., exosomes) and / or membranes can be used. Scaffold portions modified to contain the minimum fragments required for specific and effective targeting to EVs (e.g., exosomes), membranes can also be used.

[0135] In some aspects, an IL-12 portion (e.g., polypeptide) disclosed herein is expressed on the surface of an EV (e.g., exosome) as a fusion protein (e.g., a fusion protein of an IL-12 portion (e.g., polypeptide) with Scaffold X and / or Scaffold Y). For example, a fusion protein can comprise an IL-12 portion (e.g., polypeptide) disclosed herein linked to a scaffold portion (e.g., Scaffold X or Scaffold Y).

[0136] In certain aspects, scaffold X comprises PTGFRN protein, BSG protein, IGSF2 protein, IGSF3 protein, IGSF8 protein, ITGB1 protein, ITGA4 protein, SLC3A2 protein, ATP transporter, Lamp-1 protein, Lamp-2 protein, CD13 protein, CD86 protein, floater protein, syntaxin-3 protein, CD2 protein, CD36 protein, CD40 protein, CD40L protein, CD41a protein, CD44 protein, CD45 protein, ICAM-1 protein, integrin α4 protein, L1CAM protein, LFA-1 protein, Mac-1α and β protein, Vti-1A and B protein, CD3ε and ζ protein, CD9 protein, CD18 protein, CD37 protein, CD53 protein, CD63 protein, CD81 protein, CD82 protein, CXCR4 protein, FcR protein, GluR2 / 3 protein, HLA-DM (MHC II) protein, immunoglobulin, MHC-I or MHC-II component protein, TCRβ protein, tetraspanin or fragments or variants thereof.

[0137] In some aspects, the surface-engineered EVs (e.g., exosomes (e.g., Scaffold X engineered EVs, e.g., exosomes) described herein exhibit superior properties compared to EVs (e.g., exosomes) known in the art. For example, surface-engineered EVs (e.g., Scaffold X) contain modified proteins that are more highly enriched on their surfaces compared to naturally occurring EVs (e.g., exosomes) or EVs (e.g., exosomes) produced using conventional exosomal proteins. In addition, surface-engineered EVs (e.g., exosomes) (e.g., Scaffold X engineered EVs, e.g., exosomes) of the present invention may have greater, more specific, or more controlled biological activity compared to naturally occurring EVs (e.g., exosomes) or EVs (e.g., exosomes) produced using conventional exosomal proteins.

[0138] In other aspects, the EVs (eg, exosomes) of the present disclosure contain an IL-12 moiety (eg, a polypeptide) and a scaffold X, wherein the IL-12 moiety is linked to the scaffold X.

[0139] In some aspects, the support X that can be used for the present disclosure comprises prostaglandin F2 receptor negative regulator (PTGFRN polypeptide).PTGFRN protein can also be referred to as CD9 partner 1 (CD9P-1), protein F (EWI-F) containing Glu-Trp-Ile EWI motif, prostaglandin F2-α receptor regulatory protein, prostaglandin F2-α receptor associated protein or CD315.The full-length amino acid sequence of human PTGFRN protein (Uniprot accession number Q9P2B2) is shown as SEQ ID NO:1 in Table 2.PTGFRN polypeptide contains signal peptide (SEQ ID NO:1 amino acid 1 to 25), extracellular domain (SEQ ID NO:1 amino acid 26 to 832), transmembrane domain (SEQ ID NO:1 amino acid 833 to 853) and cytoplasmic domain (SEQ ID NO:1 amino acid 854 to 879).Mature PTGFRN polypeptide is by SEQ ID NO:1 without signal peptide, i.e. SEQ ID NO:1 amino acid 26 to 879 composition. In some aspects, the PTGFRN polypeptide fragments useful in the present disclosure comprise the transmembrane domain of the PTGFRN polypeptide. In other aspects, the PTGFRN polypeptide fragments useful in the present disclosure comprise the transmembrane domain of the PTGFRN polypeptide and (i) at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 amino acids located at the N-terminus of the transmembrane domain, (ii) at least five, at least 10, at least 15, at least 20 or at least 25 amino acids located at the C-terminus of the transmembrane domain, or both (i) and (ii).

[0140] In some aspects, Scaffold X comprises an amino acid sequence that is at least about 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% identical to SEQ ID NO: 33.

[0141] Table 2A. Exemplary Scaffold X Protein Sequences

[0142]

[0143] In other aspects, Scaffold X comprises an amino acid sequence that is 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% identical to SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8.

[0144] Non-limiting examples of other Scaffold X proteins that can be used to link an IL-12 moiety (e.g., a polypeptide) to the surface of an EV (e.g., an exosome) can be found in U.S. Pat. Nos. 10,195,290 B1 and 10,561,740 B2, each of which is incorporated herein by reference in its entirety.

[0145] In some aspects, the Scaffold X described herein can also be used to simultaneously attach the IL-12 portion to the luminal surface and / or the external surface of the EV (e.g., exosome). For example, in addition to the surface of the EV (e.g., exosome), the PTGFRN polypeptide can also be used to attach the IL-12 portion to the interior of the lumen. In some aspects, the Scaffold X can be used to attach the IL-12 portion and an additional therapeutic agent to the EV (e.g., exosome) (e.g., a payload). Therefore, in some aspects, the Scaffold X disclosed herein can be used for dual purposes.

