Immunoexosomes and methods of use thereof

Abstract

To provide compositions for treating diseases requiring immunomodulation, such as cancer, autoimmune disease or infectious disease, and methods of using such compositions for treating diseases.SOLUTION: A composition comprises exosomes, where the exosomes comprise a payload on their surface, where the payload is an immunomodulatory molecule. The immunomodulatory molecule includes ICOSL or OX40L, for example.SELECTED DRAWING: None

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 641,523, filed March 12, 2018, the entire contents of which are incorporated herein by reference.

[0002] 1. Field The present invention relates generally to the fields of biology, medicine, oncology, and immunology. More particularly, the present invention relates to immunomodulatory exosomes and their therapeutic uses. [Background technology]

[0003] 2. Description of Related Technology Extracellular vesicles (EVs), including exosomes, are nano-sized intracellular communication vehicles containing DNA, RNA, and proteins and are involved in several physiological processes. Many surface proteins on exosomes have been identified, with varying frequency of occurrence, but they are primarily not immunomodulatory. Dendritic cell-derived exosomes have been found to have mild immunomodulatory activity, but T cell responses are minimal. Exosomes isolated from epithelial and mesenchymal cells are generally not immunomodulatory, but they efficiently bind and enter other cells. Therefore, there is a need to develop exosome-based immunomodulatory drugs with the specific ability to activate T cells. Summary of the Invention

[0004] overview Thus, provided herein is an exosome having an immunomodulatory molecule, such as ICOSL and / or OX40L, on its surface. In one embodiment, provided herein is a composition comprising an exosome, wherein the exosome comprises a payload on its surface, and the payload is an immunomodulatory molecule. In some aspects, the immunomodulatory molecule is CD80, CD86, PD-L1, PD-L2, HVEM, GAL9, CTLA-4, PD-1, PD-1H, CD160, BTLA, TIM3, KIR, LAG3, A2aR, OX40L, CD27L, CD137L, BAFF, APRIL, CD70, CD40, B7H3, ICOSL, OX40, CD40L, BMCA, TACI, GITR, BAFFR, CD27, CD137, ICOS, and / or CD28. In some aspects, the exosome comprises OX40L on its surface. In some aspects, the exosomes contain ICOSL on their surface. In various aspects, the exosomes further contain CD47 on their surface. In some aspects, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the exosomes contain immunomodulatory molecules on their surface. In certain aspects, the exosomes are isolated from cells that overexpress immunomodulatory molecules. In some aspects, the exosomes are isolated from patients in need of treatment.

[0005] In some aspects, the exosomes further comprise a therapeutic agent as an intravesicular payload. In various aspects, the therapeutic agent is a therapeutic protein, an antibody (e.g., a full-length antibody, a monoclonal antibody, an scFv, a Fab fragment, an F(ab')2, a diabody, a triabody, or a minibody), an inhibitory RNA, a CRISPR system, or a small molecule drug. In some aspects, the therapeutic protein is a protein whose loss or inactivation is known to be associated with the disease to be treated, such as a tumor suppressor, a kinase, a phosphatase, or a transcription factor. In some aspects, the antibody binds to an intracellular antigen. Such an intracellular antigen may be a protein whose activity is necessary for cell growth and / or survival, such as an oncogene. In some cases, the antibody blocks the function of the antigen. In some cases, the antibody disrupts a protein-protein interaction. In some aspects, the inhibitory RNA is an siRNA, shRNA, miRNA, or pre-miRNA. In various aspects, the inhibitory RNA blocks the expression of a protein, such as an oncogene, whose activity is necessary for maintaining a particular disease state. If the oncogene is a mutated form of a gene, the inhibitory RNA may preferentially block expression of the mutated oncogene and not the wild-type protein. In some aspects, the CRISPR system includes a guide RNA and an endonuclease, such as a Cas endonuclease. In some aspects, the small molecule drug is an imaging agent. In some aspects, the small molecule drug is a chemotherapeutic agent.

[0006] In one embodiment, a pharmaceutical composition is provided comprising the exosome of any one of the embodiments of the present invention and an excipient. In some aspects, the composition is formulated for parenteral administration. In some aspects, the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. In some aspects, the composition further comprises an antibacterial agent. In some aspects, the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

[0007] In one aspect, a method of treating a disease in a patient in need thereof is provided, comprising administering to the patient a composition according to any one of the aspects of the present invention, thereby treating the disease in the patient. In some aspects, the administration causes immunomodulation in the patient. In some aspects, the disease is an immune disease, cancer, an infectious disease, or an autoimmune disease. In some aspects, the disease is cancer. In some aspects, the administration is systemic administration. In certain aspects, the systemic administration is intravenous administration. In some aspects, the method further comprises administering at least a second therapy to the patient. In certain aspects, the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, immune checkpoint blockade, or cytokine therapy. In some aspects, the second anti-cancer therapy comprises adoptive T cell therapy, anti-PD1 antibody, anti-CTLA-4 antibody, and / or anti-PD-L1 antibody. In some aspects, the patient is human. In some aspects, the exosomes are autologous to the patient.

[0008] In one aspect, a method for treating a disease in a patient in need thereof is provided, comprising electroporating liposomes or exosomes with a therapeutic agent (e.g., a protein payload) and providing the electroporated liposomes or exosomes to the patient, thereby treating the disease in the patient. In some aspects, the liposomes or exosomes comprise an immunomodulatory molecule on their surface. In some aspects, the disease is an immune disease, cancer, an infectious disease, or an autoimmune disease. In some aspects, the disease is cancer. In some aspects, the protein payload is a monoclonal antibody that specifically or selectively binds to an intracellular antigen.

[0009] In one aspect, a method is provided for administering a therapeutic protein to a patient in need thereof, the method comprising transfecting nucleic acid (e.g., DNA or RNA) encoding the therapeutic protein (e.g., a monoclonal antibody or antigen-binding fragment thereof) into exosomes comprising an immunomodulatory molecule on their surface, incubating the transfected exosomes under conditions that allow expression of the therapeutic protein within the exosomes, and providing the incubated exosomes to the patient, thereby administering the therapeutic protein to the patient.

[0010] In one embodiment, a composition comprising exosomes for use in treating a disease in a patient is provided, wherein the exosomes comprise a payload on their surface, and the payload is an immunomodulatory molecule. In some aspects, the immunomodulatory molecule is CD86, PD-L1, PD-L2, HVEM, GAL9, CTLA-4, PD-1, PD-1H, CD160, CD80, BTLA, TIM3, KIR, LAG3, A2aR, OX40L, CD27L, CD137L, BAFF, APRIL, CD70, CD40, B7H3, ICOSL, OX40, CD40L, BMCA, TACI, GITR, BAFFR, CD27, CD137, ICOS, and / or CD28. In some aspects, the exosomes comprise OX40L on their surface. In some aspects, the exosomes comprise ICOSL on their surface. In various aspects, the exosomes further comprise CD47 on their surface. In some aspects, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the exosomes comprise an immunomodulatory molecule on their surface. In some aspects, the exosomes further comprise an intravesicular protein payload. In some aspects, the disease is an immune disease, cancer, an infectious disease, or an autoimmune disease. In some aspects, the disease is cancer. In some aspects, the composition is formulated for parenteral or systemic administration. In some aspects, the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. In some aspects, the composition further comprises an antibacterial agent. In certain aspects, the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidin, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.In some aspects, the composition further comprises at least a second therapy.In certain aspects, the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy.In some aspects, the patient is human.In some aspects, the exosomes are autologous to the patient.

[0011] In one embodiment, the present invention provides a use of exosomes in the manufacture of a medicament for treating a disease, wherein the exosomes comprise a payload on their surface, and the payload is an immunomodulatory molecule. In some aspects, the immunomodulatory molecule is CD86, PD-L1, PD-L2, HVEM, GAL9, CTLA-4, PD-1, PD-1H, CD160, CD80, BTLA, TIM3, KIR, LAG3, A2aR, OX40L, CD27L, CD137L, BAFF, APRIL, CD70, CD40, B7H3, ICOSL, OX40, CD40L, BMCA, TACI, GITR, BAFFR, CD27, CD137, ICOS, and / or CD28. In some aspects, the exosomes comprise OX40L on their surface. In some aspects, the exosomes comprise ICOSL on their surface. In various aspects, the exosomes further comprise CD47 on their surface. In some aspects, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the exosomes comprise immunomodulatory molecules on their surface. In some aspects, the exosomes further comprise an intravesicular protein payload. In some aspects, the disease is an immune disease, cancer, an infectious disease, or an autoimmune disease. In some aspects, the disease is cancer. In some aspects, the composition is formulated for parenteral or systemic administration. In some aspects, the composition is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. In some aspects, the composition further comprises an antibacterial agent. In certain aspects, the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

[0012] As used herein, "essentially free" is used herein to mean that, in terms of the named components, none of the named components are intentionally formulated into the composition and / or are present merely as contaminants or in trace amounts. Thus, the total amount of the named components resulting from unintentional contamination of the composition is significantly less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the named components is undetectable using standard analytical methods.

[0013] As used herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0014] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or to refer to mutually exclusive alternatives, but this disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second, or more.

[0015] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects.

