Immunotherapeutic phospholipids for treatment of cancer

Abstract

A method of treating cancer in a subject is provided, the method comprising administering to the subject a therapeutic amount of a composition comprising phosphatidylglycerol nanovesicles (NVs). Also provided are a method for inhibiting polarization of M2 macrophages in a tumor microenvironment by administering phosphatidylglycerol NVs, and a pharmaceutical composition comprising phosphatidylglycerol NVs.

Description

Cross - Reference to Related Applications

[0001] This application claims the priority of U.S. Provisional Application No. 63 / 401,889, filed on August 29, 2022, the entire content of which is incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the field of cancer treatment. Specifically, the present disclosure relates to immunotherapeutic phospholipid nanovesicles for cancer treatment. Background

[0003] Cancer cells have evolved multiple mechanisms to establish an immunosuppressive tumor microenvironment, thereby facilitating their escape from host immune attacks and maintaining tumor growth. The tumor microenvironment is a complex environment containing various secreted factors and multiple cell types. During tumor progression, circulating monocytes and resident macrophages are recruited to the tumor site.

[0004] In the tumor microenvironment, macrophages polarize into M1 or M2 phenotypes. Classically activated M1 macrophages are generally considered to have antitumor effects, while alternatively activated M2 macrophages (tumor - associated macrophages, abbreviated as TAMs) promote tumor survival by immunosuppression, regulation of angiogenesis / lymphangiogenesis, induction of hypoxia, promotion of tumor cell proliferation and metastasis, etc.

[0005] Inhibiting tumor macrophage M2 polarization is an attractive therapeutic strategy in cancer treatment. There is an urgent need for therapeutic compositions and methods that can inhibit M2 macrophage polarization within tumors, thereby reducing the immunosuppression of the tumor microenvironment to treat cancer. Summary of the Invention

[0006] It has now been found that heat shock protein 70 (Hsp70) secreted by cancer cells can induce M2 macrophage polarization in the tumor microenvironment, and isolating Hsp70 by immunotherapeutic phospholipid nanovesicles can inhibit tumor growth. Accordingly, the present invention provides compositions and methods for isolating Hsp70 secreted by cancer to inhibit M2 macrophage polarization, reduce tumor volume, and treat cancer.

[0007] In one embodiment, a method for treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a composition comprising phosphatidylglycerol nanovesicles (NVs).

[0008] In another embodiment, a method for inhibiting M2 macrophage polarization in a tumor microenvironment is provided, the method comprising administering to the tumor microenvironment an effective amount of a composition comprising phosphatidylglycerol NVs.

[0009] In another embodiment, a pharmaceutical composition for treating cancer is provided, comprising: a therapeutically effective amount of phosphatidylglycerol nanovesicles; and at least one pharmaceutically acceptable excipient, wherein the phosphatidylglycerol nanovesicles do not encapsulate a second therapeutic agent and are not conjugated to a second therapeutic agent.

[0010] In another embodiment, a composition comprising phosphatidylglycerol nanovesicles (NVs) for use in a method of treating cancer is provided, the method comprising administering the composition to a subject.

[0011] These and other features, aspects, and advantages will be better understood from the following detailed description and the appended claims.

[0012] Other features and advantages of the embodiments described herein will be set forth in the following detailed description, and in part will be obvious to those of ordinary skill in the art from the description, or can be learned by practice of the embodiments described herein, including the following detailed description, the claims, and the drawings. Brief Description of the Drawings

[0013] Figure 1 It is a result graph of flow cytometry analysis of the expression of THP-1 differentiation marker CD14 under the action of CM of Gli36 cells and monoclonal antibodies against α-enolase, Hsp70, fascin, and S5A.

[0014] Figure 2 It is a result graph of flow cytometry analysis of the expression of THP-1 cell differentiation marker CD14 when the concentration of Hsp70 in the conditioned medium (CM) of Gli36 gradually increases.

[0015] Figure 3 It is a result graph of data mined from The Cancer Genome Atlas (TCGA) database, showing that the expression level of HSP70A1A mRNA in glioma cancer cells is higher than that in normal cells.

[0016] Figure 4 It is a result graph of data mined from the TCGA database, showing that the expression level of HSP70A1A mRNA in pancreatic cancer cells is higher than that in normal cells.

[0017] Figure 5 It is a result graph of Western blot analysis of whole cell lysates and CM from the indicated cell lines using anti-Hsp70 monoclonal antibody (upper row) and anti-actin monoclonal antibody (lower row).

[0018] Figure 6Shows the results of Western blot analysis of whole cell lysates of LLC-GFP cells (upper left panel) and LN229 cells (upper right panel) of wild type (control shRNA), shHsp70#1 or shHsp70#2 using anti-Hsp70 monoclonal antibody or anti-actin monoclonal antibody; and the results of flow cytometry measurement of CD14 expression in THP-1 cells cultured in the CM of wild type (control shRNA) or shHsp70#2-expressing LLC-GFP cells (lower left panel) and LN229 cells (lower right panel).

[0019] Figure 7 (Upper panel) Shows a schematic diagram of implanting tumors from LLC-GFP cells subcutaneously in the flank of mice (upper panel) and a graph of the change in subcutaneous tumor volume over time from wild type LLC-GFP cells (line 1) or Hsp70-knockdown LLC-GFP cells (line 2) (lower panel).

[0020] Figure 8 Shows the results of flow cytometry measurement of the expression of the M2 macrophage marker CD206 in macrophages isolated from subcutaneous tumors of wild type LLC-GFP cells expressing control shRNA and Hsp70-knockdown tumors (the two upper left panels and the lower left panel) and the results of flow cytometry measurement of the expression of the M1 macrophage marker INOS in macrophages isolated from subcutaneous tumors of wild type LLC-GFP cells expressing control shRNA and Hsp70-knockdown tumors (the two upper right panels and the lower right panel).

[0021] Figure 9 Shows the results of flow cytometry measurement of CD14 expression in THP-1 cells after incubation with 100 μM of the specified phospholipid nanovesicles (NVs) and the CM of MiaPaCa-2.

[0022] Figure 10 Shows the dose-dependent inhibition of THP1 cell differentiation by DOPG nanovesicles compared to DSPG nanovesicles.

[0023] Figure 11 Shows flow cytometry dot plots of propidium iodide-positive cells after incubation of THP-1 cells and the CM of MiaPaCa-2 and after co-incubation of THP-1 cells and the CM of MiaPaCa-2 with 100 μM of the specified phospholipid nanovesicles.

[0024] Figure 12Shows the result graph of quantifying Hsp70 in the precipitate (upper figure) and supernatant (lower figure) by enzyme-linked immunosorbent assay (ELISA), where the precipitate and supernatant were obtained by ultracentrifugation after co-incubating the CM of Gli36 cells with the specified phospholipid nanovesicles for 3 hours.

[0025] Figure 13 Is the Western blot detection result of Hsp70 in the supernatant and precipitate obtained after co-incubating the CM of Gli36 cells with the specified phospholipid nanovesicles for 3 hours and performing ultracentrifugation.

[0026] Figure 14 Shows a schematic diagram of implanting LLC-GFP cells into the flank of mice and the DOPG NVs treatment protocol (upper figure); and the curve graphs of the body weight of mice (lower left figure) and the tumor volume of mice (lower right figure) after PBS injection or DOPG nanovesicle injection.

[0027] Figure 15 Shows the result graph of M2-polarized macrophages inside polarized tumors in mice injected with PBS or DOPG nanovesicles.

[0028] Figure 16 Is the result graph of propidium iodide (PI) staining of nanovesicle-treated Mia-Pa-Ca-2 cells, indicating no cytotoxicity. Detailed implementation manners

[0029] This document elaborates on the details of the implementation manners of the present disclosure. After studying the information provided in this document, modifications to the implementation manners described in this document and other implementation manners will be obvious to those skilled in the art.