[0146] In some aspects, the EVs (e.g., exosomes) of the present disclosure comprise an internal space (i.e., a lumen) that is different from the internal space of a naturally occurring EV (e.g., exosome). For example, an EV (e.g., exosome) can be altered such that the composition in the lumen side of the EV (e.g., exosome) has a protein, lipid, or glycan content that is different from the protein, lipid, or glycan content of a naturally occurring EV (e.g., exosome).

[0147] In some aspects, engineered EVs (e.g., exosomes) can be produced by cells transformed with exogenous sequences encoding a scaffold portion (e.g., an exosomal protein, e.g., Scaffold Y) or a modification or fragment of a scaffold portion that alters the composition or content of the lumenal side of the EV (e.g., exosome). Various modifications or fragments of exosomal proteins that can be expressed in the lumenal side of EVs (e.g., exosomes) can be used in various aspects of the present disclosure.

[0148] In some aspects, the IL-12 portion disclosed herein is located in the lumen of an EV (e.g., an exosome) (i.e., encapsulated). In some aspects, the IL-12 portion is connected to the lumen surface of the EV (e.g., an exosome). As used herein, when a molecule (e.g., an IL-12 portion) is described as being in the "lumen" of an EV (e.g., an exosome), this means that the molecule is located within (e.g., associated with) the EV (e.g., an exosome), but is not connected to any molecule on the lumen surface of the EV. In other aspects, the IL-12 portion is expressed as a fusion molecule on the lumen surface of an EV (e.g., an exosome), e.g., a fusion molecule of the IL-12 portion and a scaffold portion (e.g., scaffold X or scaffold Y).

[0149] II.D. Connector

[0150] The EVs of the present disclosure may comprise one or more linkers that connect the IL-12 portion to the EV or to a scaffold portion (e.g., scaffold X on the outer surface of the EV). In some aspects, the IL-12 portion is connected to the EV directly or through a linker in a scaffold portion on the EV. In some aspects, the IL-12 portion is connected to the lipid bilayer of the EV, for example, through a linker. The linker can be any chemical moiety known in the art.

[0151] In some aspects, the term "joint" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or to a non-polypeptide. In some aspects, two or more joints can be connected in series. Typically, the joint provides flexibility or prevents / improves steric hindrance. The joint is usually not cut; however, in some aspects, such cutting may be desired. Therefore, in some aspects, the joint may include one or more protease cleavage sites, which may be located within the sequence of the joint or at either end of the joint sequence.

[0152] In some aspects, the joint is a peptide joint. In some aspects, the peptide joint can include at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95 or at least about 100 amino acids.

[0153] In some aspects, the peptide linker is synthetic, i.e., non-naturally occurring. In one aspect, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) comprising an amino acid sequence that connects or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids, the peptide not being naturally connected or genetically fused to the second linear sequence of amino acids in nature. For example, in one aspect, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation, such as an addition, substitution, or deletion).

[0154] The linker may be susceptible to cleavage ("cleavable linker"), thereby facilitating the release of the IL-12 portion or other payload. In some aspects, the linker is a "reduction-sensitive linker". In some aspects, the reduction-sensitive linker contains a disulfide bond. In some aspects, the linker is an "acid-labile linker". In some aspects, the acid-labile linker contains a hydrazone. Suitable acid-labile linkers also include, for example, cis-aconitine linkers, hydrazide linkers, thiocarbamoyl linkers, or any combination thereof. In some aspects, the linker comprises a non-cleavable linker.

[0155] III.E. Producer Cells and Modifications

[0156] EVs (e.g., exosomes) can be produced by cells grown in vitro or by body fluids of a subject. When EVs (e.g., exosomes) are produced from in vitro cell culture, various producer cells (e.g., HEK293 cells) can be used. Additional cell types that can be used to produce the lumen-engineered EVs (e.g., exosomes) described herein include, but are not limited to, mesenchymal stem cells, T cells, B cells, dendritic cells, macrophages, and cancer cell lines. Additional examples include: Chinese hamster ovary (CHO) cells, mesenchymal stem cells (MSCs), BJ human foreskin fibroblasts, fHDF fibroblasts, Neuronal precursor cells, Amniotic cells, adipose-derived mesenchymal stem cells, and RPTEC / TERT1 cells. In certain aspects, the producer cell is not a dendritic cell, a macrophage, a B cell, a mast cell, a neutrophil, a Kupfer-Brauvich cell, a cell derived from any of these cells, or any combination thereof.

[0157] Some aspects may also include genetically modifying EVs (e.g., exosomes) to contain one or more exogenous sequences (e.g., PTGFRN linked to IL-12) to produce modified EVs that express exogenous proteins on the surface of the vesicles.

[0158] More specifically, the EV (e.g., exosome) of the present invention can be produced by cells transformed with sequences encoding one or more additional exogenous proteins, including but not limited to ligands, cytokines or antibodies or any combination thereof. These additional exogenous proteins can enable activation or regulation of additional immunostimulatory signals. Exemplary additional exogenous proteins for use are contemplated to include proteins, ligands and other molecules described in detail in U.S. Patent Application 62 / 611,140, ​​International Publication No. WO / 2019 / 133934 and U.S. Patent Nos. 10,195,290B1 and 10,561,740B2, each of which is incorporated herein by reference in its entirety. In some aspects, EV (e.g., exosome) is further modified by a ligand comprising CD40L, OX40L or CD27L. In some aspects, EV (e.g., exosome) is further modified by a cytokine comprising IL-7 or IL-15. Any of the one or more exosomal proteins described herein can be expressed from a plasmid, an exogenous sequence inserted into the genome, or other exogenous nucleic acid, such as a synthetic messenger RNA (mRNA).