[0016] [The present invention 1001] A composition comprising an exosome, said exosome comprising a payload on its surface, said payload being an immunomodulatory molecule. [The present invention 1002] 1001. The composition of the present invention, wherein the immunomodulatory molecule is CD86, PD-L1, PD-L2, HVEM, GAL9, CTLA-4, PD-1, PD-1H, CD160, CD80, BTLA, TIM3, KIR, LAG3, A2aR, OX40L, CD27L, CD137L, BAFF, APRIL, CD70, CD40, B7H3, ICOSL, OX40, CD40L, BMCA, TACI, GITR, BAFFR, CD27, CD137, ICOS, or CD28. [The present invention 1003] The composition of claim 1002, wherein said immunomodulatory molecule is OX40L. [The present invention 1004] The composition of claim 1002, wherein the immunomodulatory molecule is ICOSL. [The present invention 1005] The composition of any of claims 1001 to 1004, wherein the exosome further comprises CD47 on its surface. [The present invention 1006] 1006. The composition of any of claims 1001 to 1005, wherein at least 50% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1007] 1006. The composition of claim 1006, wherein at least 60% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1008] 1007. The composition of claim 1007, wherein at least 70% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1009] 1008. The composition of claim 8, wherein at least 80% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1010] 1009. The composition of claim 10, wherein at least 90% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1011] 1011. The composition of any of claims 1001 to 1010, wherein the exosome further comprises an intravesicular protein payload. [The present invention 1012] A pharmaceutical composition comprising the exosome according to any one of claims 1001 to 1010 of the present invention and an excipient. [The present invention 1013] A composition of the present invention 1012 formulated for parenteral administration. [The present invention 1014] The composition of the present invention 1013, which is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [The present invention 1015] The composition of claim 1013, further comprising an antibacterial agent. [The present invention 1016] The composition of the present invention 1015, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidin, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. [The present invention 1017] A method for treating a disease in a patient in need thereof, comprising the step of administering any one of the compositions of present inventions 1012 to 1016 to the patient, thereby treating the disease in the patient. [The present invention 1018] The method of claim 1017, wherein said administering results in immunomodulation in said patient. [The present invention 1019] 1017. The method of claim 1017, wherein said disease is an immune disease, cancer, an infectious disease, or an autoimmune disease. [The present invention 1020] The method of claim 1019, wherein the disease is cancer. [The present invention 1021] 1017. The method of claim 1017, wherein said administration is systemic administration. [The present invention 1022] 1021. The method of claim 1021, wherein said systemic administration is intravenous administration. [The present invention 1023] 1017. The method of claim 1017, further comprising administering at least a second therapy to said patient. [The present invention 1024] 1024. The method of claim 1023, wherein said second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. [The present invention 1025] 1025. The method of claim 1024, wherein said second anti-cancer therapy comprises adoptive T cell therapy, an anti-PD1 antibody, an anti-CTLA-4 antibody, and / or an anti-PD-L1 antibody. [The present invention 1026] 1017. The method of claim 1017, wherein said patient is a human. [The present invention 1027] 1017. The method of claim 1017, wherein said exosomes are autologous to said patient. [The present invention 1028] 1. A composition comprising an exosome for use in treating a disease in a patient, wherein the exosome comprises a payload on its surface, the payload being an immunomodulatory molecule. [The present invention 1029] The composition for use of the present invention 1028, wherein the immunomodulatory molecule is CD86, PD-L1, PD-L2, HVEM, GAL9, CTLA-4, PD-1, PD-1H, CD160, CD80, BTLA, TIM3, KIR, LAG3, A2aR, OX40L, CD27L, CD137L, BAFF, APRIL, CD70, CD40, B7H3, ICOSL, OX40, CD40L, BMCA, TACI, GITR, BAFFR, CD27, CD137, ICOS, or CD28. [The present invention 1030] The composition for use of the present invention 1029, wherein said immunomodulatory molecule is OX40L. [The present invention 1031] The composition for use of the present invention 1029, wherein said immunomodulatory molecule is ICOSL. [The present invention 1032] The composition for use according to any one of claims 1028 to 1031, wherein the exosomes further comprise CD47 on their surface. [The present invention 1033] 1032. The composition for use according to any of claims 1028 to 1032, wherein at least 50% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1034] A composition for use according to the present invention 1033, wherein at least 60% of said exosomes comprise an immunomodulatory molecule on their surface. [This invention 1035] A composition for use according to the present invention 1034, wherein at least 70% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1036] A composition for use according to the present invention 1035, wherein at least 80% of said exosomes comprise an immunomodulatory molecule on their surface. [This invention 1037] 1036. A composition for use according to the present invention, wherein at least 90% of said exosomes comprise an immunomodulatory molecule on their surface. [The present invention 1038] The composition for use of the present invention 1028, wherein the disease is an immune disease, cancer, an infectious disease, or an autoimmune disease. [This invention 1039] The composition for use of the present invention 1038, wherein the disease is cancer. [The present invention 1040] A composition for use according to the present invention 1028, which is formulated for parenteral administration. [This invention 1041] A composition for use of the present invention 1040 formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [The present invention 1042] A composition for use according to the present invention 1040 further comprising an antimicrobial agent. [This invention 1043] A composition for use according to the present invention 1042, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidin, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. [This invention 1044] A composition for use according to the present invention 1028 further comprising at least a second therapy. [This invention 1045] The composition for use of the present invention 1044, wherein said second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, or immunotherapy. [The present invention 1046] The composition for use according to the present invention 1028, wherein said patient is a human. [This invention 1047] The composition for use of the present invention 1028, wherein said exosomes are autologous to said patient. [This invention 1048] 1. Use of exosomes in the manufacture of a medicament for treating a disease, wherein the exosomes comprise a payload on their surface, and the payload is an immunomodulatory molecule. [This invention 1049] The use of the present invention 1048, wherein the immunomodulatory molecule is CD86, PD-L1, PD-L2, HVEM, GAL9, CTLA-4, PD-1, PD-1H, CD160, CD80, BTLA, TIM3, KIR, LAG3, A2aR, OX40L, CD27L, CD137L, BAFF, APRIL, CD70, CD40, B7H3, ICOSL, OX40, CD40L, BMCA, TACI, GITR, BAFFR, CD27, CD137, ICOS, or CD28. [The present invention 1050] The use of claim 1049, wherein said immunomodulatory molecule is OX40L. [This invention 1051] The use of claim 1049, wherein said immunomodulatory molecule is ICOSL. [This invention 1052] The use of any of claims 1048 to 1051, wherein the exosomes further comprise CD47 on their surface. [This invention 1053] 1052. The use of any of claims 1048 to 1052, wherein at least 50% of said exosomes comprise an immunomodulatory molecule on their surface. [This invention 1054] The use of claim 1053, wherein at least 60% of said exosomes comprise an immunomodulatory molecule on their surface. [This invention 1055] The use of the present invention 1054, wherein at least 70% of said exosomes comprise an immunomodulatory molecule on their surface. [This invention 1056] The use of the present invention 1055, wherein at least 80% of said exosomes comprise an immunomodulatory molecule on their surface. [This invention 1057] The use of the present invention 1056, wherein at least 90% of said exosomes comprise an immunomodulatory molecule on their surface. [This invention 1058] The use of the present invention 1048, wherein the disease is an immune disease, cancer, an infectious disease, or an autoimmune disease. [This invention 1059] The use of the present invention 1058, wherein the disease is cancer. [The present invention 1060] The use of invention 1048, wherein said medicament is formulated for parenteral administration. [The present invention 1061] The use of invention 1048, wherein said medicament is formulated for systemic administration. [The present invention 1062] The use of invention 1060, wherein said medicament is formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection. [The present invention 1063] The use of the present invention 1048, wherein the medicament comprises an antibacterial agent. [This invention 1064] The use of invention 1063, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidin, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of example only, as various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0017] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Real-time PCR analysis of 293T cells stably transfected with ICOSLG- and OX40L-expressing plasmids. [Figure 2A] Western blot analysis of 293T cells stably transfected with ICOSLG expression plasmid and OX40L expression plasmid and exosomes isolated therefrom. Figure 2A shows the expression of ICOSLG. [Figure 2B] Western blot analysis of 293T cells stably transfected with ICOSLG and OX40L expression plasmids and exosomes isolated therefrom. Figure 2B shows the expression of control vinculin. [Figure 3-1]Flow cytometry analysis of 293T cells stably transfected with ICOSLG expression plasmid and OX40L expression plasmid and exosomes isolated therefrom. [Figure 3-2] Flow cytometry analysis of 293T cells stably transfected with ICOSLG expression plasmid and OX40L expression plasmid and exosomes isolated therefrom. [Figure 4-1] Schematic diagram of the experiment to determine the activity of ICOSLG+exosomes and OX40L+exosomes. [Figure 4-2] Schematic diagram of the experiment to determine the activity of ICOSLG+exosomes and OX40L+exosomes. [Figure 5-1] Flow cytometry analysis of the effect of ICOSLG + exosomes on T cell proliferation using naive T cells. [Figure 5-2] Flow cytometry analysis of the effect of ICOSLG + exosomes on T cell proliferation using naive T cells. [Figure 6-1] Flow cytometry analysis of the effect of ICOSLG + exosomes on T cell proliferation using splenic T cells derived from tumor-bearing mice. [Figure 6-2] Flow cytometry analysis of the effect of ICOSLG + exosomes on T cell proliferation using splenic T cells derived from tumor-bearing mice. [Figure 7A] Analysis of the effects of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7A shows tumor burden from mice in each treatment group on days 9, 11, 13, and 15 after implantation. [Figure 7B] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7B shows tumor burden in mice from treatment groups G1 and G7 over time up to day 19. [Figure 7C]Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7C shows tumor burden in mice from treatment groups G2 and G7 over time up to day 19. [Figure 7D] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7D shows tumor burden in mice from treatment groups G3 and G7 over time up to day 19. [Figure 7E] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7E shows tumor burden in mice from treatment groups G4 and G7 over time up to day 19. [Figure 7F] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7F shows tumor burden in mice from treatment groups G5 and G7 over time up to day 19. [Figure 7G] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7G shows tumor burden in mice from treatment groups G6 and G7 over time up to day 19. [Figure 7H] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7H shows tumor burden in mice from treatment groups G3 and G6 over time up to day 19. [Figure 7I] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7I shows the tumor burden in mice from treatment groups G1 and G5 over time up to day 19. [Figure 7J] Analysis of the effect of various treatment regimens using ICOSLG+exosomes and OX40L+exosomes alone and in combination with anti-CTLA-4 on tumor burden in mice. Figure 7J shows tumor burden in mice from treatment groups G4 and G6 over time up to day 19. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description Provided herein is a novel and efficient method for generating exosomes capable of modulating the adaptive immune system, with applications in cancer and other diseases. As a proof-of-concept, 239T-derived exosomes expressing ICOSL or OX40L were generated. Using exosomes, in vitro and in vivo activities demonstrating T cell activation properties and antitumor immunity were demonstrated. These imExosomes represent next-generation immunomodulatory drugs that can function with properties similar to or superior to agonist and antagonist antibodies that modulate tumor immunity, as well as with properties similar to or superior to potential small molecules that modulate the immune system. Naive T cells and splenic T cells from tumor-bearing mice were transfected with imExosomes. ICOSL and imExosomes OX40L Treatment with imExosomes activates T cells and produces INF-γ and IL-2. ICOSL and imExosomes OX40L inhibits B16F10 melanoma tumors when injected in combination with anti-CTLA4 or alone. The imExosomes platform has the ability to generate exosomes with surface and intraluminal protein payloads that modulate the immune system.