[0030] Although the following terms are considered to be well understood in the art, their definitions are given for the convenience of explaining the present disclosure. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0031] Unless otherwise specified, all numbers representing properties such as the amount of components, reaction conditions, etc. used in this specification and the claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in this specification and the claims are approximate values and may vary depending on the desired properties obtained from the present disclosure.

[0032] As used herein, the term "about," when referring to a numerical value, mass, weight, time, volume, concentration, or percentage, means a variation of ±20%, in certain embodiments, ±10%, in certain embodiments, ±5%, in certain embodiments, ±1%, in certain embodiments, ±0.5%, and in certain embodiments, ±0.1% relative to the specified amount, so long as such variations are suitable for carrying out the disclosed methods.

[0033] It should be understood that every upper numerical limit given in this specification includes every lower numerical limit, as if such lower numerical limits were expressly written herein. Every lower numerical limit given in this specification will include every upper numerical limit, as if such upper numerical limits were expressly written herein. Every numerical range given in this specification will include every narrower numerical range that falls within the broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0035] As used herein, the term "subject" generally refers to an organism (e.g., an animal or a human) capable of developing cancer. In a specific embodiment, the subject is a mammal, such as a human, rat, mouse, monkey, horse, cow, pig, dog, cat, guinea pig, etc. In a more specific embodiment, the subject is a human subject, rat, or mouse. In a more specific embodiment, the subject is a human.

[0036] As used herein, the terms "treating," "treatment," and "being treated" refer to methods of alleviating or eliminating a disease, disorder, and its symptoms. In a specific embodiment, the disease or disorder is cancer. In a more specific embodiment, the cancer is a cancer that secretes heat shock protein 70 (Hsp70), wherein Hsp70 polarizes macrophages in the tumor microenvironment to an M2 phenotype.

[0037] As used herein, the term "administering" or "administration" may include routes of administration such as enteral (e.g., oral, sublingual, buccal, or rectal), parenteral (e.g., intravenous, intramuscular, subcutaneous, intraarterial, intrathecal, intratumoral, etc.), intranasal, inhalation, vaginal, transdermal, etc., so long as the route of administration is capable of treating cancer. In a specific embodiment, the route of administration is parenteral. In a very specific embodiment, the route of administration is intravenous or intratumoral.

[0038] As used herein, "phosphatidylglycerol" or "PG" refers to a phospholipid having two acyl chains esterified to glycerol, which in turn is linked to a head structure containing one phosphate group and no other groups with compensating positive charges (the remaining structure being glycerol), such that PG bears a net negative charge. PG has two chiral centers (the sn-2 position of the phosphatidyl group and the central carbon atom of the alcohol glycerol). In some embodiments, the PG lipid contains a more unsaturated chain occupying the sn-1 position. An exemplary structure of PG is shown in Formula I. General chemical structure of phosphatidylglycerol, where R 1 and R 2 are fatty acid side chains

[0039] In specific embodiments, phosphatidylglycerol lipids suitable for the methods and compositions of the present disclosure include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (18:1(Δ9-cis)PG); 1,2-dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (18:1(Δ9-trans)PG); 1,2-dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (18:2PG); 1,2-dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (18:3PG); 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (20:4PG); 1,2-docosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (22:6PG); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (16:0-18:1PG); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (16:0-18:2PG); 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (18:0-18:1PG); 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (18:0-18:2PG); 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol) (17:1Lyso PG); 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol) (18:1Lyso PG), and combinations thereof. In a very specific embodiment, the nanovesicles are DOPG nanovesicles. In embodiments, the nanovesicles.

[0040] "Nanovesicles" are generally spherical lipid bilayer vesicles composed of a lipid such as phospholipid. In some embodiments, the nanovesicles used in the methods and compositions of the present disclosure are phosphatidylglycerol nanovesicles. In some embodiments, the diameter of the nanovesicles is about 20 nm to about 200 nm.

[0041] As used herein, "co - administration" refers to the administration of the phosphatidylglycerol nanovesicle composition of the present disclosure and a second therapeutic agent such that the two drugs can produce a physiological effect simultaneously, e.g., in a subject being treated. However, the two drugs do not necessarily have to be administered at the same time. In certain embodiments, the administration of one drug can precede that of the other. Simultaneously producing a physiological effect does not necessarily require the two drugs to be present in the subject's blood circulation at the same time. However, in certain embodiments, co - administration generally results in the two drugs being present in the subject's body at the same time. Thus, in certain embodiments, the phosphatidylglycerol nanovesicle composition and the second therapeutic agent can be administered simultaneously or sequentially.

[0042] As used herein, "effective amount" refers to the amount of an agent sufficient to achieve a desired biological effect. The effective amount can vary depending on factors such as the age, weight, health status, etc. of the subject, and those skilled in the art can determine the effective amount based on the present disclosure. The compositions in the present disclosure can be administered by single or multiple administrations of an effective amount. In some embodiments, the effective amount of an agent refers to the amount sufficient to treat cancer. In a specific embodiment, the effective amount refers to the amount sufficient to sequester cancer - secreted Hsp70 in the tumor microenvironment, thereby affecting the polarization of M2 macrophages to the M1 phenotype.

[0043] The development of strategies to block the generation of immunosuppressive M2 macrophages requires clarification of the mechanisms by which tumor - derived factors polarize macrophages to the M2 type and the disruption of these molecules by therapeutic means, thereby breaking the immunosuppressive communication between tumor cells and immune cells.

[0044] Heat shock protein 70 is one of the numerous proteins that promote cancer cell growth. Although initially discovered as an intracellular chaperone protein involved in the cellular stress response, it is now known to be overexpressed in a variety of cancers. In pancreatic ductal adenocarcinoma, glioblastoma, colon cancer, prostate cancer, and hepatocellular carcinoma, reducing the level of heat shock protein 70 has been shown to slow tumor growth. In addition, both plasma membrane - bound and circulating heat shock protein 70 levels are elevated in patients with glioblastoma, pancreatic cancer, and lung cancer. Heat shock protein 70 lacks a traditional secretion signal. Its secretion is thought to occur by non - traditional means, involving lysosomal vesicles or binding to membrane rafts and other secreted proteins. Post - translational modifications of heat shock protein 70, such as phosphorylation, play a key role in chaperone function. Due to enhanced kinase activity, the phosphorylation level of heat shock protein 70 at multiple sites is thought to be increased in cancer cells.

[0045] Unlike intracellular heat shock protein 70 (Hsp70), the role of secreted Hsp70 in tumor growth has not been fully understood, partly because the mechanism of its extracellular receptor-mediated action is not clear. Although Hsp70 is known to bind to Toll-like receptors (TLRs), a family of receptors with well-defined pro-inflammatory functions, its functional significance remains unclear. TLRs are highly expressed on macrophages and can specifically bind to various bacterial or pathogen structures, thereby triggering an inflammatory response. The role of macrophage TLRs in regulating tumor-induced immunosuppression is also not clear. Tyro3, Axl, and Mer receptor tyrosine kinases play a key role in macrophage M2 polarization in the tumor microenvironment, but it is not known whether TLRs interact with these receptors in the tumor microenvironment to induce macrophage M2 polarization.

[0046] Now, researchers have found that the major macrophage polarization activity in conditioned medium (CM) can be explained by secreted Hsp70. Knockdown of intracellular Hsp70 led to a slowdown in tumor growth and a decrease in M2 polarized macrophages within the tumor. Infusion of PG NVs that can bind secreted Hsp70 into mice inhibited cancer-induced macrophage differentiation into the M2 phenotype, intratumoral M2 macrophages, and tumor growth in mice.

[0047] Usage Method

[0048] One embodiment of the present invention provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising phosphatidylglycerol nanovesicles (NVs). In some embodiments, the phosphatidylglycerol nanovesicles are capable of sequestering secreted heat shock protein 70 (Hsp70), particularly cancer-secreted Hsp70 in the tumor microenvironment (TME) and / or secreted Hsp70 in the circulation.