[0159] III. EV Purification

[0160] EVs (e.g., exosomes) prepared for the present disclosure can be isolated from producer cells. It is considered that all known ways of isolating EVs (e.g., exosomes) are considered applicable to this article. For example, the physical properties of EVs (e.g., exosomes) can be used to separate the EVs from culture media or other source materials, including separations based on charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieves, etc.), density (e.g., conventional or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc.). Alternatively or additionally, separation can be based on one or more biological properties, and include methods that can use surface markers (e.g., for precipitation, reversible binding to a solid phase, FACS separation, specific ligand binding, non-specific ligand binding, etc.). In yet another contemplated method, EVs (e.g., exosomes) can also be fused using chemical and / or physical methods, including PEG-induced fusion and / or ultrasonic fusion.

[0161] EVs (e.g., exosomes) can also be purified after incubation with a STING agonist to remove free, unencapsulated STING agonist from the composition. All methods previously disclosed are also considered applicable herein, including separation based on physical or biological properties of EVs (e.g., exosomes).

[0162] Separation, purification and enrichment can be performed in a general and non-selective manner (usually including continuous centrifugation). Alternatively, separation, purification and enrichment can be performed in a more specific and selective manner (e.g., using producer cell-specific surface markers). For example, specific surface markers can be used for immunoprecipitation, FACS sorting, affinity purification, bead-bound ligands for magnetic separation, etc.

[0163] In some aspects, size exclusion chromatography can be used to separate or purify EVs (e.g., exosomes). Size exclusion chromatography techniques are known in the art. Exemplary non-limiting techniques are provided herein. In some aspects, the void volume fraction is separated and contains the EVs of interest (e.g., exosomes). In some aspects, for example, density gradient centrifugation can be used to further separate EVs (e.g., exosomes). Still further, in some aspects, it may be desirable to further separate producer cell-derived EVs (e.g., exosomes) from EVs from other sources. For example, by using an antigen-antibody specific to producer cells for immunoabsorption capture, producer cell-derived EVs (e.g., exosomes) can be separated from non-producer cell-derived EVs (e.g., exosomes).

[0164] In some aspects, the separation of EVs (e.g., exosomes) may involve size exclusion chromatography or ion chromatography, such as anion exchange, cation exchange, or mixed mode chromatography. In some aspects, the separation of EVs (e.g., exosomes) may involve desalting, dialysis, tangential flow filtration, ultrafiltration, or diafiltration, or any combination thereof. In some aspects, the separation of EVs (e.g., exosomes) may involve a combination of methods, including but not limited to differential centrifugation, size-based membrane filtration, concentration, and / or rate zonal centrifugation. In some aspects, the separation of EVs (e.g., exosomes) may involve one or more centrifugation steps. Centrifugation may be performed at about 50,000 to 150,000 xg. Centrifugation may be performed at about 50,000 xg, 75,000 xg, 100,000 xg, 125,000 xg, or 150,000 xg.

[0165] IV. Therapeutic Administration

[0166] IV.A. Methods of treating cancer

[0167] Provided herein are methods for treating cancer in a subject. The method comprises 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. In some aspects, the IL-12 portion (e.g., polypeptide) is administered intratumorally to the subject. In some aspects, the IL-12 portion (e.g., polypeptide) is administered parenterally, orally, intravenously, intramuscularly, intraperitoneally, or by any other suitable route of administration.

[0168] Also provided herein is a method for preventing cancer metastasis in a subject. The method comprises administering a dose of an IL-12 polypeptide to the subject, wherein the amount of the IL-12 polypeptide in the dose is about 0.1 μg to about 30 μg. In some aspects, an IL-12 portion (e.g., a polypeptide) is administered intratumorally in a first tumor in one location, and administration of an IL-12 portion (e.g., a polypeptide) in the first tumor prevents metastasis of one or more tumors at a second location.

[0169] In some aspects, administration of an IL-12 portion (e.g., polypeptide) disclosed herein inhibits and / or reduces tumor growth in a subject. In some aspects, tumor growth (e.g., tumor volume or weight) is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., a tumor volume of a corresponding subject after administration of an EV (e.g., exosome) without an IL-12 portion).

[0170] In some aspects, the cancer being treated is characterized by infiltration of leukocytes (T cells, B cells, macrophages, dendritic cells, monocytes) into the tumor microenvironment, or so-called "hot tumors" or "inflammatory tumors". In some aspects, the cancer being treated is characterized by infiltration of leukocytes into the tumor microenvironment, or low or undetectable levels in so-called "cold tumors" or "non-inflammatory tumors". In some aspects, the amount and time of EV (e.g., exosomes) administered are sufficient to convert "cold tumors" into "hot tumors", that is, the administration causes leukocytes (such as T cells) to infiltrate into the tumor microenvironment. In some aspects, cancer comprises bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer and ovarian, lymphoma, liver cancer, glioblastoma, melanoma, myeloma, leukemia, pancreatic cancer or a combination thereof. As used herein, the term "distant tumor", "distant tumor" or "secondary tumor" refers to a tumor that spreads from the original (or primary) tumor to a distant organ or distant tissue (e.g., lymph node). In some aspects, the EVs (e.g., exosomes) disclosed herein treat tumors after metastatic spread.