[0019] I. Lipid-Based Nanoparticles In some embodiments, the lipid-based nanoparticle is a liposome, an exosome, a lipid preparation, or another lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP:cholesterol vesicle). The lipid-based nanoparticle can be positively charged, negatively charged, or neutral.

[0020] B. Liposomes "Liposome" is a generic term that includes various unilamellar and multilamellar lipid vesicles formed by forming a closed lipid bilayer or aggregate. Liposomes may be characterized as having a vesicular structure with a bilayer membrane generally comprising phospholipids and an internal medium generally comprising an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged.

[0021] Multilamellar liposomes contain multiple lipid layers separated by aqueous medium. Such liposomes form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution. The lipid components self-reorganize to form a closed structure, trapping water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0022] In certain aspects, the polypeptide, nucleic acid, or small molecule drug may be, for example, placed in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule that binds both the liposome and the polypeptide / nucleic acid, entrapped within the liposome, or complexed with the liposome.

[0023] Liposomes used in accordance with this embodiment can be prepared by various methods known to those skilled in the art. For example, phospholipids, such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC), are dissolved in tert-butanol. The lipids are then mixed with polypeptides, nucleic acids, and / or other components. Tween 20 is added to the lipid mixture so that it accounts for approximately 5% of the composition's weight. Excess tert-butanol is added to this mixture so that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized preparation can be stored at -20°C and used for up to 3 months. When needed, the lyophilized liposomes are reconstituted by dissolving them in 0.9% saline.

[0024] Alternatively, liposomes can be prepared by dissolving lipids in a solvent and mixing them in a container, such as a glass pear-shaped flask. The volume of the container should be 10 times the volume of the expected liposome suspension. The solvent is removed using a rotary evaporator under negative pressure at approximately 40°C. The solvent is usually removed within approximately 5 minutes to 2 hours, depending on the desired liposome volume. The composition can be further dried under reduced pressure in a desiccator. Dried lipids tend to deteriorate over time, so they are generally discarded after approximately one week.

[0025] The dried lipids can be dissolved and hydrated in sterile, pyrogen-free water at approximately 25-50 mM phospholipid by shaking until the lipid film is fully resuspended. The aqueous liposome solution can then be divided into aliquots, each placed in a vial, lyophilized, and sealed under vacuum.

[0026] The dried lipid or lyophilized liposomes prepared as described above can be dehydrated, reconstituted by dissolving in a protein or peptide solution, and diluted to an appropriate concentration with an appropriate solvent, such as DPBS. The mixture is then vigorously shaken in a vortex mixer. Any additional unencapsulated materials, such as active substances including, but not limited to, hormones, drugs, nucleic acid constructs, etc., are removed by centrifugation at 29,000 x g, and the liposome pellet is washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., approximately 50-200 mM. The amount of encapsulated additional materials or active substances can be determined according to standard methods. After determining the amount of additional materials or active substances encapsulated in the liposome preparation, the liposomes can be diluted to an appropriate concentration and stored at 4°C until use. Pharmaceutical compositions containing liposomes typically contain a pharmaceutically acceptable sterile carrier or diluent, such as water or saline.

[0027] Additional liposomes that may be useful with this embodiment include cationic liposomes, such as those described in WO02 / 100435A1, U.S. Patent No. 5,962,016, U.S. Patent Application No. 2004 / 0208921, WO03 / 015757A1, WO04029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068, all of which are incorporated by reference in their entirety without disclaimer.

[0028] When preparing such liposome, can use any protocol described herein or known to those skilled in the art.Other non-limiting examples of preparing liposome are described in U.S. Patent No. 4,728,578, U.S. Patent No. 4,728,575, U.S. Patent No. 4,737,323, U.S. Patent No. 4,533,254, U.S. Patent No. 4,162,282, U.S. Patent No. 4,310,505 and U.S. Patent No. 4,921,706; International Application No. PCT / US85 / 01161 and U.S. Patent No. PCT / US89 / 05040, each of which is incorporated herein by reference.

[0029] In certain embodiments, the lipid-based nanoparticles are neutral liposomes (e.g., DOPC liposomes). As used herein, "neutral liposomes" or "uncharged liposomes" are defined as liposomes having one or more lipid components that result in an essentially neutral net charge (substantially uncharged). "Essentially neutral" or "essentially uncharged" means that within a particular population (e.g., a population of liposomes), some, if any, lipid components contain a charge that is not counterbalanced by the opposite charge of another component (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1% of the components contain an uncounterbalanced charge). In certain embodiments, neutral liposomes may contain a majority of lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., about pH 7).

[0030] The liposomes and / or lipid-based nanoparticles of this embodiment may contain phospholipids. In certain embodiments, one type of phospholipid may be used to prepare liposomes (e.g., a neutral phospholipid, such as DOPC, may be used to prepare neutral liposomes). In other embodiments, multiple types of phospholipids may be used to prepare liposomes. Phospholipids may be derived from neutral or synthetic sources. Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. Because phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., about pH 7), these compounds may be particularly useful for preparing neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce uncharged liposomes. In certain embodiments, a lipid other than a phospholipid (e.g., cholesterol) may be used.

[0031] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("MPPC"), 1-myris ... lysine monophosphate ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoylsphingomyelitis ("DSPG"), and glycerol monophosphate ("PDPG"). Dioleoylphosphatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoylphosphatidic acid ("DMPA"), dipalmitoylphosphatidic acid ("DPPA"), dimyristoylphosphatidylethanolamine ("DMPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), di Myristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristoyl phosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,These include, but are not limited to, 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoylphosphatidylcholine ("POPC"), palmitoyloeoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0032] C. Exosomes "Extracellular vesicles" and "EVs" as a class include exosomes, exosome-like vesicles, ectosomes (resulting from vesicle budding directly from the plasma membrane), microparticles, microvesicles, shedding microvesicles (SMVs), nanoparticles, and are microvesicles derived from and secreted by cells, including (large) apoptotic blebs or apoptotic bodies (resulting from cell death) or even membrane particles.

[0033] As used herein, the terms "microvesicle" and "exosome" refer to membranous particles having a diameter (or largest dimension if the particle is not a spheroid) of about 10 nm to about 5,000 nm, more typically 30 nm to 1,000 nm, and most typically about 50 nm to 750 nm, where at least a portion of the exosome's membrane is obtained directly from a cell. Most commonly, the size (average diameter) of an exosome is up to 5% of the size of the donor cell. Thus, exosomes specifically contemplated include exosomes shed from cells.

[0034] Exosomes may be detected in or isolated from any suitable sample type, such as, for example, a bodily fluid. As used herein, the term "isolated" refers to separation from the natural environment and is intended to include at least partial purification, and may include substantial purification. As used herein, the term "sample" refers to any sample suitable for the methods provided by the present invention. The sample may be any sample containing exosomes suitable for detection or isolation. Sample sources include blood, bone marrow, pleural fluid, ascites, cerebrospinal fluid, urine, saliva, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, sweat, tears, synovial fluid, and bronchial washings. In one aspect, the sample is a blood sample, including, for example, whole blood or any fraction or component thereof. Blood samples suitable for use with the present invention can be extracted from any known source containing blood cells or components thereof, such as veins, arteries, peripheries, tissues, cords, etc. For example, samples can be obtained and processed using well-known and routine clinical methods (e.g., techniques for collecting and processing whole blood). In one aspect, an exemplary sample can be peripheral blood collected from a subject with cancer.

[0035] Exosomes can also be isolated from tissue samples, such as surgical samples, biopsy samples, tissues, feces, and cultured cells.When isolating exosomes from tissue sources, it may be necessary to obtain a single cell suspension and then homogenize the tissue to dissolve the cells and release exosomes.When isolating exosomes from tissue samples, it is important to select a homogenization and lysis method that does not destroy exosomes.The exosomes intended herein are preferably isolated from body fluids dissolved in a physiologically acceptable solution, such as buffered saline, growth medium, various aqueous media, etc.