[0049] While not wishing to be bound by theory, it is believed that the degree of unsaturation of phosphatidylglycerol and the area per lipid (APL), in combination with the net negative charge of phosphatidylglycerol, are factors for the successful clearance of secreted Hsp70 by phosphatidylglycerol nanovesicles. In some embodiments, the phosphatidylglycerol is unsaturated. In certain embodiments, the area per lipid (APL) of phosphatidylglycerol is greater than about

[0050] In some embodiments, the composition comprising phosphatidylglycerol nanovesicles does not comprise a second therapeutic agent. In some embodiments, the phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent. That is, in some embodiments, the phosphatidylglycerol nanovesicles are the sole therapeutic agent in the composition for treating cancer. In some embodiments, the composition consists essentially of phosphatidylglycerol nanovesicles. In a very specific embodiment, the phosphatidylglycerol is DOPG.

[0051] In some embodiments, the subject of the method provided by the present invention is a mammal, such as a human, rat, mouse, monkey, horse, cow, pig, dog, cat, guinea pig, etc. In a more specific embodiment, the subject is a human, rat or mouse. In a more specific embodiment, the subject is a human.

[0052] In some embodiments, the route of administration of the composition comprising phosphatidylglycerol nanovesicles can be an enteral route (such as oral, sublingual, buccal or rectal), parenteral injection or infusion (such as intravenous, intramuscular, subcutaneous, intra-arterial, intrathecal, intratumoral, etc.), intranasal, inhalation, vaginal, transdermal, etc., as long as the route of administration can treat cancer and / or sequester secreted Hsp70 in the tumor microenvironment. In a specific embodiment, the route of administration is a parenteral route. In a more specific embodiment, the route of administration is injection or infusion.

[0053] The cancers that the method disclosed in the present application is applicable to treat include those characterized by heat shock protein 70 (Hsp70) secreted and / or elevated in the tumor microenvironment. In some embodiments, such cancers produce tumors, more specifically solid tumors. In certain embodiments, the cancers include but are not limited to pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. It should be understood that other types of cancers can also be treated by the methods and compositions disclosed in the present application.

[0054] In another embodiment, a method of inhibiting M2 macrophage polarization in the tumor microenvironment is provided, the method comprising administering to the tumor microenvironment an effective amount of a composition comprising phosphatidylglycerol nanovesicles (NVs). In some embodiments, the phosphatidylglycerol is unsaturated. In a further embodiment, the area per lipid (APL) of the phosphatidylglycerol is greater than about

[0055] In some embodiments, the method of inhibiting M2 macrophage polarization can be carried out in vitro or in vivo. In some embodiments, phosphatidylglycerol nanovesicles can sequester secreted Hsp70, particularly cancer-secreted Hsp70 in circulation and / or cancer-secreted Hsp70 in the tumor microenvironment. Advantageously, sequestering secreted Hsp70 in the tumor microenvironment can shift macrophage polarization predominantly towards the M1 phenotype, thereby reducing the immunosuppressive nature of the tumor microenvironment. In some embodiments, the methods disclosed herein sequester secreted Hsp70, resulting in a decrease in tumor volume and / or inhibition of tumor growth.

[0056] In some embodiments, the composition comprising phosphatidylglycerol nanovesicles does not comprise a second therapeutic agent. In some embodiments, the phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent. That is, in some embodiments, the phosphatidylglycerol nanovesicles are the sole therapeutic agent in the composition for inhibiting macrophage M2 polarization. In some embodiments, the composition consists of or consists essentially of phosphatidylglycerol nanovesicles. In a very specific embodiment, the composition consists of or consists essentially of DOPG nanovesicles.

[0057] In some embodiments, the tumor microenvironment is associated with cancer and includes, but is not limited to, pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. In a specific embodiment, the tumor is a solid tumor.

[0058] In some embodiments, a second therapeutic agent can be co-administered with the composition comprising phosphatidylglycerol nanovesicles. In some embodiments, the phosphatidylglycerol nanovesicle composition and the second therapeutic agent can be administered simultaneously or sequentially.

[0059] Regarding sequential administration, the phosphatidylglycerol nanovesicles and the second therapeutic agent can be administered within one hour, two hours, four hours, eight hours, twenty-four hours, two days, three days, four days, five days, six days, or one week of each other. In some embodiments, the phosphatidylglycerol nanovesicles are administered first, followed by the second therapeutic agent. In some embodiments, the second therapeutic agent is administered first, followed by the phosphatidylglycerol nanovesicles.

[0060] Suitable therapeutic agents that can be co-administered with the phosphatidylglycerol nanovesicles described in the present disclosure include any therapeutic agents that can be used in cancer treatment patients. Exemplary therapeutic agents are described in U.S. Patent No. 10,787,440 issued to Keilhack et al. on September 29, 2020, which is hereby incorporated by reference in its entirety. The therapeutic agents listed below are for illustrative purposes only and are not intended to be limiting. It should be understood that for the treatment of a particular cancer, any suitable therapeutic agent can be co-administered with a composition comprising phosphatidylglycerol nanovesicles.

[0061] In some embodiments, the second therapeutic agent is an anti-cancer agent. In one embodiment, the anti-cancer agent is a compound that affects histone modification, such as a histone deacetylase inhibitor. In certain embodiments, the anti-cancer agent is selected from the following: chemotherapeutic drugs (such as 2CdA, 5-FU, 6-mercaptopurine, 6-TG, Abraxane TM , actinomycin D, all-trans retinoic acid, methotrexate, cytarabine, azacitidine, carmustine, clofarabine, Clolar TM , daunorubicin hydrochloride, DIC, etoposide phosphate, hexamethylmelamine, ixabepilone, L-asparaginase, liposomal cytarabine, melphalan, mitotane, actinomycin D, mitomycin C, nilotinib, nitrogen mustard, carmustine implant Proliferan20, thiotepa, Vidaza TM , vincristine sulfate, VM 26, and ); biological agents (such as α-interferon, BCG, erlotinib, interleukin-2, lenalidomide, Tarceva TM , and Zevalin TM);Corticosteroids (such as sodium dexamethasone phosphate, and Delta- );Hormone therapy (such as Plenaxis TM and );And radiopharmaceuticals (such as and samarium SM-153).

[0062] In another embodiment, the second therapeutic agent is a chemotherapeutic drug (also known as an anti-tumor drug or anti-proliferative drug), selected from the group consisting of: alkylating agents; antibiotics; antimetabolites; antidotes; interferons; polyclonal or monoclonal antibodies; epidermal growth factor receptor inhibitors; human epidermal growth factor receptor 2 inhibitors; histone deacetylase inhibitors; hormones; mitosis inhibitors; mammalian target of rapamycin inhibitors; multi-kinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; vascular endothelial growth factor / vascular endothelial growth factor receptor inhibitors; taxanes or taxane derivatives; aromatase inhibitors; anthracyclines; microtubule-targeting drugs; topoisomerase inhibitors; molecular target or enzyme (e.g., kinase or protein methyltransferase) inhibitors; cytosine analog drugs; or any chemotherapeutic, anti-tumor or anti-proliferative drug.

[0063] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin (Zanosar).

[0064] Exemplary antibiotics include, but are not limited to, Adriamycin; Doxil; Novantrone; Blenoxane; Cerubidine; DaunoXome; Cosmegen; Ellence; Idamycin; Mithracin; Mutamycin; Nipent; or Valstar.

[0065] Exemplary antimetabolites include, but are not limited to: Adrucil; Xeloda; Hydrea; Purinethol; Alimta; Fludara; Arranon; Cladribine Novaplus; Clolar; Cytosar-U; Dacogen; DepoCyt; Droxia; Folotyn; FUDR; Gemzar; Leustatin; Oforta; MTX; Rheumatrex; Trexall; Tabloid; TS-1 or Tarabine PFS.

[0066] Exemplary antidotes include, but are not limited to, Ethyol and Mesnex.

[0067] Exemplary interferons include, but are not limited to, interferon α-2b (Intro A) and interferon α-2a (Roferon-A).