[0171] Non-limiting examples of cancers (or tumors) that can be treated with the methods disclosed herein include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, pancreatic cancer, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer (or carcinomas). , germ cell tumors, pediatric sarcomas, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer (e.g., choriocarcinoma and non-seminoma), fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer (e.g., gastroesophageal junction cancer), small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, ureteral cancer, renal pelvis cancer, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma (Kaposi's sarcoma), epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including cancers induced by asbestos), virus-related cancers or cancers of viral origin (e.g., human papillomavirus (HPV-related or HPV-derived tumors)), and hematological malignancies arising from one of the two major blood cell lineages, the myeloid cell lineage (which produces granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid cell lineage (which produces B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, for example, acute, chronic, lymphocytic and / or myeloid leukemias, such as acute leukemia (A LL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; in which the cells are mature), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas, such as Hodgkin's lymphoma (Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1 +) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), lymphoid lineage hematopoietic neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma (Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTCL) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma myeloma) (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; myeloid hematopoietic tumors, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including but not limited to T-cell disorders, such as T-prolymphocytic leukemia (T-PLL), including small cell and cerebriform cell types; large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma and any combination thereof.

[0172] In some aspects, the cancer (or tumor) that can be treated includes breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, peritoneal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma or its combination. In some aspects, the cancer that can be treated with the disclosure is pancreatic cancer and / or peritoneal cancer. In some aspects, the cancer (or tumor) that can be treated includes bladder cancer. In some aspects, the composition is delivered to the subject by intravesical administration, wherein the subject suffers from bladder cancer.

[0173] In some aspects, the methods described herein can also be used to treat metastatic cancer, unresectable refractory cancer (eg, cancer that is refractory to previous cancer therapy), and / or recurrent cancer.

[0174] In some aspects, the EVs (e.g., exosomes) disclosed herein can be used in combination with one or more additional anticancer and / or immunomodulatory agents. Such agents can include, for example, chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some aspects, the methods described herein are used in combination with standard of care treatments (e.g., surgery, radiation, and chemotherapy).

[0175] Some aspects of the present disclosure relate to methods of administering (i) a dose of an IL-12 polypeptide and (ii) an additional therapy to a subject in need thereof, wherein the amount of the IL-12 polypeptide in the dose is about 0.1 μg to about 30 μg. In some aspects, the additional therapy is an additional anticancer agent and / or immunomodulator. Such agents may include, for example, chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some aspects, the methods described herein are used in combination with standard of care treatments (e.g., surgery, radiation, and chemotherapy).

[0176] In some aspects, the IL-12 moieties disclosed herein, e.g., associated with EVs (e.g., exosomes), can be used in combination with one or more additional therapeutic agents (e.g., immuno-oncology agents) so that multiple elements of the immune pathway can be targeted. Non-limiting examples of such combinations include: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, cellular vaccines that secrete GM-CSF, CpG oligonucleotides, imiquimod); therapies that inhibit negative immunoregulation, e.g., by inhibiting CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Tregs or other immunosuppressive cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immunoregulation, e.g., using agonists that stimulate CD-137, OX-40 and / or CD40 or GITR pathways and / or stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells; therapies that deplete or inhibit Tregs (e.g., Tre in tumors); g), for example, using antagonists of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect suppressive myeloid cell function in the tumor; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T-cell or NK-cell transfer comprising genetically modified cells, e.g., cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenases, arginase, or nitric oxide synthase; therapies that reverse / prevent T-cell anergy or exhaustion; therapies that trigger innate immune activation and / or inflammation at the tumor site; administration of immunostimulatory cytokines; or blockade of immunosuppressive cytokines.

[0177] In some aspects, additional anticancer agents include immune checkpoint inhibitors (i.e., blocking signal transduction through specific immune checkpoint pathways). Non-limiting examples of immune checkpoint inhibitors that can be used for the methods of the present invention include CTLA-4 antagonists (e.g., anti-CTLA-4 antibodies), PD-1 antagonists (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies), TIM-3 antagonists (e.g., anti-TIM-3 antibodies) or combinations thereof.

[0178] In some aspects, the immuno-oncology agent comprises an immune checkpoint activator (ie, promoting signal transduction through a specific immune checkpoint pathway). In certain aspects, the immune checkpoint activator comprises an OX40 agonist (eg, an anti-OX40 antibody), a LAG-3 agonist (eg, an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (eg, an anti-CD137 antibody), a GITR agonist (eg, an anti-GITR antibody), or any combination thereof.

[0179] In some aspects, the additional anticancer agent comprises a standard of care chemotherapy. In some aspects, the standard of care chemotherapy comprises a platinum-based chemotherapy. In some aspects, the standard of care chemotherapy comprises a platinum-based dual chemotherapy.

[0180] In some aspects, other anticancer agents are standard of care therapies for treating colorectal cancer. In some aspects, other anticancer agents include 5-fluorouracil (FU), leucovorin (LV), oxaliplatin (oxaliplatin), irinotecan (irinotecan), anti-vascular endothelial growth factor (VEGF) antibodies, anti-epidermal growth factor receptor (EGFR) antibodies or any combination thereof. In some aspects, other anticancer agents include FU, LV and oxaliplatin (FOLFOX). In some aspects, other anticancer agents include FU, LV and irinotecan (FOLFIRI). In some aspects, the subject suffers from colorectal cancer with high MSI. In some aspects, the subject suffers from colorectal cancer presenting one or more, for example, at position Gly12, Gly13, Glu61 or any combination thereof KRAS mutations. In some aspects, the subject suffers from colorectal cancer presenting one or more BRAF mutations (e.g., BRAFV600E). 。

[0181] In some aspects, other anticancer agents are standard of care therapies for treating HCC. In some aspects, other anticancer agents include sorafenib. In some aspects, other anticancer agents include lenvatinib. In some aspects, other anticancer agents include atezolizumab and bevacizumab. In some aspects, other anticancer agents include pembrolizumab, nivolumab, ipilimumab or a combination thereof. In some aspects, other anticancer agents include FU, LV and oxaliplatin (FOLFOX). In some aspects, other anticancer agents include regorafenib, cabozantinib, ramucirumab or any combination thereof.