[0036] Exosomes may be isolated from freshly collected samples or from samples that have been stored frozen or refrigerated. In some embodiments, exosomes may be isolated from cell culture medium. Although not necessary, even higher purity exosomes may be obtained if the fluid sample is clarified before precipitation with a volume-excluding polymer to remove debris from the sample. Clarification methods include centrifugation, ultracentrifugation, filtration, or ultrafiltration. Most typically, exosomes can be isolated by numerous methods well known in the art. One preferred method is differential centrifugation from body fluids or cell culture supernatant. Exemplary methods for exosome isolation are described in (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Alternatively, exosomes may also be isolated by flow cytometry as described in (Combes et al., 1997).

[0037] One commonly accepted protocol for isolating exosomes involves ultracentrifugation, often combined with a sucrose density gradient or sucrose cushion to float the relatively low-density exosomes. Isolation of exosomes by differential centrifugation is complicated by the potential overlap in size distribution with other microvesicles or macromolecular complexes. Furthermore, centrifugation can provide an insufficient means of separating vesicles based on size. However, when differential centrifugation is combined with sucrose gradient ultracentrifugation, exosomes can be highly enriched.

[0038] Size-based exosome isolation using alternatives to ultracentrifugation is another option. Successful exosome purification using ultrafiltration, a method less time-consuming than ultracentrifugation and requiring no specialized equipment, has been reported. Similarly, a commercially available kit (EXOMIR™, Bio Scientific) is available that uses positive fluid pressure to remove cells, platelets, and cellular debris in one microfilter and capture vesicles larger than 30 nm in a second microfilter. However, this process does not recover exosomes; instead, their RNA content can be extracted directly from the material captured in the second microfilter and then used for PCR analysis. Using HPLC-based protocols, these processes require specialized equipment and are difficult to scale up, but potentially yield highly pure exosomes. A significant problem is that both blood and cell culture media contain numerous nanoparticles (some of which are non-vesicles) in the same size range as exosomes. For example, some miRNAs may be contained within extracellular protein complexes rather than exosomes. However, treatment with a protease (eg, proteinase K) can be carried out to eliminate any contamination with "extraexosomal" proteins.

[0039] In another embodiment, cancer cell-derived exosomes may be captured by techniques commonly used to enrich exosomes in a sample, such as techniques involving immunospecific interactions (e.g., immunomagnetic capture). Immunomagnetic capture, also known as immunomagnetic cell separation, typically involves attaching antibodies directed against proteins found on the surface of a specific cell type to small paramagnetic beads. When the antibody-coated beads are mixed with a sample, such as blood, the beads attach to and surround the specific cells. The sample is then placed in a strong magnetic field, causing the beads to pellet to one side. After the blood is removed, the captured cells are retained along with the beads. Many variations of this general method are known in the art and are suitable for use in isolating exosomes. In one example, exosomes may be attached to magnetic beads (e.g., aldehyde / sulfate beads), and then an antibody is added to the mixture to recognize epitopes on the surface of the exosomes attached to the beads. Exemplary proteins known to be found on cancer cell-derived exosomes include ATP-binding cassette sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), and glypican-1 (GPC1). Cancer cell-derived exosomes can be isolated, for example, using antibodies or aptamers against one or more of these proteins.

[0040] As used herein, analysis includes any method that allows for direct or indirect visualization of exosomes, and may be in vivo or ex vivo. For example, analysis may include, but is not limited to, ex vivo detection and visualization by microscopy or cytometry of exosomes bound to a solid support, flow cytometry, fluorescence imaging, etc. In an exemplary aspect, the cancer cell-derived exosomes contain ATP-binding cassette sub-family A member 6 (ABCA6), tetraspanin-4 (TSPAN4), SLIT and NTRK-like protein 4 (SLITRK4), putative protocadherin beta-18 (PCDHB18), myeloid cell surface antigen CD33 (CD33), glypican-1 (GPC1), histone H2A type 2-A (HIST1H2AA), histone H2A type 1-A (HIST1H1AA), histone H3.3 (H3F3A), histone H3.1 (HIST1H3A), zinc finger protein 37 homolog (ZFP37), laminin subunit beta-1 (LAMB1), Tubulointerstitial nephritis antigen-like (TINAGL1), peroxiredeoxin-4 (PRDX4), collagen α-2(IV) chain (COL4A2), putative protein C3P1 (C3P1), hemicentin-1 (HMCN1), putative rhophilin-2-like protein (RHPN2P1), ankyrin repeat domain-containing protein 62 (ANKRD62), tripartite motif-containing protein 42 (TRIM42), junction plakoglobin (JUP), tubulin β-2B chain (TUBB2B), endoribonuclease Dicer (DICER1), E3 ubiquitin-protein ligase TRIM71 (TRIM71), and Katanin p60 ATPase-containing subunit A-like 2 (KATNAL2), protein S100-A6 (S100A6), 5'-nucleotidase domain-containingProtein 3 (NT5DC3), valine-tRNA ligase (VARS), kazrin (KAZN), ELAV-like protein 4 (ELAVL4), ring finger protein 166 (RNF166), FERM and PDZ domain-containing protein 1 (FRMPD1), 78 kDa glucose-regulated protein (HSPA5), trafficking protein particle complex subunit 6A (TRAPPC6A), squalene monooxygenase (SQLE), tumor susceptibility gene 101 protein (TSG101), vacuolar protein sorting 28 homolog (VPS28), prostaglandin F2 receptor negative regulator (PTGFRN), isobutyryl-CoA dehydrogenase, mitochondrial (ACAD8), 26S protease regulatory subunit 6B (PSMC4), elongation factor 1-gamma (EEF1G), titin (TTN), tyrosine-protein phosphatase type 13 (PTPN13), triosephosphate isomerase (TPI1), or carboxypeptidase E (CPE), which may then be bound to a solid support and / or visualized using microscopic or cytometric detection.

[0041] It should be noted that not all proteins expressed in cells are found in the exosomes secreted by those cells.For example, calnexin, GM130, and LAMP-2 are all proteins expressed in MCF-7 cells, but are not found in the exosomes secreted by MCF-7 cells (Baietti et al., 2012).As another example, one study found that 190 / 190 pancreatic ductal adenocarcinoma patients had higher levels of GPC1+ exosomes than healthy controls (Melo et al., 2015, the entire contents of which are incorporated herein by reference).Notably, on average, only 2.3% of healthy controls had GPC1+ exosomes.

[0042] 2. Exemplary Protocol for Harvesting Exosomes from Cell Cultures On day 1, seed a sufficient number of cells (e.g., approximately 5 million cells) into a T225 flask containing medium containing 10% FBS so that the cells are approximately 70% confluent the next day. On day 2, aspirate the medium from the cells, wash them twice with PBS, and then add 25–30 mL of basal medium (i.e., without PenStrep or FBS) to the cells. Incubate the cells for 24–48 hours. While a 48-hour incubation is preferred, some cell lines are sensitive to serum-free medium, so the incubation time must be reduced to 24 hours. Note that FBS contains exosomes, which can strongly skew NanoSight results.

[0043] On day 3 / 4, collect the medium and centrifuge it at 800 x g for 5 minutes at room temperature to pellet dead cells and large debris. Transfer the supernatant to a new conical tube and recentrifuge the medium at 2000 x g for 10 minutes to remove other large debris and large vesicles. Pass the medium through a 0.2 μm filter and then aliquot it into ultracentrifuge tubes (e.g., 25 x 89 mm Beckman Ultra-Clear) using 35 mL per tube. If the volume of medium per tube is less than 35 mL, fill the remainder of the tube with PBS to make 35 mL. Ultracentrifuge the medium at 28,000 rpm for 2-4 hours at 4°C using an SW 32 Ti rotor (k-factor 266.7, RCF max 133,907). Carefully aspirate the supernatant until approximately 1 inch of liquid remains. Tilt the tube and slowly transfer the remaining medium into an aspirator pipette. If desired, the exosome pellet can be resuspended in PBS and ultracentrifuged at 28,000 rpm for 1-2 h repeatedly to further purify the exosome population.

[0044] Finally, resuspend the exosome pellet in 210 μL of PBS. If multiple ultracentrifuge tubes are used for each sample, resuspend each exosome pellet consecutively using the same 210 μL of PBS. For each sample, remove 10 μL and add it to 990 μL of HO for use in nanoparticle tracking analysis. Use the remaining 200 μL of exosome-containing suspension for downstream processing or immediately store at -80 °C.

[0045] 3. Exemplary Protocol for Extracting Exosomes from Serum Samples First, thaw the serum sample on ice. Then, dilute 250 μL of the cell-free serum sample with 11 mL of PBS and filter it through a 0.2 μm pore filter. Ultracentrifuge the diluted sample at 150,000 × g at 4 ° C overnight. The next day, carefully discard the supernatant and wash the exosome pellet with 11 mL of PBS. Ultracentrifuge for a second time at 150,000 × g at 4 ° C for 2 hours. Finally, carefully discard the supernatant and resuspend the exosome pellet in 100 μL of PBS for analysis.

[0046] D. Exemplary Protocols for Electroporating Exosomes and Liposomes 1×10 8 100 nm exosomes (measured by NanoSight analysis) or 100 nm liposomes (e.g., purchased from Encapsula Nano Sciences) were mixed with 1 μg of siRNA (Qiagen) or shRNA in 400 μL of electroporation buffer (1.15 mM potassium phosphate, pH 7.2, 25 mM potassium chloride, 21% Optiprep). The exosomes or liposomes were electroporated using a 4 mm cuvette (see, e.g., Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). After electroporation, the exosomes or liposomes were treated with protease-free RNase followed by the addition of 10x concentrated RNase inhibitor. Finally, the exosomes or liposomes were washed with PBS using ultracentrifugation as described above.