[0068] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, Herceptin; Arzerra; Avastin; Rituxan; Erbitux; Vectibix; Bexxar; Campath; Zevalin; In-111; Y-90 Zevalin; Mylotarg; and Soliris denosumab.

[0069] Exemplary epidermal growth factor receptor (EGFR) inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab (Emd7200); and EKB-569.

[0070] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb), and AC-480.

[0071] Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0072] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); medroxyprogesterone (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone acetate (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); and testosterone lactobionate (Teslac).

[0073] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); eribulin (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); and amastatin or laminin D (LAM-D).

[0074] Exemplary mTOR inhibitors include, but are not limited to, everolimus (Afmitor) or temsirolimus (Torisel); rapamycin, ridaforolimus; and AP23573.

[0075] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; and vandetanib.

[0076] Exemplary microtubule-targeting agents include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilone, and vinorelbine.

[0077] Exemplary topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunorubicin, actinomycin D, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0078] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxel.

[0079] Exemplary systemic chemotherapy, anti-tumor, anti-proliferative drugs include, but are not limited to: altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak); porfimer sodium (Photofin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine (Leucenol); (1M tegafur - 0.4M 5-chloro-2,4-dihydroxypyrimidine - 1M potassium oxide), and lovastatin.

[0080] In another aspect, the second therapeutic agent is a chemotherapeutic drug or cytokine, such as G-CSF (granulocyte colony-stimulating factor).

[0081] On the other hand, the second therapeutic agent can be a standard chemotherapy combination, such as, but not limited to, CMF (cyclophosphamide, methotrexate, and 5-fluorouracil), CAF (cyclophosphamide, doxorubicin, and 5-fluorouracil), AC (doxorubicin and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC (doxorubicin, cyclophosphamide, and paclitaxel), rituximab, capecitabine, cisplatin (CDDP), carboplatin, TS-1 (tegafur, gimestat, and otastat potassium in a molar ratio of 1:0.4:1), irinotecan-11 (CPT-11, Irinotecan or Camptosar TM ), CHOP (cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone or prednisolone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone or prednisolone), and CMFP (cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone).

[0082] On the other hand, the second therapeutic agent can be an enzyme inhibitor, such as a receptor or non-receptor kinase inhibitor. Receptor and non-receptor kinases are, for example, tyrosine kinases or serine / threonine kinases. The kinase inhibitors described herein can be small molecules, polynucleotides, polypeptides, or antibodies.

[0083] Exemplary kinase inhibitors include, but are not limited to: bevacizumab (targeting VEGF), BIBW2992 (targeting EGFR and Erb2), cetuximab / Erbitux (targeting Erb 1), imatinib / Gleevec (targeting Bcr-Abl), trastuzumab (targeting Erb2), gefitinib / Iressa (targeting EGFR), ranibizumab (targeting VEGF), pegaptanib (targeting VEGF), erlotinib / Tarceva (targeting Erb 1), nilotinib (targeting Bcr-Abl), lapatinib (targeting Erb1 and Erb2 / Her2), GW-572016 / lapatinib ditosylate (targeting HER2 / Erb2), panitumumab / Vectibix (targeting EGFR), vandetanib (targeting RET / VEGFR), E7080 (multiple targets including RET and VEGFR), Herceptin (targeting HER2 / Erb2), PKI-166 (targeting EGFR), canertinib / CI-1033 (targeting EGFR), sunitinib / SU-11464 / Sutent (targeting EGFR and FLT3), matuzumab / Emd7200 (targeting EGFR), EKB-569 (targeting EGFR), Zd6474 (targeting EGFR and VEGFR), PKC-412 (targeting VEGR and FLT3), vatalanib / Ptk787 / ZK222584 (targeting VEGR), CEP-701 (targeting FLT3), SU5614 (targeting FLT3). MLN518 (targeting FLT3), XL999 (targeting FLT3), VX-322 (targeting FLT3), Azd0530 (targeting SRC), BMS-354825 (targeting SRC), SKI-606 (targeting SRC), CP-690 (targeting JAK), AG-490 (targeting JAK), WHI-P154 (targeting JAK), WHI-P 131 (targeting JAK), sorafenib / Nexavar (targeting RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3 and RET), dasatinib / Sprycel (targeting BCR / ABL and SRC), AC-220 (targeting Flt3), AC-480 (targeting all HER proteins, "pan-HER"), motesanib diphosphate (targeting VEGF 1-3, PDGFR and c-kit), denosumab (targeting RANKL, inhibiting SRC), AMG888 (targeting HER3) and AP24534 (multiple targets including Flt3).

[0084] Exemplary serine / threonine kinase inhibitors include, but are not limited to: rapamycin (acts on mTOR / FRAP1), deforolimus (acts on mTOR), Certican / Everolimus (acts on mTOR / FRAP1), AP23573 (acts on mTOR / FRAP1), Eril / fasudil hydrochloride (acts on RHO), xanthinol (acts on CDK), seliciclib / CYC202 / roscovitine (acts on CDK), SNS-032 / BMS-387032 (acts on CDK), ruboxistaurin (acts on PKC), Pkc412 (acts on PKC), bryostatin (acts on PKC), KAI-9803 (acts on PKC), SF 1126 (acts on PI3K), VX-680 (acts on Aurora kinase), Azd1152 (acts on Aurora kinase), Arry-142886 / AZD-6244 (acts on MAP / MEK), SCIO-469 (acts on MAP / MEK), GW681323 (acts on MAP / MEK), CC-401 (acts on JNK), CEP-1347 (acts on JNK), and PD 332991 (acts on CDK).

[0085] Exemplary tyrosine kinase inhibitors include, but are not limited to: erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; and AMG888.

[0086] Drug Composition

[0087] In another embodiment, a pharmaceutical composition for treating cancer is provided, the pharmaceutical composition comprising: a therapeutically effective amount of phosphatidylglycerol nanovesicles (NVs); and at least one pharmaceutically acceptable excipient. In some embodiments, the phosphatidylglycerol nanovesicles do not encapsulate a second therapeutic agent. These phosphatidylglycerol nanovesicles are not conjugated to a second therapeutic agent. In a specific embodiment, the phosphatidylglycerol nanovesicles are the sole therapeutic agent in the pharmaceutical composition.

[0088] In some embodiments, the phosphatidylglycerol is unsaturated. In further embodiments, the area per lipid (APL) of the phosphatidylglycerol is greater than about

[0089] The phosphatidylglycerol nanovesicles can be prepared according to methods known in the art. In some embodiments, the desired amount of phosphatidylglycerol phospholipid is dissolved in an organic solvent and then the solvent is removed by drying, for example, using nitrogen drying. Subsequently, the dried phospholipid film is mixed with a pharmaceutically acceptable carrier (such as PBS) and sonicated in a sonicator to generate the phosphatidylglycerol nanovesicles as described herein.

[0090] In some embodiments, the pharmaceutical composition comprising phosphatidylglycerol nanovesicles can be formulated into enteral, parenteral, intranasal, inhaled, vaginal, transdermal or other dosage forms. In a specific embodiment, the pharmaceutical composition is formulated for parenteral administration. In a more specific embodiment, the pharmaceutical composition is formulated for intravenous administration by injection or infusion. In another specific embodiment, the pharmaceutical composition is formulated for intratumoral injection.

[0091] These compositions can be prepared by any known method in the pharmaceutical art, for example, using the methods described in Remington: The Science and Practice of Pharmacy (23rd Edition, edited by Adeboye Adejare et al., 2020, see Chapter 7: Pharmaceutical Materials and Equipment / Industrial Pharmacy). Suitable pharmaceutical carriers are well known in the art. For example, see Handbook of Pharmaceutical Excipients (6th Edition, edited by Raymond C. Rowe, 2009). Those skilled in the art will recognize that certain carriers may be more suitable or appropriate for a particular mode of administration of the active ingredient. Selecting the appropriate carrier for a given pharmaceutical composition is within the skill of those in the art.