[0182] In some aspects, other anticancer agents are standard of care therapies for treating colorectal cancer. In some aspects, other anticancer agents include (i) epirubicin, cisplatin and 5-FU ("ECF"); (ii) or epirubicin, cisplatin and capecitabine (capecitabine) ("ECX"); or (iii) docetaxel, oxaliplatin and 5-FU / folinic acid ("FLOT"). In some aspects, other anticancer agents include HER2 targeting agents.

[0183] In some aspects, the combination of, for example, an IL-12 portion associated with an EV (e.g., exosome) and a second agent discussed herein (e.g., an immune checkpoint inhibitor) disclosed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other aspects, the combination of, for example, an IL-12 portion associated with an EV (e.g., exosome) and a second agent discussed herein (e.g., an immune checkpoint inhibitor) can be administered simultaneously as a separate composition. In other aspects, the combination of, for example, an IL-12 portion associated with an EV (e.g., exosome) and a second agent discussed herein (e.g., an immune checkpoint inhibitor) can be administered sequentially. In some aspects, the IL-12 portion associated with an EV (e.g., exosome) is administered prior to administering the second agent (e.g., an immune checkpoint inhibitor).

[0184] IV.A.1. Cutaneous T-cell lymphoma

[0185] In some aspects of the present disclosure, cancers (tumors) that can be treated by the methods disclosed herein include cutaneous T-cell lymphoma (CTCL). As used herein, CTCL refers to a type of non-Hodgkin T-cell lymphoma that affects the skin. There are several subtypes of CTCL, including but not limited to mycosis fungoides (MF), Sézary syndrome (SS), and a series of T-cell tumors known as CD30-positive lymphoproliferative disorders. Therefore, in some aspects, the methods disclosed herein treat MF in a subject in need thereof. In some aspects, the methods disclosed herein treat SS in a subject in need thereof. In some aspects, the methods disclosed herein treat CD30-positive lymphoproliferative disorders.

[0186] In some aspects, CTCL comprises an aggressive phenotype. In some aspects, CTCL comprises an indolent, i.e., slow-growing, phenotype.

[0187] CTCL has four main stages, such as MF, SS and / or CD30-positive lymphoproliferative disorders. Phase 1 CTCL is characterized by a phenotype that affects only the skin, presenting as plaques or plaques. During phase 1A, less than one-tenth of the skin is affected; during phase 1B, one-tenth or more of the skin is affected. Phase 2 CTCL is characterized by the appearance of plaques or plaques and lymphadenopathy (which lacks abnormal lymphoma cells) on the skin (phase 2A); or one or more raised lumps or tumors are presented on the skin, with or without lymphadenopathy (phase 2B). Phase 3 CTCL is characterized by the appearance of plaques or plaques on four-fifths or more of the skin, accompanied by general redness, swelling, itching, and sometimes pain (erythroderma); lymphadenopathy (which lacks abnormal lymphoma cells); few or no lymphoma cells in the bloodstream (erythrodermic mycosis fungoides; phase 3A) or a moderate number of lymphoma cells in the bloodstream (Sezary syndrome; phase 3B). Stage 4 CTCL affects the skin and has also spread to the bloodstream, lymph nodes, or other organs. Stage 4A means there are large numbers of abnormal lymphoma cells in the bloodstream (Sézary syndrome) or lymph nodes; Stage 4B means the lymphoma has spread to other organs.

[0188] In some aspects, the methods disclosed herein treat stage 1A CTCL. In some aspects, the methods disclosed herein treat stage 1B CTCL. In some aspects, the methods disclosed herein treat stage 2A CTCL. In some aspects, the methods disclosed herein treat stage 2B CTCL. In some aspects, the methods disclosed herein treat stage 3A CTCL. In some aspects, the methods disclosed herein treat stage 3B CTCL. In some aspects, the methods disclosed herein treat stage 4A CTCL. In some aspects, the methods disclosed herein treat stage 4B CTCL.

[0189] IV.A.2. Merkel Cell Carcinoma

[0190] In some aspects of the present disclosure, cancers (tumors) that can be treated by the methods disclosed herein include Merkel cell carcinoma (MCC). As used herein, MCC or cutaneous neuroendocrine carcinoma refers to a rare type of skin cancer characterized by flesh-colored or bluish-red nodules that usually appear on the face, head, or neck. MCC is typically an aggressive form of skin cancer with a high risk of recurrence and metastasis.

[0191] Standard of care treatments for MCC include radiation therapy, surgery, systemic therapy or chemotherapy, or a combination thereof. In addition, antibodies that inhibit the interaction of programmed death 1 (PD-1) and its ligand (PD-L1) have been approved for the treatment of MCC, including avelumab.