[0047] II. Immunomodulatory molecules Provided herein is a novel and efficient method for generating exosomes capable of modulating the adaptive immune system, which may have applications in cancer and other diseases. To this end, exosomes bearing immunomodulatory molecules on their surface were generated. As proof-of-concept, 239T-derived exosomes expressing ICOSL or OX40L were generated. These exosomes were used to demonstrate in vitro and in vivo activity demonstrating T cell activation properties and antitumor immunity. The imExosomes platform has the ability to generate exosomes with surface and intraluminal protein payloads that modulate the immune system. Immunomodulatory molecules may enhance or inhibit immune responses. The following references discuss immune checkpoint modulation and various ligand:receptor pairs and are incorporated herein by reference in their entirety for all purposes: Pardoll (2014); Wykes & Lewin (2018); Pico de Coana et al. (2015).

[0048] For example, without being bound by theory, it may be desirable to increase signaling via inhibitory molecules by using exosomes containing ligands for immunoinhibitory receptors to directly stimulate signaling via inhibitory receptors present on the surface of immune cells. Examples of inhibitory receptor ligands that can be delivered as exosome payloads include, but are not limited to, CD80, CD86, PD-L1, PD-L2, HVEM, and GAL9. Alternatively, the exosome payload may contain an antibody that acts as an agonist of an immunoinhibitory receptor, as discussed below.

[0049] For example, without being bound by theory, it may be desirable to reduce signal transduction mediated by inhibitory molecules by using exosomes containing immunoinhibitory receptors to act as a sponge for the ligands of the immunoinhibitory receptors, preventing the ligands from binding to the inhibitory receptors present on the surface of immune cells. Examples of inhibitory receptors that can be delivered as exosome payloads include, but are not limited to, CTLA-4, PD-1, PD-1H, CD160, CD80, BTLA, TIM3, KIR, LAG3, and A2aR. Alternatively, the exosome payload may include an antibody that acts as an antagonist of the immunoinhibitory receptor, as discussed below, and may include an antibody that binds to the ligand and thereby prevents the ligand from binding to its receptor.

[0050] For example, without being bound by theory, it may be desirable to increase signal transduction via stimulatory molecules by using exosomes containing ligands for immunostimulatory receptors to directly stimulate signal transduction via stimulatory receptors present on the surface of immune cells. Examples of stimulatory receptor ligands that can be delivered as exosome payloads include, but are not limited to, OX40L, CD27L, CD137L, BAFF, APRIL, CD70, CD40, B7H3, ICOSL, CD80, and CD86. Alternatively, the exosome payload may contain an antibody that acts as an agonist of an immunostimulatory receptor, as discussed below.

[0051] For example, without being bound by theory, it may be desirable to reduce signal transduction mediated by stimulatory molecules by using exosomes containing immunostimulatory receptors to act as a sponge for the ligands of the immunostimulatory receptors, preventing the ligands from binding to the stimulatory receptors present on the surface of immune cells. Examples of stimulatory receptors that can be delivered as exosome payloads include, but are not limited to, OX40, CD40L, BMCA, TACI, GITR, BAFFR, CD27, CD137, ICOS, and CD28. Alternatively, the exosome payload may contain an antibody that acts as an antagonist of the immunostimulatory receptor, as discussed below, and may include an antibody that binds to the ligand and thereby prevents the ligand from binding to its receptor.

[0052] III. Disease Treatment A specific aspect of the present invention provides for the treatment of patients in need of immunomodulation using exosomes containing immunomodulatory molecules, such as OX40L or ICOSL, on their surface. The immunomodulatory molecules may be membrane-bound. The exosomes may induce immunomodulation in patients. That is, the exosomes may enhance or inhibit immune responses as needed. Therefore, any disease in which modulation of immune responses is desired is intended to be treated. For example, the disease may be, but is not limited to, an immune disease, cancer, an infectious disease, or an autoimmune disease.

[0053] In addition to immune modulatory molecules on the surface of exosomes, exosomes are known to contain the machinery necessary to complete mRNA transcription and protein translation (see PCT / US2014 / 068630, which is incorporated herein by reference in its entirety), so mRNA or DNA nucleic acids encoding therapeutic proteins may be transfected into exosomes by transfection. Alternatively, the therapeutic protein itself may be introduced into exosomes by electroporation or directly incorporated into liposomes.

[0054] As used herein, "subject" refers to any individual or patient to whom the method is carried out.Generally, the subject is human, but as will be understood by those skilled in the art, the subject can also be an animal.Therefore, other animals, including mammals, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas), are included in the definition of subject.

[0055] "Treatment" and "treating" refer to the administration or application of a therapeutic substance to a subject, or the performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment may include administering exosomes comprising OX40L or ICOSL on their surface, administering chemotherapy, administering immunotherapy, or administering radiation therapy, performing surgery, or any combination thereof.

[0056] As used herein, the term "therapeutic benefit" or "therapeutically effective" refers to anything that enhances or improves the health of a subject with respect to the medical treatment of the condition. This term includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer may include, for example, reducing tumor invasiveness, reducing the rate of cancer growth, or preventing metastasis. Treating cancer may also refer to extending the survival of a subject with cancer. Treating autoimmune diseases may involve, for example, inducing tolerance to or inhibiting an immune response against an autoantigen that is responsible for an unwanted immune response. Treating infectious diseases may involve, for example, eliminating an infectious agent, reducing the level of an infectious agent, or maintaining the level of an infectious agent at a certain level.

[0057] As used herein, the term "cancer" may be used to describe a solid tumor, a metastatic cancer, or a non-metastatic cancer. In certain aspects, the cancer may arise in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testicles, tongue, or uterus.

[0058] Cancers include, specifically, the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; adenocarcinoma of the auditory canal adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell tumor; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; hemangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus melanoma in giant pigmented nevus); epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Mullerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonic carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor Tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory nerve tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease The present invention may be, but is not limited to, Hodgkin's disease, granulomatous lymphoma, malignant lymphoma, small lymphocytic lymphoma, malignant lymphoma, diffuse large cell lymphoma, malignant lymphoma, follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia. Nevertheless, the present invention may also be used to treat non-cancer diseases (e.g., fungal infections, bacterial infections, viral infections, neurodegenerative diseases, autoimmune diseases, and / or genetic disorders).

[0059] Autoimmune diseases include those in which a subject's own antibodies react with host tissues, or in which immune effector T cells autoreact with endogenous self-peptides and destroy tissues. Autoimmune diseases for which the treatment methods of the present invention are useful include Addison's disease, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, atherosclerosis, autoimmune diabetes (e.g., type 1 diabetes; insulin-dependent diabetes mellitus), autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune liver disease, autoimmune thrombocytopenic purpura, autoimmune thyroid disease, bullous pemphigoid, celiac disease, Crohn's disease, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), Goodpasture's syndrome, graft-versus-host disease, Graves' disease, host-versus-graft disease, idiopathic autoimmune-associated infertility, inflammatory bowel disease, insulin resistance, irritable bowel disease, arthritis (e.g., early arthritis, enteropathic arthritis), and the like. Arthritis, psoriatic arthritis, reactive arthritis, viral arthritis, familial Mediterranean fever, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus (e.g., pemphigus vulgaris), pernicious anemia, polymyositis, psoriasis, rheumatoid arthritis, juvenile rheumatoid arthritis, scleroderma with anti-collagen antibodies, Sjogren's syndrome, spondyloarthropathy, systemic lupus erythematosus (SLE), transplant rejection, and ulcerative colitis. Diagnosis and treatment of these diseases are well described in the literature.

[0060] Infectious diseases for which the treatment methods of the present invention are useful include, but are not limited to, bacterial infections, viral infections, fungal infections, parasitic infections, and sepsis. Exemplary viral infections include hepatitis B virus, hepatitis C virus, human immunodeficiency virus 1, human immunodeficiency virus 2, human papillomavirus, herpes simplex virus 1, herpes simplex virus 2, shingles, varicella zoster, coxsackievirus A16, cytomegalovirus, Ebola virus, enterovirus, Epstein-Barr virus, hantavirus, Hendra virus, viral meningitis, respiratory syncytial virus, rotavirus, West Nile virus, adenovirus, and influenza virus infections. Exemplary bacterial infections include Chlamydia trachomatis, Listeria monocytogenes, Helicobacter pylori, Escherichia coli, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria species (e.g., M. tuberculosis, M. avium, M. intraseriuiare, M. kansaii, M. gordonae), and the like.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Streptococcus pyogenes (group A streptococci), Streptococcus agalactiae (group B streptococci), Streptococcus spp. (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus spp. (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus anthrax anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum pallidium, Treponema pertenue, Leptospira, Rickettsia, Actinomyces israelli, Shigella species (e.g., S. flexneri, S.Exemplary fungal infections include infections with S. flexneri, S. sonnei, S. dysenteriae, and Salmonella species. Exemplary fungal infections include infections with Candida albicans, Candida glabrata, Aspergillus fumigatus, Aspergillus terreus, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, and Chlamydia irachomatis. .

[0061] The terms "contacted" and "exposed," when applied to cells, are used herein to describe the process by which a therapeutic agent is delivered to or placed in direct juxtaposition with a target cell. To kill the cell, for example, one or more agents are delivered to the cell in an amount effective to kill the cell or prevent the cell from dividing.

[0062] An effective patient response or a patient's "responsiveness" to a treatment refers to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder. Such benefit may include a cellular or biological response, a complete response, a partial response, stable disease (no progression or recurrence), or a response that subsequently recurs. For example, an effective response may be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer.