[0092] For parenteral administration, suitable compositions include aqueous and non-aqueous sterile suspensions for intravenous administration. These compositions can be provided in single-dose or multi-dose containers, such as sealed vials and ampoules.

[0093] In some embodiments, the dosage or concentration range of phosphatidylglycerol nanovesicles in the compositions disclosed herein can be from 1 μM to 500 μM; from 25 μM to 500 μM; from 25 μM to 400 μM; from 25 μM to 350 μM; from 25 μM to 300 μM; from 25 μM to 250 μM; from 25 μM to 200 μM; from 25 μM to 150 μM; from 25 μM to 100 μM; from 50 μM to 350 μM; from 50 μM to 300 μM; from 50 μM to 250 μM; from 50 μM to 200 μM; from 50 μM to 150 μM; from 50 μM to 125 μM; from 50 μM to 100 μM; from 50 μM to 75 μM; from 100 μM to 350 μM; from 100 μM to 300 μM; from 100 μM to 250 μM; from 100 μM to 200 μM; from 100 μM to 150 μM; from 150 μM to 350 μM; from 150 μM to 300 μM; from 150 μM to 250 μM; from 150 μM to 200 μM; from 200 μM to 350 μM; from 200 μM to 300 μM; from 200 μM to 250 μM; from 250 μM to 350 μM; from 250 μM to 300 μM; or any range within the above ranges.

[0094] Those skilled in the art will appreciate that for any particular subject, the specific dosage level will depend on a variety of factors, including the activity of the drug used; the age, weight, general health, and gender of the individual being treated; the time and route of administration; the rate of excretion; and factors such as these. Examples

[0095] The following examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0096] Example 1. Materials and Methods

[0097] Cell Line. The cell lines studied included human pancreatic cancer cell line (MiaPaCa-2), lung cancer cell lines (H1299, LLC-GFP), glioblastoma cell line (Gli36), human monocyte cell line (THP-1), human macrophage cell line (SC), and mouse macrophage cell line (J774), all from ATCC.

[0098] Cell Culture. Wild-type / TLR2-deficient THP-1 cells, human SC cells, and J774 macrophages were cultured in RPMI containing 25 mM HEPES. All other cell lines were cultured in DMEM medium, and all media were supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were cultured in a 5% carbon dioxide incubator at 37 °C. Cells were routinely tested for mycoplasma contamination. Cross-contamination between cell lines was determined by cell morphology and growth parameters and was not found.

[0099] Preparation of Serum-Free, Exosome / Particle-Free CM from Human / Mouse Cancer Cell Lines and Differentiation of THP-1 Assay. Grow human / mouse cancer cell lines to 70% confluence in their respective media in 10-cm Corning tissue culture dishes (ThermoFisher, MA), and then remove the media at this time. Wash the cells twice with serum-free media to remove serum residues and dead cells, and then supplement with serum-free media. After 24 hours, collect the CM, centrifuge at 10,000 g to remove cell debris, and then ultracentrifuge at 100,000 g to remove extracellular exosomes and microparticles. Culture 2×10 5 THP-1 cells in 1 ml of CM, and normalize the total cellular protein from the designated cancer cell line in the CM to 100 μg. Control THP-1 cells are grown in DMEM. After 24 hours, centrifuge the control cells and CM-treated cells, and then incubate them with CD14-PE-conjugated antibody (eBioscience, CA) and propidium iodide (PI; BD Biosciences, NJ) in 100 μL of FACS buffer on ice for 30 minutes. Wash the cells with flow cytometry buffer (PBS containing 2% FBS), and measure CD14 expression by flow cytometry. In studies involving the evaluation of exosomes / microparticles in THP-1 differentiation, THP-1 cells are cultured in unfractionated CM, CM without exosomes / microparticles, or the exosome / microparticle fraction of CM for 24 hours, and then the differentiation is evaluated by detecting CD14 expression by flow cytometry, as described above.

[0100] Flow Cytometry. Wash the cells once with PBS, and after centrifugation, incubate the cell pellet with the corresponding antibody in flow cytometry buffer (PBS containing 2% fetal bovine serum) on ice for 45 minutes. After incubation, wash the cells twice with flow cytometry buffer, and then perform detection. THP-1 cell differentiation is evaluated by detecting CD14 conjugated to PE (eBioscience, CA) by flow cytometry. Add propidium iodide (PI) to all stained cells to exclude dead cells. Analyze the stained cells using a BD Fortessa flow cytometer.

[0101] Intracellular Staining for Flow Cytometry Analysis. Wash the cells twice with PBS, and then fix them with 2% formaldehyde for 30 minutes. After fixation, centrifuge the cells to precipitate, and then wash them twice with PBS and precipitate again. Incubate the cell pellet in permeabilization buffer (cBioscience, CA) for 30 minutes, followed by centrifugation. Incubate these cell pellets with an antibody against intracellular antigen in permeabilization buffer for 1 hour. Wash twice with permeabilization buffer and once with flow cytometry buffer, and then analyze the cells.

[0102] Lentivirus-Mediated Hsp70 Knockdown.The pLKO.1 vector expressing shRNA targeting Hsp70 was from Sigma-Aldrich, MO. The pLKO.1 lentivirus was packaged in HEK-293T cells by co-transfecting the pMD2.G (VSV G) packaging plasmid and the Gag, Pol, psPAX2 packaging plasmid expressing plasmid. After transfection, the cells were cultured for 48 hours, and the lentiviral particles were collected from the supernatant for transduction of LLC-GFP and LN229 cells. The gene silencing efficiency was analyzed by detecting Hsp70 by immunoblotting 36 hours after infection.

[0103] Quantification of Hsp70 in CM of Cancer Cells by ELISA. Human / mouse cancer cell lines were grown to 70% confluence in their respective media in 10-cm tissue culture plates (ThermoFisher, MA). After removing the medium, the cells were rinsed twice with serum-free medium to remove serum residues and dead cells, and then supplemented with serum-free medium. After 24 hours, the CM was collected by ultracentrifugation at 100,000 g to remove extracellular exosomes and microparticles. Hsp70 in the CM was quantitatively detected using an Hsp70 ELISA kit (ThermoFisher, MA) according to the manufacturer's protocol.

[0104] Preparation of Phospholipid (PL) Nanovesicles and Treatment of THP-1 Cells. The required amounts of different phospholipids (DOPG, DOPA, DOPS, DSPC, or DSPG; Avanti Polar Lipids, AL) were dissolved in the organic solvent chloroform to prepare a 1 M phospholipid nanovesicle stock solution in a glass tube. The organic solvent was dried with nitrogen gas. 1 mL of PBS was added to the dried lipid film, and the phospholipid nanovesicles were prepared by sonication in an ice-water bath sonicator for 30 minutes. THP-1 cells were co-incubated with 50 to 100 μM of the designated phospholipid nanovesicles and the CM of the designated cancer cells for 24 hours.

[0105] Affinity Purification of Hsp70 and Evaluation of Its Differentiation Activity on THP-1.The CM of Gli36 (10, 20, and 30 mL) was concentrated to 1 mL, incubated overnight with anti-Hsp70 specific monoclonal antibody at 4 °C, and then protein A / G magnetic beads were added and incubation continued overnight at 4 °C. Subsequently, the magnetic beads were collected by centrifugation and washed twice with PBS. The bound proteins were eluted using Tris (20 mM)-glycine (200 mM) buffer (pH 2.5, 3.0, 3.5, or 4.0). The eluate was neutralized by adding an equal volume of pH 8.5 buffer. Alternatively, the magnetic beads were eluted with 3.5 MgCl2 and the eluate was renatured by dialysis against PBS using a dialysis membrane with a 10 kDa molecular weight cut-off. In addition, proteins were removed from the antibody with 8 M urea and the eluate was dialyzed successively against buffers containing 6, 4, and 2 M urea and finally against PBS. 100 μL of the eluate was added to THP-1 cells together with 1 mL of DMEM medium, and the differentiation of THP-1 cells was evaluated by detecting CD14 expression by flow cytometry 24 hours later.