[0192] Stage 0 MCC refers to the presence of abnormal Merkel cells on the surface layer of the skin. Stage 1 MCC refers to a tumor that is 2 cm or less in diameter. Stage 2 MCC refers to a tumor that is larger than 2 cm (stage 2A) or has spread to nearby connective tissue, muscle, cartilage, or bone (stage 2B). Stage 3A MCC refers to a tumor of any size that (i) has spread to nearby connective tissue, muscle, cartilage, or bone, and in which cancer is found in the lymph nodes; or (ii) spread to the lymph nodes can be observed by physical examination and confirmed by biopsy. Stage 3B MCC refers to a tumor of any size that has (i) spread to nearby connective tissue, muscle, cartilage, or bone, and in which cancer is found in the lymph nodes; or (ii) spread to the lymphatic vessels between the primary tumor and the proximal and / or distal lymph nodes. Stage IV MCC refers to cancer that has metastasized to a distant location on the skin or to other organs, such as the liver, lungs, bones, or brain.

[0193] In some aspects, the methods disclosed herein treat stage 0 MCC. In some aspects, the methods disclosed herein treat stage 1 MCC. In some aspects, the methods disclosed herein treat stage 2A MCC. In some aspects, the methods disclosed herein treat stage 2B MCC. In some aspects, the methods disclosed herein treat stage 3A MCC. In some aspects, the methods disclosed herein treat stage 3B MCC. In some aspects, the methods disclosed herein treat stage 4 MCC. In some aspects, the methods disclosed herein treat recurrent MCC.

[0194] IV.A.3. Kaposi sarcoma

[0195] In some aspects of the present disclosure, cancers (tumors) that can be treated by the methods disclosed herein include Kaposi's sarcoma. As used herein, Kaposi's sarcoma refers to a cancer that develops from cells located in lymphatic or blood vessels, and the cancer usually manifests as tumors (purple, red or brown spots) on the skin, on mucosal surfaces (such as the inside of the mouth), or less frequently in other parts of the body (including lymph nodes).

[0196] There are at least four types of Kaposi sarcoma: epidemic Kaposi sarcoma (which occurs in people infected with HIV), classical Kaposi sarcoma, endemic Kaposi sarcoma, and iatrogenic (transplant-associated) Kaposi sarcoma. Often, Kaposi sarcoma is associated with a weakened immune system, for example, in malnourished or elderly patients or those with chronic viral infections (e.g., HIV or HSV).

[0197] Kaposi's sarcoma is often treated by immune reconstitution, especially for HIV patients. Chemotherapy may also be administered, including but not limited to doxorubicin, daunorubicine, paclitaxel, and combinations thereof.

[0198] IV.A.4. Triple-negative breast cancer

[0199] In some aspects of the present disclosure, the cancer (tumor) that can be treated by the methods disclosed herein includes triple negative breast cancer (TNBC). As used herein, TNBC refers to breast cancer characterized by the expression of estrogen receptors (ER negative), progesterone receptors (PR negative) and HER2 (HER2 negative) as test negative tumors. TNBC is considered to be an invasive cancer that grows rapidly, is more likely to have spread at the time of diagnosis, and has a higher recurrence rate than other forms of cancer after therapy. Therefore, the survival rate of TNBC is lower than that of other breast cancers, with a 5-year survival rate of about 91% for local TNBC, about 65% for regional TNBC, and about 11% for distant TNBC.

[0200] Since TNBC lacks expression of ER, PR and HER2, the treatments available for TNBC are limited. For non-metastatic TNBC, excision surgery after chemotherapy is the main treatment option. Metastatic TNBC is usually treated with chemotherapy (e.g., PARP inhibitors, platinum-based chemotherapy and / or immunotherapy).

[0201] Phase 0 TNBC refers to non-invasive breast cancer, such as DCIS (ductal carcinoma in situ). In Phase 0, there is no evidence that cancer cells or non-cancerous abnormal cells break from the part of the breast where they start, or pass through or invade adjacent normal tissue. Phase I TNBC refers to invasive breast cancer (cancer cells are observed to break through or invade normal surrounding breast tissue). Phase 1A refers to invasive breast cancer, in which the tumor measures up to 2 centimeters (cm), and the cancer does not spread to the outside of the breast (for example, lymph nodes are not affected). Phase 1B refers to invasive breast cancer, in which (i) small cancer cell groups (diameter is 0.2mm to 2mm) are found in the lymph nodes, but no tumor is presented in the breast, or (ii) a tumor not greater than 2cm is presented in the breast, and there are small cancer cell groups (diameter is 0.2mm to 2mm) in the lymph nodes. In Phase 1 breast cancer, microscopic infiltration is possible. In microscopic infiltration, cancer cells have just begun to infiltrate the tissue outside the lining of the duct or lobule, but the infiltrated cancer cell measurement does not exceed 1mm. For HER2-negative breast cancer (e.g., TNBC), a tumor that is larger than 2 cm but not larger than 5 cm and has not spread to the lymph nodes is considered stage 1. Stage 2A refers to breast cancer in which (i) no tumor is found in the breast, but cancer (larger than 2 mm) is found in one to three axillary lymph nodes or in lymph nodes near the breastbone; or (ii) the tumor measurement is 2 cm or less and has spread to the axillary lymph nodes. Stage 2B refers to invasive breast cancer in which (i) the tumor size is 2 cm to 5 cm, with small groups of breast cancer cells (size 0.2 to 2 mm) present in the lymph nodes; (ii) the tumor size is 2 cm to 5 cm; and metastases to one to three axillary lymph nodes or lymph nodes near the breastbone; or (iii) the tumor is larger than 5 cm, but has not spread to the axillary lymph nodes. Stage 3A refers to invasive breast cancer in which (i) no tumor is found in the breast or the tumor can be any size and has spread to four to nine axillary lymph nodes or lymph nodes near the breastbone; (ii) the tumor is larger than 5 cm and small groups of breast cancer cells (0.2 mm to 2 mm in size) are found in the lymph nodes; or (iii) the tumor is larger than 5 cm and the cancer has spread to one to three axillary lymph nodes or lymph nodes near the breastbone. Stage 3B describes invasive breast cancer in which the tumor is any size and has spread to the chest wall and / or breast skin, causing swelling or ulceration, and the tumor has spread to (i) up to nine axillary lymph nodes or (ii) lymph nodes near the breastbone.Stage 3C refers to invasive breast cancer in which there may be no signs of cancer in the breast, or if there is a tumor, it may be any size and may have spread to the chest wall and / or skin of the breast, and the cancer has spread to (i) ten or more axillary lymph nodes, (ii) lymph nodes above or below the collarbone, or (iii) lymph nodes near the breastbone. Stage 4 or advanced breast cancer refers to invasive breast cancer that has spread beyond the breast and nearby lymph nodes to other organs of the body, such as the lungs, distant lymph nodes, skin, bones, liver, or brain.