[0063] Treatment outcomes can be predicted and monitored, and / or patients who would benefit from such treatment can be identified or selected by the methods described herein.

[0064] Regarding the treatment of neoplastic conditions, depending on the stage of the neoplastic condition, the treatment of the neoplastic condition involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiation therapy, and chemotherapy. Other therapeutic regimens may be combined with the administration of anti-cancer agents, such as therapeutic compositions and chemotherapeutic agents. For example, patients who are to be treated with such anti-cancer agents may also undergo radiation therapy and / or surgery.

[0065] When treating a disease, the appropriate dosage of the therapeutic composition will depend on the type of disease being treated, as defined above, the severity and course of the disease, the patient's medical history and response to the agent, and the judgment of the attending physician. The agent is suitably administered to the patient at one time or over a series of treatments.

[0066] Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect. Tissues, tumors, or cells can be contacted with one or more compositions or pharmacological preparations containing one or more of the active substances, or the tissues, tumors, and / or cells can be contacted with two or more separate compositions or preparations. It is also contemplated that such combination therapy can be used with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

[0067] Coadministration may include simultaneous administration of two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the therapeutic composition and another therapeutic agent can be formulated together in the same dosage form and administered simultaneously. Alternatively, the therapeutic composition and another therapeutic agent can be administered simultaneously, where both agents are in separate formulations. Alternatively, one therapeutic agent can be administered immediately followed by the other therapeutic agent, or vice versa. In other administration protocols, the therapeutic composition and another therapeutic agent may be administered minutes apart, hours apart, or days apart.

[0068] The first anti-cancer treatment (e.g., exosomes comprising OX40L or ICOSL on their surface) may be administered prior to, during, or after the second anti-cancer treatment, or in various combinations relative to the second anti-cancer treatment. Administration may occur simultaneously or at intervals ranging from minutes to days to weeks. In embodiments in which the first treatment is provided to a patient separately from the second treatment, the effective period will generally not expire between the respective deliveries, so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is contemplated that the first and second therapies may be provided to a patient within about 12 to 24 hours or 72 hours of administering either therapy, more particularly, within about 6 to 12 hours of administering either therapy. In some circumstances, significantly longer treatment periods may be desirable. In this case, the period between each administration can be anywhere from a few days (2, 3, 4, 5, 6, or 7 days) to a few weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0069] In certain embodiments, the course of treatment lasts from 1 to 90 days or longer (such ranges are inclusive). It is contemplated that one agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof, and another agent may be given on any day from day 1 to day 90 (such ranges are inclusive), or any combination thereof. Within a single day (24-hour period), the patient may receive one or more administrations of the agent. Furthermore, it is contemplated that after the course of treatment, there will be a period during which no anti-cancer treatment is administered. This period may last from 1 to 7 days, and / or from 1 to 5 weeks, and / or from 1 to 12 months or longer (such ranges are inclusive), depending on the patient's condition, e.g., the patient's prognosis, strength, health, etc. It is anticipated that the treatment cycle will be repeated as necessary.

[0070] Various combinations can be used. For the example below, the first anti-cancer therapy is "A" and the second anti-cancer therapy is "B". TIFF0007801290000001.tif17128

[0071] Administration of any compound of the invention or administration of any therapy of the invention to a patient will follow standard protocols for administering such compounds, taking into account the toxicity, if any, of the agents, and thus, in some embodiments, there will be a step of monitoring for toxicity resulting from the combination therapy.

[0072] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to the mode of activity within cells, for example, whether or at what stage of the cell cycle they affect. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or affect nucleic acid synthesis to induce chromosomal and mitotic abnormalities.

[0073] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. ethylenimines and methylamelamines, including ylolomelamime; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and biceresin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; Duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichi n), phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosurea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I); dynemicins, including dynemicin A; bisphosphonates, e.g., clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxo doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zino Statins, or zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, propionibacterium, thiampicillin ... Dromostanolone onate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate;Hydroxyurea; Lentinan; Lonidynin; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin n); sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C") ); cyclophosphamide; taxoids, such as paclitaxel and docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CP T-11); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; cisplatin, carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0074] 2. Radiation therapy Other widely used agents that cause DNA damage include gamma rays, X-rays, and / or what are commonly known as specific delivery of radioisotopes to tumor cells. Other forms of DNA damage, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and UV irradiation, are also contemplated. All of these agents likely affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50–200 roentgens for prolonged periods (3–4 weeks) to single doses of 2000–6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0075] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy can be used in combination with or in conjunction with the methods of the present invention. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (Rituxan®) is one such example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. This antibody may act alone as an effector of therapy or may recruit other cells to actually affect cell killing. This antibody may also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) or simply act as a targeting agent. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0076] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., that is not present on the majority of other cells. Many tumor markers exist, and any of them may be suitable for targeting in the context of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. Another aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0077] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2013; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0078] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either strengthen signals (e.g., costimulatory molecules) or weaken signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibition include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0079] The immune checkpoint inhibitor may be a drug, e.g., a small molecule, a recombinant ligand or receptor, or, in particular, an antibody, e.g., a human antibody (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both of which are incorporated herein by reference). Known immune checkpoint protein inhibitors or analogs thereof may be used, in particular chimeric, humanized, or human antibodies. As those skilled in the art will recognize, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and / or equivalent names MK-3475 and pembrolizumab.

[0080] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its ligand binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its ligand binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Publication Nos. 20140294898, 2014022021, and 20110008369, all of which are incorporated herein by reference.

[0081] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular portion or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0082] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28; both molecules bind to CD80, also known as B7-1, and CD86, also known as B7-2, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 increases the expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0083] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0084] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art, or art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The disclosure of each of the aforementioned publications is incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0085] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to an antibody described above (e.g., at least about 90%, at least about 95%, or at least about 99% variable region identity to ipilimumab).

[0086] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins, such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0087] In some embodiments, immunotherapy may involve adoptive immunotherapy, which involves the transfer of ex vivo-generated autoantigen-specific T cells. T cells used in adoptive immunotherapy can be generated by expanding antigen-specific T cells or by redirecting T cells through genetic engineering (Park, Rosenberg et al. 2011). Isolation and transfer of tumor-specific T cells have been shown to be successful in treating melanoma. Novel specificities in T cells have been successfully generated by gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety linked to one or more signaling domains in a fusion molecule. Typically, the binding moiety of a CAR consists of the light chain fragment of a monoclonal antibody and the antigen-binding domain of a single-chain antibody (scFv), in which the variable fragment is connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains of first-generation CARs are derived from the cytoplasmic region of CD3ζ or the Fc receptor γ chain. CARs have been successfully used to redirect T cells to antigens expressed on the surface of tumor cells from a variety of neoplasms, including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0088] In one embodiment, the present application provides a combination therapy for treating cancer comprising adoptive T cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another aspect, the autologous and / or allogeneic T cells are targeted against tumor antigens.

[0089] 4.Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventative, diagnostic, or staging surgery, curative, and palliative surgery. Curative surgery involves resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs surgery).

[0090] When cancer cells, tissues, or tumors are partially or completely removed, a cavity may be formed in the body.Treatment can be carried out by perfusion, direct injection, or local application of additional anticancer therapy to the area.Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.These treatments can also be various dosages.

[0091] 5. Other agents It is contemplated that other agents may be used in combination with certain aspects of the present invention to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that sensitize hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions enhances the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present invention to improve the anti-hyperproliferative efficacy of treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that sensitize hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present invention to improve the efficacy of treatment.

[0092] IV. Pharmaceutical Compositions It is contemplated that exosomes containing OX40L or ICOSL on their surface can be administered systemically or locally to inhibit tumor cell growth, most preferably to kill cancer cells in cancer patients with locally advanced or metastatic cancer. Exosomes expressing or containing CRISPR systems can be administered intravenously, intrathecally, and / or intraperitoneally. Exosomes expressing or containing CRISPR systems can be administered alone or in combination with antiproliferative drugs. In one embodiment, exosomes expressing or containing CRISPR systems are administered to reduce a patient's cancer burden before surgery or other treatment. Alternatively, exosomes expressing or containing CRISPR systems can be administered after surgery to ensure that any remaining cancer (e.g., cancer not removed by surgery) does not survive.

[0093] The present invention is not intended to be limited by the specific nature of the therapeutic preparation.For example, such compositions can be provided in the form of a formulation together with a physiologically acceptable liquid, gel, solid carrier, diluent, or excipient.These therapeutic preparations, like other therapeutic substances, can be administered to mammals for veterinary use, such as veterinary use with livestock, and clinical use in humans.Generally, the dosage required for therapeutic effectiveness varies according to the type of use and administration method, and the specific requirements of each individual subject.