[0106] Subcutaneous Cancer Cell Implantation and DOPG NV Treatment. Wild-type and Hsp70 knockdown Lewis lung carcinoma cells expressing green fluorescent protein (LLC-GFP) (1×10 5 ) were subcutaneously implanted into 6-8-week-old C57BL / 6 mice (5 mice per group). The treatment group (DOPG NVs in 200 μL PBS, 1.6 mg / kg per mouse) and the control group (200 μL PBS per mouse) were administered by tail vein injection. Treatment started 2 days after implantation, 3 times a week until the end of treatment. Tumor growth was evaluated by measuring tumor volume daily. Tumors were collected for histological and tumor macrophage analysis when the tumor volume reached 500 cubic millimeters. Tumor volume was measured with vernier calipers and calculated using the formula V = (π / 6)LW 2 (V is volume; L is length; W is width).

[0107] Tumor Dissociation into Single Cells and Flow Cytometry Detection. Freshly excised tumor tissues were cut into small pieces and minced with a scalpel. The minced tumor tissues were incubated in RPMI medium containing 100 units of type IV collagenase (Worthington Biochemical Corporation, NJ) at 37 °C for 45 minutes. The collagenase-treated tumor tissues were filtered through a 40 μM cell strainer (ThermoFisher, MA). The isolated tumor cells were washed twice with PBS, and 5×10 5Cells were incubated with mouse Fc blocker for 30 min on ice in 100 μL flow cytometry buffer (PBS with 2% fetal bovine serum), followed by incubation with anti-mouse F4 / 80-PE (eBioscience, CA) and anti-mouse CD206 APC (BioLegend, CA) for 30 min on ice. Cells were washed and then fixed with fixative (eBioscience, CA) for 30 min. Fixed cells were centrifuged and washed twice with PBS. Cells were permeabilized with permeabilization buffer (eBioscience, CA) according to the manufacturer's protocol and then incubated with anti-mouse iNOS antibody (eBioscience, CA) in permeabilization buffer for 45 min. Cells were washed twice with permeabilization buffer and finally washed with flow cytometry buffer. M2 CD206+ cells and M1 iNOS+ cells in macrophages were analyzed by gating F4 / 80 positive cells.

[0108] Cell Sorting. The indicated cancer cell lines were stained with Annexin V-FITC (Invitrogen, CA) and propidium iodide (PI) according to the manufacturer's protocol. Briefly, 1 × 10 6 The cells were incubated with Annexin V binding buffer (Invitrogen, CA) and PI for 30 minutes at room temperature. The cells were washed with Annexin V buffer, resuspended in it, and cells with high and low Annexin V signals were gated and sorted by flow cytometry.

[0109] SDS-PAGE and Western Blot Analysis. For SDS-PAGE, 50 μg of whole cell lysate in RIPA buffer (Sigma-Aldrich, MO) was denatured in SDS loading buffer (Bio-Rad, CA) and then loaded onto a 4-15% denaturing gradient gel (Bio-Rad, CA). The proteins in the gel were transferred to a nitrocellulose membrane and the blot was blocked in PBS containing 5% nonfat dry milk and 0.1% Tween-20, followed by the addition of specific protein antibodies and incubation overnight at 4°C. The blot was washed three times with PBS-Tween-20 and further incubated with a secondary antibody conjugated to horseradish peroxidase (HRP). After washing three more times with PBS-Tween-20, the blot was developed with SuperSignal West Dura (ThermoFisher, MA). For the immunoprecipitate, protein A+G agarose beads were boiled in SDS loading buffer and then loaded onto the gel for western blot analysis.

[0110] Lipid Binding Assay.The preparation method of PL NVs was the same as above, except that Hepes buffered saline was used instead of PBS. Gli36CM was incubated with 100 μM PL NVs by rotation at room temperature for 3 hours and then centrifuged at 170,000 g for 1 hour. The supernatant part was used for ELISA detection. Both the precipitate and the supernatant were used for Western blot analysis.

[0111] Statistical Analysis. All statistical analyses were performed using GraphPad Prism 6 software. Data were expressed as mean ± standard error, and two-group comparisons were made using the t-test (paired nonparametric). Details of the statistical analysis are provided in the legends. In vitro experiments were performed two to three times with consistent results. In vivo experiments were performed at least three times with consistent results. A P value less than 0.05 was considered statistically significant.

[0112] Example 2. Cancer-Secreted Hsp70 Stimulates THP-1 Cell Differentiation in a Dose-Dependent Manner

[0113] The ability of monoclonal antibodies (mAbs) against alpha enolase, Hsp70, moesin, and S5A to inhibit the differentiation of THP-1 cells under the action of Gli36 CM was evaluated. The differentiation marker CD14 of THP-1 cells under the combined action of Gli36 CM and the specified mAbs against the specified target proteins was analyzed by flow cytometry. In THP-1 cell cultures containing Gli36 CM equivalent to 7.4 ng of Hsp70, 200 ng of mAbs were used.

[0114] The monoclonal antibody against Hsp70 showed the strongest inhibitory effect on Gli36 CM-induced THP-1 cell differentiation, which strongly indicated that cancer-secreted Hsp70 could stimulate THP-1 cell differentiation ( Figure 1 ).

[0115] Subsequently, flow cytometry analysis of the THP-1 differentiation marker CD14 in response to increasing concentrations of Hsp70 in Gli36 CM was performed. The results demonstrated the Hsp70 concentration-dependent THP-1 differentiation activity of Gli36 CM ( Figure 2 ).

[0116] Example 3. Hsp70 is Expressed in Glioma and Pancreatic Cancer

[0117] To evaluate the expression of Hsp70 mRNA in glioma (n = 153) and pancreatic cancer (n = 176) cells compared with normal cells (n = 121 and n = 248, respectively), data from The Cancer Genome Atlas project were queried. The results are as Figure 3 (glioma) and Figure 4(Pancreatic cancer) as shown, indicating that the expression of Hsp70 A1A mRNA in glioma and pancreatic cancer cells is relatively higher than that in their corresponding normal tissue cells.

[0118] Example 4. Cancer-Secreted Hsp70 is a Mediator of THP-1 Cell Differentiation

[0119] To rule out that THP-1 and SC cells are the source of Hsp70, Western blot analysis was performed on the lysates and CM of these cells cultured alone. Compared with the CM of Gli36 cells, Hsp70 was not detected in the CM of THP-1 and SC cells ( Figure 5 ). These results indicate that the observed differentiation of THP-1 cells is caused by Hsp70 in the CM secreted by cancer.

[0120] Example 5. Knockdown of Hsp70 in Cancer Cells Shifts Macrophage Differentiation towards the M1 Phenotype

[0121] Considering tumor growth and the polarization of macrophages within the tumor, the effect of CM-derived Hsp70 on macrophage differentiation was investigated. Hsp70 knockdown (KD) was performed by lentivirus-mediated expression of control shRNA (WT) or Hsp70-targeted shRNA in two cancer cell lines, LC-GFP and LN229. Illustrated are Western blot analyses of whole cell lysates of WT (control shRNA), shHsp70#1, or shHsp70#2 from LLC-GFP cells ( Figure 6 , upper left panel) and LN229 cells ( Figure 6 , upper right panel) probed with anti-Hsp70 monoclonal antibody or anti-actin monoclonal antibody. The expression of CD14 on THP-1 cells co-cultured with the CM of LLC-GFP cells ( Figure 6 , lower left panel) and LN229 cells ( Figure 6 , lower right panel) expressing control shRNA (WT) or shHsp70#2 was detected by flow cytometry.

[0122] In in vitro experiments, Hsp70-knockdown LLC-GFP and LN229 cells survived and grew normally, comparable to wild-type cells, most likely because the expression of Hsp70 was not completely eliminated ( Figure 6 , upper left and upper right panels). Knockdown of Hsp70 in LLC-GFP and LN229 cells led to a significant reduction in the macrophage differentiation activity of the CM obtained from LLC-GFP and LN229 cells compared with the CM obtained from cells expressing control shRNA ( Figure 6 , lower left and lower right panels).