[0202] In some aspects, the methods disclosed herein treat stage 0 TNBC. In some aspects, the methods disclosed herein treat stage 1A TNBC. In some aspects, the methods disclosed herein treat stage 1B TNBC. In some aspects, the methods disclosed herein treat stage 2A TNBC. In some aspects, the methods disclosed herein treat stage 2B TNBC. In some aspects, the methods disclosed herein treat stage 3A TNBC. In some aspects, the methods disclosed herein treat stage 3B TNBC. In some aspects, the methods disclosed herein treat stage 3C TNBC. In some aspects, the methods disclosed herein treat stage 4 TNBC. In some aspects, the methods disclosed herein treat recurrent TNBC.

[0203] IV.A.5. Glioblastoma

[0204] In some aspects of the present disclosure, the cancer (tumor) that can be treated by the methods disclosed herein comprises a glioblastoma. As used herein, a glioblastoma is also referred to as "glioblastoma multiforme" or "GBM", and refers to a type of cancer that is a stage IV astrocytoma. Glioblastoma has several variants, including giant cell glioblastoma, gliosarcoma, classic glioblastoma, neuroblastoma, proglioblastoma, and mesenchymal glioblastoma. Glioblastoma can be primary (de novo) or secondary.

[0205] There is currently no cure for glioblastoma. The standard of care treatment for glioblastoma is surgical resection, followed by radiation therapy and chemotherapy (e.g., with temozolomide). Bevacizumab can also be used as a second-line therapy for glioblastoma. The median survival after diagnosis is 15-18 months, and the five-year survival rate for the disease is about 10%. Although all glioblastomas recur, initial treatment may keep the tumor under control for several months or even years.

[0206] Examples

[0207] Example 1: Administration of exosomes containing surface-displayed IL-12 in a mouse cancer model

[0208] Exosomes with surface displayed IL-12 were prepared by expressing a fusion construct in exosome-producing cells, comprising a single peptide IL-12 linked to PTGFRN (p35 linked via a peptide linker p40) ( Figure 1 ). Efficacy was evaluated in vitro using human PBMC or murine splenocytes and in vivo using a mouse subcutaneous tumor model. Local versus systemic pharmacology was determined by intratumoral injection in mice and subcutaneous injection in monkeys. All studies were based on recombinant IL-12 (rIL-12).

[0209] In the MC38 mouse tumor model, intratumoral administration of exosomes with surface displayed IL-12 (exoIL-12) showed enhanced PK and sustained PD compared to free recombinant IL-12 and untreated controls. Mice administered exoIL-12 showed increased tumor retention (approximately 15-fold) at 3, 12, 24, and 48 hours post-administration compared to free recombinant IL-12, as measured by IL-12p70 concentration / tumor ( Figure 2A In addition, exoIL-12 administration resulted in an approximately 4-fold increase in the intratumoral IFN-γ AUC compared with recombinant IL-12 ( Figure 2B ). In addition, intratumoral administration of exoIL-12 resulted in a dose-dependent reduction in MC38 tumor growth in mice. In terms of tumor growth inhibition, exoIL-12 was 100 times more potent than rIL-12, with mice receiving 100ng exoIL-12 showing little or no tumor growth (Figure 2C). In the MC38 tumor model, a 63% complete response was observed in mice treated with exoIL-12; in contrast, rIL-12 caused a 0% complete response at an equivalent IL-12 dose. This was associated with a dose-dependent increase in tumor antigen-specific CD8+ T cells, which increased nearly 4-fold in mice treated with exoIL-12 (Figure 2E).

[0210] After MC38 re-challenge, inhibition of tumor growth was observed again (Figure 2D). Re-challenge studies in mice with complete exoIL-12 response showed no tumor regeneration, and depletion of CD8+T cells completely eliminated the anti-tumor activity of exoIL-12. After intratumoral administration, exoIL-12 exhibited 10 times the intratumoral exposure of rIL-12 and prolonged IFNγ production for up to 48 hours. The retained local pharmacology of exoIL-12 was further confirmed using subcutaneous injection in non-human primates.

[0211] Toxicological analysis revealed that the highest dose tested (3 μg exoIL-12) was the NOAEL (no observable adverse effect level), showing limited plasma levels and dose-dependent tissue levels ( Figure 3A ). CXCL10 / IP-10 expression was observed to persist in the skin after a single dose but was undetectable in plasma ( Figure 3B-3C ).

[0212] The tumor-restricted pharmacology of exoIL-12 elicits superior in vivo efficacy and immune memory without systemic IL-12 exposure and associated toxicity. Thus, exoIL-12 overcomes key limitations of rIL-12.