[0094] Where clinical application is intended, it may be necessary to prepare pharmaceutical compositions containing recombinant proteins and / or exosomes in a form appropriate for the intended use. Generally, pharmaceutical compositions may be parenteral formulations and may contain an effective amount of one or more recombinant proteins and / or exosomes and / or additional active substances dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals, such as humans, as appropriate. The preparation of pharmaceutical compositions containing the recombinant proteins and / or exosomes disclosed herein, or additional active ingredients, is exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference in its entirety for all purposes. Furthermore, for administration to animals (e.g., humans), it is understood that preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0095] Furthermore, according to certain aspects of the present invention, the composition suitable for administration may be provided dissolved in a pharmaceutically acceptable carrier with or without an inert diluent. As used herein, "pharmaceutically acceptable carrier" refers to any and all aqueous solvents known to those skilled in the art (e.g., water, alcoholic / aqueous solutions, ethanol, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., fats, oils, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and injectable organic esters such as ethyl oleate), lipids, liposomes, dispersion media, coatings (e.g., lecithin), surfactants, anti-inflammatory agents, and the like. These include oxidizing agents, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, noble gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof), isotonic agents (e.g., sugars and sodium chloride), absorption delaying agents (e.g., aluminum monostearate and gelatin), salts, drugs, drug stabilizers, gels, resins, fillers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutrient replenishers, and combinations thereof. The carrier must be assimilable and include liquid, semi-solid, i.e., paste, or solid carriers. Additionally, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffering agents. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0096] Although pharmaceutically acceptable carriers are formulated specifically for administration to humans, in certain embodiments, it may be desirable to use pharmaceutically acceptable carriers formulated for administration to non-human animals but not acceptable for administration to humans (e.g., due to government regulations). Except where a conventional carrier is incompatible with the active ingredient (e.g., harmful to the recipient or detrimental to the therapeutic effect of the composition contained in the carrier), its use in therapeutic or pharmaceutical compositions is contemplated. According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such techniques are routine for those skilled in the art.

[0097] Certain embodiments of the present invention may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, and whether they need to be sterile for routes of administration such as injection. The compositions may be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion directly bathing target cells, via catheter, by lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the foregoing, as described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference.

[0098] The active compound can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or intraperitoneal route.Therefore, embodiments include parenteral formulations.Typically, such compositions can be prepared as either liquid solutions or suspensions.Solid dosage forms suitable for use in preparing solutions or suspensions by adding liquid before injection can also be prepared.The preparation can also be emulsified.

[0099] According to this embodiment, the parenteral formulation may comprise the exosomes disclosed herein together with one or more solutes and / or solvents, one or more buffers and / or one or more antimicrobial agents, or any combination thereof. In some aspects, the solvent may comprise water, a water-miscible solvent, such as ethyl alcohol, liquid polyethylene glycol, and / or propylene glycol, and / or a water-immiscible solvent, such as a fixed oil, including corn oil, cottonseed oil, peanut oil, and / or sesame oil. In certain versions, the solute may comprise one or more antimicrobial agents, buffers, antioxidants, tonicity agents, cryoprotectants, and / or lyoprotectants.

[0100] Antimicrobial agents according to the present disclosure may include those provided elsewhere in this disclosure as well as benzyl alcohol, phenol, mercurials, and / or parabens. Antimicrobial agents may include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidin, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal, or any combination thereof. In various aspects, the antimicrobial agent may be present at a concentration necessary to ensure sterility, such as that required for pharmaceutical agents. For example, the agent may be present in a preparation, for example, in a multi-dose container, at a bacteriostatic or fungistatic concentration. In various embodiments, the agent may be a preservative and / or may be present at a concentration sufficient at the time of use to prevent the growth of microorganisms, such as those unintentionally introduced into the preparation, for example, during withdrawal of a portion of the contents using a hypodermic needle and syringe. In various aspects, the agent has a maximum volume and / or concentration limit (e.g., phenylmercuric nitrate and thimerosal 0.01%, benzethonium chloride and benzalkonium chloride 0.01%, phenol or cresol 0.5%, and chlorobutanol 0.5%). In various cases, an agent such as phenylmercuric nitrate is used at a concentration of 0.002%. A combination of 0.18% methyl p-hydroxybenzoate and 0.02% propyl p-hydroxybenzoate, and 2% benzyl alcohol can also be applied according to embodiments. Antimicrobial agents may also include 0.5% hexylresorcinol, 0.1% phenylmercuric benzoate, and / or a therapeutic compound.

[0101] Antioxidants according to the present disclosure may include ascorbic acid and / or its salts and / or the sodium salt of ethylenediaminetetraacetic acid (EDTA). Tonicity agents described herein may include electrolytes and / or mono- or disaccharides. Cryoprotectants and / or lyoprotectants are additives that protect biological products from the adverse effects of freezing and / or drying of the product during the freeze-drying process. Cryoprotectants and / or lyoprotectants may include sugars (non-reducing), such as sucrose or trehalose, amino acids, such as glycine or lysine, polymers, such as liquid polyethylene glycol or dextran, and polyols, such as mannitol or sorbitol. All of these are potential cryoprotectants or lyoprotectants. This embodiment may also include an antifungal agent, such as butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid, or any combination thereof. Additional solutes and antimicrobial agents, buffers, antioxidants, tonicity agents, cryoprotectants and / or lyoprotectants that may be used in accordance with the present disclosure, as well as characteristics thereof and aspects of methods of making the present parenteral formulations, are described, for example, in Remington's Pharmaceutical Sciences, 21st Ed., 2005, e.g., Chapter 41, which is incorporated herein by reference in its entirety for all purposes.

[0102] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and must be fluid to the extent that easy syringability exists. The dosage form must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0103] The therapeutic agent may be formulated into the composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with the free amino groups of the protein composition, or with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be obtained from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, for example, formulated for parenteral administration, such as injections or aerosols for pulmonary delivery, or formulated for alimentary administration, such as drug-release capsules.

[0104] In a specific embodiment of the present invention, the composition is combined with a semi-solid or solid carrier or thoroughly mixed with a semi-solid or solid carrier.Mixing can be performed by any convenient method, such as grinding.Stabilizers can also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., from denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0105] In a further aspect, the present invention may relate to the use of pharmaceutical lipid vehicle compositions comprising one or more lipids and an aqueous solvent. As used herein, the term "lipid" is defined to include a wide variety of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long-chain aliphatic hydrocarbons and their derivatives. Lipids can be natural or synthetic (i.e., engineered or produced by humans). However, lipids are typically biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that would be understood by those skilled in the art to be lipids are also encompassed by the compositions and methods described above.

[0106] Those skilled in the art will be familiar with the range of techniques that can be used to disperse a composition in a lipid vehicle. For example, the therapeutic substance may be dispersed in a solution containing lipids, dissolved with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained as a suspension in lipids, contained or complexed with micelles or liposomes, or otherwise bound to lipids or lipid structures by any means known to those skilled in the art. The dispersion may or may not result in the formation of liposomes.

[0107] The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response, i.e., the administration, i.e., the appropriate route and treatment regimen, discussed above. The amount to be administered will depend on the desired effect, depending on the number of treatments and the unit dose. The actual dosage of the compositions of the present invention administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health, and sex, the type of disease being treated, the extent of disease invasion, previous or concurrent therapeutic interventions, the patient's idiopathic disease, the route of administration, and the efficacy, stability, and toxicity of the particular therapeutic agent. For example, dosages may also include doses from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (such ranges inclusive), or higher, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include ranges of about 5 μg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. The administering physician will, in any given situation, determine the concentration of active ingredient(s) in a composition and the dose appropriate for the individual subject.

[0108] The actual dosage of the composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the frequency of administration of an effective amount may vary according to the subject's response. The physician in charge of administration will, in any given situation, determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject.

[0109] In certain embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise, for example, from about 2% to about 75% or from about 25% to about 60% of the weight of the unit, and any range derivable therein. Naturally, the amount of active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage amount of the compound is obtained in a particular unit dose. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are considered by those skilled in the art when preparing such pharmaceutical formulations, and various dosages and treatment regimens may be desirable accordingly.

[0110] In other non-limiting examples, dosages may also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1000 milligrams / kg / body weight, or more, per administration, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numbers recited herein include ranges based on the above numbers, such as about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc.

[0111] V. Nucleic Acids and Vectors In certain aspects of the present invention, nucleic acid sequences encoding therapeutic proteins or fusion proteins containing therapeutic proteins may be disclosed. Depending on which expression system is used, nucleic acid sequences can be selected based on conventional methods. For example, each gene or its variant may be codon-optimized for expression in a specific system. Various vectors can also be used to express the protein of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0112] VI. Recombinant Proteins and Inhibitory RNAs Some embodiments relate to recombinant proteins and polypeptides. In a further aspect, the proteins or polypeptides may be modified to enhance serum stability. Thus, when the present application refers to the function or activity of a "modified protein" or "modified polypeptide," it will be understood by those skilled in the art that this includes, for example, proteins or polypeptides that have additional advantages over the unmodified protein or polypeptide. It is specifically intended that embodiments relating to a "modified protein" may also be implemented with respect to a "modified polypeptide," and vice versa.

[0113] Recombinant proteins may have amino acid deletions and / or substitutions. Thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may further comprise inserted or added amino acids, e.g., fusion proteins or proteins with linkers. A "modified deletion protein" lacks one or more residues of the native protein but may retain the specificity and / or activity of the native protein. A "modified deletion protein" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is one that has amino acid residues deleted from at least one antigenic region, a region of the protein that has been shown to be antigenic in a particular organism, e.g., the type of organism to which the modified protein may be administered.

[0114] Substitution or replacement variants typically contain the replacement of one amino acid with another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may be conservative, i.e., one amino acid is replaced with an amino acid of similar shape or charge; substitutions may be non-conservative. Conservative substitutions are well known in the art and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0115] In addition to deletions or substitutions, modified proteins may have residue insertions, which typically involve the addition of at least one residue to the polypeptide. This may include the insertion of a targeting peptide or polypeptide, or may simply involve the insertion of a single residue. Terminal additions, called fusion proteins, are discussed below.

[0116] The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Thus, it includes about 70% to about 80% of the amino acid sequence, or about 81% to about 90% of the amino acid sequence, or even about 91% to about 99% of the amino acid sequence that is identical to or functionally equivalent to the amino acid sequence of a reference polypeptide, provided that the biological activity of the protein is maintained. A recombinant protein may, in certain aspects, be biologically functionally equivalent to its native counterpart.