[0123] Subsequently, LLC-GFP cells were subcutaneously implanted into the flanks of mice ( Figure 7, as shown in the upper figure), and observed for 16 days. Compared with LLC-GFP cells carrying control shRNA ( Figure 7 , lower figure, row 1), tumor growth from Hsp70 knockdown LLC-GFP cells ( Figure 7 , lower figure, row 2) was severely inhibited.

[0124] Flow cytometry was performed to detect the expression of the M2 macrophage marker CD206 in subcutaneous tumor macrophages isolated from wild-type LLC-GFP cells expressing control shRNA and Hsp70 knockdown tumors ( Figure 8 , upper left two figures). Flow cytometry was performed to detect the expression of the M1 macrophage marker iNOS in subcutaneous tumor macrophages isolated from wild-type LLC-GFP cells expressing control shRNA and Hsp70 knockdown tumors ( Figure 8 , upper right two figures).

[0125] The results showed that tumors from LLC-GFP cells with control shRNA (WT) mainly contained tumor-promoting M2 polarized macrophages (MO), while tumors from Hsp70 knockdown cells were very small and contained M1 polarized macrophages. The number of M1 macrophages in wild-type tumors was less than that in Hsp70 knockdown tumors ( Figure 8 , lower left and right lower figures).

[0126] Example 6. DOPG NVs can Block Cancer Cell CM-Induced Monocyte Differentiation and Inhibit Tumors in Mice Growth

[0127] Since studies have shown that cancer-secreted Hsp70 has a strong immunosuppressive effect on macrophages in the tumor microenvironment, the discovery of this new immunosuppressive pathway provides the possibility for innovative therapies. Hsp70 has been described as being able to bind to phospholipids (PLs), and this property has the potential to develop reagents that can interfere with the interaction between Hsp70 and TLR2, thereby inhibiting macrophage differentiation. The ability of nanovesicles derived from a series of phospholipids with different saturations and head groups to block the differentiation of THP-1 cells induced by cancer cell CM was evaluated.

[0128] Interestingly, after THP-1 cells treated with MiaPaCa-2 cell CM were co-incubated with DOPG NVs, the expression of CD14 on the surface of THP-1 cells was significantly reduced and showed a dose-dependent manner ( Figure 9 and Figure 10 ). In contrast, dioleoyl phosphatidic acid (DOPA) NVs, dioleoyl phosphatidylserine (DOPS) NVs, distearoyl phosphatidylcholine (DSPC) NVs, or distearoyl phosphatidylglycerol (DSPG) NVs had a weaker inhibitory effect on CD14 expression ( Figure 9 and Figure 10) The effect of DOPG NVs on THP-1 cell differentiation is unlikely to be due to toxicity, as can be seen from the results of propidium iodide (PI) staining of THP-1 cells treated with DOPG NVs, which is comparable to the PI staining results of THP-1 cells not treated with DOPG NVs. Figure 11 ) The non-cytotoxic effect of NVs was confirmed by treating MiaPaCa-2 cells with DOPG, DOPS, DOPA, DSPC, and DSPG NVs (100 μM) and staining with propidium iodide. The results showed that the tested PG NVs did not exhibit toxic effects on MiaPaCa-2 cells. Figure 16 )

[0129] Given the observed inhibitory effect of DOPG NVs on CM-induced THP-1 cell differentiation, the binding of DOPG NV to Hsp70 secreted by cancer cells was next evaluated. After co-incubating Gli36 CM with PL NV, NV-bound Hsp70 was separated into the pellet by ultracentrifugation, while free-form Hsp70 remained in the supernatant. ELISA assays showed that DOPG NV had a stronger binding affinity to Hsp70 compared to other PL NVs, as evidenced by the significant increase in Hsp70 in the pellet Figure 12 , upper panel) and the decrease in Hsp70 in the supernatant Figure 12 , lower panel). In addition, the binding of DOPG NV to Hsp70 was further confirmed by Western blot analysis. Figure 13 ) These results support the conclusion that DOPG NV can capture Hsp70 secreted by cancer cells and inhibit macrophage differentiation.

[0130] Next, the effects of DOPG NV on tumor growth and M2 polarization in vivo were evaluated. Intravenous injection of DOPG NV did not alter the body weight of mice, but significantly slowed tumor growth compared to sham-operated mice. Figure 14 ) Importantly, the inhibitory effect of DOPG NV on tumor growth was associated with a significant reduction in M2-polarized macrophages within the tumor. Figure 15 )

[0131] Aspects of the present disclosure may be described with reference to the following numbered clauses, with the preferred features listed in the dependent clauses. 1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising phosphatidylglycerol nanovesicles (NVs). 2. The method according to clause 1, wherein the per-lipid area of the phosphatidylglycerol is greater than about 3. The method according to any one of the preceding clauses, wherein the phosphatidylglycerol nanovesicles sequester heat shock protein 70 (Hsp70) secreted by cancer. 4. The method according to any one of the preceding clauses, wherein the composition does not comprise a second therapeutic agent. 5. The method according to any one of the preceding clauses, wherein the phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent. 6. The method according to any one of the preceding clauses, wherein the composition is administered by injection or infusion. 7. The method according to any one of the preceding clauses, wherein the cancer is selected from pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. 8. The method according to any one of the preceding clauses, wherein the composition consists essentially of phosphatidylglycerol nanovesicles. 9. The method according to any one of the preceding clauses, wherein the phosphatidylglycerol is selected from: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1,2-docosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol); and 1-Oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol), and combinations thereof. 10. A method for inhibiting M2 macrophage polarization in a tumor microenvironment, the method comprising administering to the tumor microenvironment an effective amount of a composition comprising phosphatidylglycerol nanovesicles (NVs). 11. The method according to clause 10, wherein the phosphatidylglycerol is unsaturated and the area per lipid is greater than about 12. The method according to clause 10 or clause 11, wherein the method is in vitro or in vivo. 13. The method according to any one of clauses 10 to 12, wherein the phosphatidylglycerol nanovesicles sequester heat shock protein 70 (Hsp70) secreted by cancer. 14. The method according to any one of clauses 10 to 13, wherein the sequestration reduces tumor volume and / or inhibits tumor growth. 15. The method according to any one of clauses 10 to 14, wherein the composition does not comprise a second therapeutic agent. 16. The method according to any one of clauses 10 to 15, wherein the phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent. 17. The method according to any one of clauses 10 to 16, wherein the composition consists essentially of phosphatidylglycerol nanovesicles. 18. The method according to any one of clauses 10 to 17, wherein the tumor is selected from pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. 19. The method according to any one of clauses 10 to 18, wherein the phosphatidylglycerol is selected from: 1,2-Dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-Dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-Dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-Dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-Dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1,2-docosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol); and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol), and combinations thereof. 20. The method according to any one of the preceding clauses, wherein the composition comprises phosphatidylglycerol nanovesicles at a concentration of from about 50 μM to about 350 μM. 21. The method according to any one of the preceding clauses, wherein the phosphatidylglycerol is 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG). 22. The method according to any one of the preceding clauses, wherein the cancer or tumor is characterized by increased Hsp70 secretion. 23. A pharmaceutical composition for treating cancer, comprising: a therapeutically effective amount of phosphatidylglycerol nanovesicles (NVs); and at least one pharmaceutically acceptable excipient, wherein the phosphatidylglycerol nanovesicles do not encapsulate a second therapeutic agent and are not conjugated to a second therapeutic agent. 24. The pharmaceutical composition according to clause 23, wherein the phosphatidylglycerol is unsaturated and the area per lipid (APL) of the phosphatidylglycerol is greater than about 25. The pharmaceutical composition according to clause 23 or clause 24, wherein the composition is formulated for injection or infusion. 26. The pharmaceutical composition according to any one of clauses 23 to 25, wherein the phosphatidylglycerol nanovesicles are the only therapeutic agent in the pharmaceutical composition. 27. The pharmaceutical composition according to any one of clauses 23 to 26, wherein the phosphatidylglycerol is selected from: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-Dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-Dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-Dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-Dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-Diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1,2-Docosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-(10Z-Heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol); and 1-Oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol), and combinations thereof. 28. The pharmaceutical composition according to any one of clauses 23 to 27, wherein the phosphatidylglycerol is 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG). 29. The pharmaceutical composition according to any one of clauses 23 to 28, wherein the composition comprises phosphatidylglycerol nanovesicles at a concentration of about 50 μM to about 350 μM. 30. A composition comprising phosphatidylglycerol nanovesicles (NVs) for use in a method of treating cancer, the method comprising administering the composition to a subject. 31. The composition according to clause 30, wherein the cancer is characterized by increased Hsp70 secretion. 32. The composition according to any one of clauses 30 to 31, wherein the per-lipid area of the phosphatidylglycerol is greater than about 33. The composition according to any one of clauses 30 to 32, wherein the phosphatidylglycerol nanovesicles sequester heat shock protein 70 (Hsp70) secreted by cancer. 34. The composition according to any one of clauses 30 to 33, wherein the composition does not contain a second therapeutic agent. 35. The composition according to any one of clauses 30 to 34, wherein the phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent. 36. The composition according to any one of clauses 30 to 35, wherein the composition is administered by injection or infusion. 37. The composition according to any one of clauses 30 to 36, wherein the cancer is selected from pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma. 38. The composition according to any one of clauses 30 to 37, wherein the composition consists essentially of phosphatidylglycerol nanovesicles. 39. The composition according to any one of clauses 30 to 38, wherein the phosphatidylglycerol is selected from: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1,2-docosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol); and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol), and combinations thereof. 40. The composition according to any one of clauses 30 to 39, wherein the phosphatidylglycerol is DOPG. 41. The composition according to any one of clauses 30 to 40, wherein the composition comprises phosphatidylglycerol nanovesicles at a concentration of about 50 μM to about 350 μM.