[0213] Example 2: Clinical trial investigating the safety and efficacy of administration of exosomes containing surface displayed IL-12

[0214] A clinical study is underway to test the safety and efficacy of administering engineered exosomes containing surface-displayed IL-12 to treat cancer in human subjects ( Figures 4A-4B ). In part A, different doses of exoIL-12 will be administered to healthy volunteers, and adverse events and biomarkers will be monitored (Figure 9A). Patients will be administered exoIL-12 at doses of 0.3μg, 1.0μg, 3.0μg, 6.0μg, and 12.0μg of IL-12 polypeptide. In part B, different doses of exoIL-12 will be administered to subjects diagnosed with CTCL (stage IA-IIB), and safety and biomarkers will be monitored ( Figure 4B ). Clinical activity will be monitored with one or more CT scans. Subjects with CTCL, TNBC, melanoma, GBM, MCC, and / or Kaposi's sarcoma will be eligible to participate in Part B of the trial.

[0215] In the CTCL portion of the study, two patients were treated, each of whom received twenty doses of exoIL-12 (6.0 μg of IL-12 polypeptide) across multiple lesions. The duration of treatment was greater than six months, and no grade 3 or higher treatment-emergent adverse events or SAEs were observed, and no dose adjustment was required. Notably, no chills, fever, fatigue, dizziness, myalgia, headache, or back pain were reported. These symptoms have been observed in previous clinical studies of subcutaneous administration of rIL-12, where the doses (ranging from 2 to 12 μg) were comparable to those used in the current study of exoIL-12].

[0216] exoIL-12 demonstrated overall disease improvement with a range of 20-60% regression in all injected and non-injected lesions, making patient 001 a partial response according to the CAIL score as an enrollment endpoint. Patient 001 received 17+ injections across 3 lesions: all lesions have resolved and no additional injections are deemed necessary by the treating physician. Patient 002 received 16+ injections across 2 lesions and is on study and continuing to receive injections.

[0217] Plasma pharmacokinetic (PK) measurements in both healthy volunteers and patients treated with exoIL-12 showed no systemic exposure when IL-12 levels were below the limit of quantification. In contrast, previous clinical studies of rIL-12 showed dose-dependent systemic exposure at doses of 5 and 12 μg resulting in C max Plasma levels are approximately 15 to 45 pg / ml.

[0218] Example 3: Measurement of IL-12

[0219] With exosome payload (IL-12 is endogenously loaded by HEK293 producer cell line)

[0220] Free and total IL-12 determined by αLISA

[0221] αLISA ("Amplified Luminescent Proximity Homogeneous Assay" Ligated Immunosorbent Assay) technology is commonly used to measure protein-protein interactions or the presence of protein analytes in a homogeneous, no-wash format. In this assay, acceptor beads conjugated to anti-IL-12 antibodies are mixed with a solution of biotinylated anti-IL-12 antibodies and streptavidin-coated donor beads. The presence of IL-12 in the CB-102 drug substance sample brings the beads into proximity through the interaction of each antibody with IL-12. Excitation of the donor beads at 680 nm causes singlet oxygen to be transferred to the acceptor beads, and subsequently fluoresces at 615 nm. The size of the α signal at 615 nm is directly related to the concentration of IL-12 in the sample being tested.

[0222] The sample signal was measured relative to a standard curve of recombinant human IL-12 to determine the IL-12 content in the CB-102 sample. The CB-102 drug substance sample was diluted to 5 × 10 10 The total IL-12 content was measured directly or by centrifugation through a 96-well Agilent 300 kDa filter plate to measure the free IL-12 content in the flow-through.

[0223] Incorporated by Reference

[0224] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0225] Equivalent

[0226] The present disclosure provides, among other things, compositions of exosomes encapsulating STING agonists for use as therapeutic agents. The present disclosure also provides methods of producing exosomes encapsulating STING agonists and methods of administering such exosomes as therapeutic agents. Although various specific aspects have been illustrated and described, the above description is not limiting. It should be understood that various changes can be made without departing from the spirit and scope of the present invention. Many changes will become apparent to those skilled in the art after reading this specification.

Claims

1. A method for preventing or treating a tumor in a subject in need thereof, the method 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 via extracellular vesicles.

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. 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, to 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 about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every six weeks, or about once 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 according to any one of claims 1 to 12, wherein the extracellular vesicle comprises a scaffold portion.

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 shown 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 via a peptide bond.

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

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

23. The method of any one of claims 1 to 22, wherein the IL-12 is located 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 according to any one of claims 13 to 24, wherein the IL-12 polypeptide is linked to the C-terminus of the PTGFRN protein or the portion thereof.

26. The method of any one of claims 1 to 25, wherein the amount of IL-12 polypeptide is measured by fluorimetry, IL-12α LISA, 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 administration reduces the volume of the tumor.

29. The method of any one of claims 1 to 28, wherein the administration 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 volume of the tumor following administration of the IL-12 polypeptide in the absence of extracellular vesicles.

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

31. The method of claim 30, wherein the administration 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 monotherapy after day 14 of the administration.

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

33. The method of any one of claims 1 to 32, wherein the administration 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 following administration of extracellular vesicles comprising a 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 reencapsulated vesicle, or an extruded cell.

39. The method of any one of claims 2 to 38, wherein the EVs are exosomes.

40. The method of any one of claims 2 to 39, wherein the extracellular vesicles are produced by cells that overexpress 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.

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