[0117] It is also understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, as long as they meet the criteria set forth above, including the maintenance of biological protein activity associated with protein expression, but remain essentially as set forth in one of the sequences disclosed herein. The addition of terminal sequences applies particularly to nucleic acid sequences that may, for example, contain various non-coding sequences adjacent to the 5' or 3' portion of the coding region, or may contain various internal sequences, i.e., introns, which are known to exist within genes.

[0118] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids up to the full-length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0119] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous, and the amino acid residue sequence is not interrupted by non-amino acids. In other embodiments, the sequence may contain one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0120] Thus, the term "protein or peptide" includes amino acid sequences that include at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0121] Certain aspects of the present invention relate to fusion proteins. These molecules may have the N- or C-terminus of a therapeutic protein linked to a heterologous domain. For example, fusions may also use leader sequences from other species to enable recombinant expression of the protein in a heterologous host. Other useful fusions include the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably a cleavable antibody epitope, to facilitate fusion protein purification. Non-limiting affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0122] Methods for making fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein or by attaching a DNA sequence encoding a heterologous domain followed by expression of the intact fusion protein.

[0123] The production of fusion proteins that restore the functional activity of the parent proteins can be facilitated by joining the genes with a bridging DNA segment that encodes a peptide linker that is spliced ​​between the tandemly connected polypeptides, the linker being of sufficient length to allow correct folding of the resulting fusion protein.

[0124] VII. Kits and Diagnostics In various aspects of the present invention, kits are envisioned that contain the necessary components for purifying exosomes from body fluids or tissue culture media.In other aspects, kits are envisioned that contain the necessary components for isolating exosomes that contain OX40L or ICOSL on their surface.The kit may include one or more sealed vials containing any of these components.In some embodiments, the kit may also include suitable container means, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes, that are containers that do not react with the components of the kit.The container may be made of a sterilizable material, such as plastic or glass.

[0125] The kit may further comprise instructions outlining the procedural steps of the methods described herein, following substantially the same procedures as those described herein or known to those of skill in the art. The instruction information may be in a computer-readable medium comprising machine-readable instructions that, when executed using a computer, display a real or virtual method for purifying exosomes from a sample. [Example]

[0126] VIII. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples below demonstrate techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for practicing the invention. However, in light of the present disclosure, it should be understood by those of skill in the art that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0127] Example 1 - ICOSL + Exosomes and OX40L + Exosome isolation and purification HEK293T cells were transfected with OX40L or ICOSLG expression plasmids by treatment with Lipofectamine for 72 hours. To obtain stably transfected cells, the cells were then selected with 1 μg / ml puromycin for 10 days. The stable cells were then cultured in selective medium containing 1 μg / ml puromycin.

[0128] Exosomes were collected from untransfected HEK293T cells and stable HEK293T ICSOLG and HEK293T OX40L cells. Exosomes were purified by differential centrifugation as previously described (Alvarez-Erviti et al., 2011; El-Andaloussi et al., 2012). Supernatants were collected from cells cultured in exosome-depleted FBS-containing medium for 48 hours, followed by sequential centrifugation steps at 800 g for 5 minutes and 2000 g for 10 minutes. The resulting supernatant was then filtered through a 0.2 μm filter in a culture bottle, and the pellet was collected after 2 hours of ultracentrifugation at 28,000 g in a SW 32 Ti rotor (Beckman). The supernatant was aspirated, and the pellet was resuspended in PBS and then ultracentrifuged for another 2 hours. The purified exosomes were then analyzed and used for experimental procedures.

[0129] To measure the levels of ICOSL and OX40L transcripts in stably transfected 293T cells, total RNA was purified using TRIzol® (Invitrogen) according to the manufacturer's instructions, followed by retro-transcription of the RNA using MultiScribe Reverse Transcriptase (Applied Biosystems) and oligo-d(T) primers. Real-time PCR analysis was performed on an ABI PRISM® 7300HT Sequence Detection System Instrument using SYBR® Green Master Mix (Applied Biosystems). The transcripts of interest were normalized to the 18S transcript level. Each measurement was performed in triplicate. The threshold cycle, the fractional cycle number at which the amount of amplified target reaches a certain threshold, was determined, and expression was assessed by 2 -ΔCt As shown in Figure 1, wild-type 293T cells showed low basal levels of ICOSLG and OX40L transcripts, whereas levels in OX40L-overexpressing cells were approximately 10,000-fold higher, and levels in ICOSLG-overexpressing cells were approximately 2,500-fold higher.

[0130] To assess cellular and exosomal protein expression, cells and exosomes were harvested in RIPA buffer, and protein lysates were normalized using Bradford quantification. For electrophoretic separation of proteins under denaturing conditions, 40 μg of lysate was loaded onto an acrylamide gel and transferred to a PVDF membrane (Immobilon P) by wet electrophoretic transfer. The membrane was then blocked with 5% nonfat dry milk in PBS / 0.05% Tween®-20 for 1 hour at room temperature and incubated with the appropriate primary antibody overnight at 4°C. Secondary antibodies were incubated for 1 hour at room temperature. After antibody incubation, washes were performed three times at 15-minute intervals with 1x PBS 0.05% Tween®-20 on an orbital shaker. The membrane was developed using Pierce's chemiluminescence reagent according to the manufacturer's instructions, and chemiluminescence on the membrane was captured. As shown in Figure 2A, both stably transfected ICOSLG 293T cells and exosomes isolated from them exhibited high expression of ICOSLG. Vinculin expression was measured as a control (Figure 2B). Finally, cells and exosomes expressing OX40L or ICOSLG were detected using flow cytometry (Figure 3).

[0131] Example 2 – Treatment of T cells with exosomes Exosomes collected from HEK293T blank cells and HEK293T ICOSLG were used to treat naive T cells derived from C57BI / 6 mice or splenic T cells derived from C57BI / 6 mice bearing 689KPC GEMM tumors. As outlined in Figure 4, cells were isolated from the spleen of each mouse and subjected to negative selection to enrich for T cells. Isolated cells were labeled with carboxyfluorescein succinimidyl ester (CSFE) and stimulated with CD3 / CD28. Additionally, cells were treated with control exosomes, ICOSLG, or splenic T cells. + Exosomes, or OX40L + The cells were stimulated with exosomes. After stimulation, proliferation, INF-γ production, and IL-2 production were measured. Figure 5 shows the results of ICOSLG +Figure 6 shows that after stimulation with exosomes, the number of naive T cells producing IL-2 and IFN-γ increased compared to control exosomes. + Figure 1 shows that after stimulation with exosomes, the number of IL-2- and IFN-γ-producing splenic T cells from tumor-bearing mice was increased compared to control exosomes.

[0132] Example 3 - Treatment of implanted B16F10 tumors in vivo B16F10 cells were implanted subcutaneously into the back of each mouse. The mice were divided into seven groups. Group 1 was treated with exosomes isolated from wild-type HEK293T cells. Group 2 was treated with exosomes isolated from wild-type HEK293T cells and anti-CTLA-4. Group 3 was treated with exosomes isolated from ICOSL-overexpressing HEK293T cells and anti-CTLA-4. Group 4 was treated with exosomes isolated from OX40L-overexpressing HEK293T cells and anti-CTLA-4. Group 5 was treated with exosomes isolated from ICOSL-overexpressing HEK293T cells. Group 6 was treated with anti-CTLA-4 alone. Group 7 was treated with PBS. Mice in each group were intravenously injected daily for two weeks. Tumor burden was measured. As shown in Figures 7A-J, the lowest tumor burden was observed in mice treated with exosomes isolated from ICOSL-overexpressing HEK293T cells and anti-CTLA-4.

[0133] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods described herein and in the steps or order of steps thereof without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0134] References The following references, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007801290000002.tif183150TIFF0007801290000003.tif230150TIFF0007801290 000004.tif231151TIFF0007801290000005.tif224150TIFF0007801290000006.tif66150

Claims

1. 1. A pharmaceutical composition for use in treating cancer in a patient in need thereof, comprising an exosome comprising OX40L on its surface, wherein the pharmaceutical composition is used in combination with an anti-CTLA-4 antibody.

2. 10. The pharmaceutical composition of claim 1, wherein the exosome further comprises CD47 on its surface.

3. 3. The pharmaceutical composition of claim 1, wherein at least 50% of the exosomes comprise OX40L on their surface.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the exosomes further comprise an intravesicular protein payload.

5. 5. The pharmaceutical composition of claim 1, further comprising an excipient.

6. 6. The pharmaceutical composition of claim 5, formulated for parenteral administration.

7. 7. The pharmaceutical composition of claim 6, formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

8. 7. The pharmaceutical composition of claim 6, further comprising an antibacterial agent.

9. 9. The pharmaceutical composition of claim 8, wherein the antibacterial agent is benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, centrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, exetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, or thimerosal.

10. The pharmaceutical composition of claim 5, which induces immunomodulation in the patient.

11. 6. The pharmaceutical composition of claim 5, formulated for systemic administration.

12. 12. The pharmaceutical composition of claim 11, wherein the systemic administration is intravenous administration.

13. 6. The pharmaceutical composition of claim 5, further comprising at least a second therapy.

14. 14. The pharmaceutical composition of claim 13, wherein the second therapy comprises surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

15. The pharmaceutical composition of claim 1, wherein the patient is a human.

16. 10. The pharmaceutical composition of claim 1, wherein the exosomes are autologous to the patient.

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