[0132] It should be noted that the terms "substantially" and "about" are used herein to represent the degree of uncertainty inherent in any quantitative comparison, numerical value, measurement, or other representation. These terms are also used to represent the degree of difference between a quantitative representation and the reference value, such that the basic function of the subject matter involved does not change. The term "substantially" is also used herein to represent the degree of difference between a quantitative representation and the reference value, such that the basic function of the subject matter involved does not change. Thus, it is used to represent the degree of uncertainty inherent in any quantitative comparison, numerical value, measurement, or other representation, referring to the arrangement of elements or features which, while theoretically expected to exhibit exact correspondence or behavior, may in practice manifest as not being entirely precise.

[0133] It should be noted that one or more of the following claims use the transitional phrase "wherein". For purposes of defining the technology herein, it should be noted that this word is introduced in the claim as an open transitional word for introducing a description of a series of features of a structure and should be interpreted in the same manner as the more common open preamble "comprising".

[0134] It should be understood that when a first component is described as "comprising" or "including" a second component, it is contemplated that in some embodiments, the first component "consists of" or "consists essentially of" the second component. Additionally, in the present disclosure, the term "consisting essentially of" is used to refer to those quantitative values that do not materially affect the basic and novel features of the present disclosure.

[0135] It should be understood that for any two quantitative values that assign an attribute or measurement value, a range of that attribute or measurement value can be formed, and the present disclosure encompasses all combinations of ranges formed by all the described quantitative values of the given attribute or measurement value.

[0136] Although specific embodiments have been illustrated and described in this specification, it should be understood that various other changes and modifications can be made without departing from the scope of the claimed subject matter. Additionally, although the various aspects of the claimed subject matter are described herein, these aspects need not be used in combination. Thus, the appended claims are intended to cover all such changes and modifications within the scope of the claimed subject matter.

Claims

1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising phosphatidylglycerol nanovesicles (NVs).

2. The method according to claim 1, wherein The per-lipid area of the phosphatidylglycerol is greater than about 3. The method according to claim 1, wherein, The phosphatidylglycerol nanovesicles sequester heat shock protein 70 (Hsp70) secreted by cancer.

4. The method according to claim 1, wherein The composition does not comprise a second therapeutic agent.

5. The method according to claim 1, wherein, The phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent.

6. The method according to claim 1, wherein, The composition is administered by injection or infusion.

7. The method according to claim 1, wherein The cancer is selected from pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma.

8. The method according to claim 1, wherein The composition consists essentially of phosphatidylglycerol nanovesicles.

9. The method according to claim 1, wherein The phosphatidylglycerol is selected from: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1,2-docosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol); and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol), and combinations thereof.

10. A method of inhibiting M2 macrophage polarization in a tumor microenvironment, the method comprising administering to the tumor microenvironment an effective amount of a composition comprising phosphatidylglycerol nanovesicles (NVs).

11. The method according to claim 10, wherein, The phosphatidylglycerol is unsaturated and the per-lipid area of the phosphatidylglycerol is greater than about 12. The method according to claim 10, wherein, The method is in vitro or in vivo.

13. The method according to claim 10, wherein, The phosphatidylglycerol nanovesicles sequester heat shock protein 70 (Hsp70) secreted by cancer.

14. The method according to claim 13, wherein, The sequestration reduces tumor volume and / or inhibits tumor growth.

15. The method according to claim 10, wherein, The composition does not comprise a second therapeutic agent.

16. The method according to claim 10, wherein The phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent.

17. The method according to claim 10, wherein, The composition consists essentially of phosphatidylglycerol nanovesicles.

18. The method according to claim 10, wherein, The tumor is selected from pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma.

19. The method according to claim 10, wherein, The phosphatidylglycerol is selected from: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1,2-docosahexaenoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol); and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol), and combinations thereof.

20. The method according to any one of the preceding claims, wherein The composition comprises phosphatidylglycerol nanovesicles at a concentration of about 50 μM to about 350 μM.

21. The method according to any one of the preceding claims, wherein, The phosphatidylglycerol is 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG).

22. The method according to any one of the preceding claims, wherein, The cancer or tumor is characterized by increased Hsp70 secretion.

23. A pharmaceutical composition for treating cancer, comprising: a therapeutically effective amount of phosphatidylglycerol nanovesicles (NVs); and at least one pharmaceutically acceptable excipient, Among them, wherein the phosphatidylglycerol nanovesicles do not encapsulate a second therapeutic agent and are not conjugated to a second therapeutic agent.

24. The pharmaceutical composition according to claim 23, wherein, The phosphatidylglycerol is unsaturated and the area per lipid (APL) of the phosphatidylglycerol is greater than about 25. The pharmaceutical composition according to claim 23, wherein, The composition is formulated for injection or infusion.

26. The pharmaceutical composition according to claim 23, wherein, The phosphatidylglycerol nanovesicles are the sole therapeutic agent in the pharmaceutical composition.

27. The pharmaceutical composition according to claim 23, wherein, The phosphatidylglycerol is selected from: 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dielaidoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinoleoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-dilinolenoyl-sn-glycero-3-phosphate-(1'-rac-glycerol); 1,2-diarachidonoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1,2-docosahexaenoyl-sn-glycerol-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-(10Z-heptadecenoyl)-sn-glycerol-3-phosphate-(1'-rac-glycerol); and 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate-(1'-rac-glycerol), and combinations thereof.

28. The pharmaceutical composition according to any one of claims 23 to 27, wherein The phosphatidylglycerol is 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG).

29. The pharmaceutical composition according to any one of claims 23 to 28, wherein, The composition comprises phosphatidylglycerol nanovesicles at a concentration of about 50 μM to about 350 μM.

30. A composition comprising phosphatidylglycerol nanovesicles (NVs) for use in a method of treating cancer, the method comprising administering the composition to a subject.

31. The composition according to claim 30, wherein, The cancer is characterized by increased Hsp70 secretion.

32. The composition according to any one of claims 30 to 31, wherein, The per-lipid area of the phosphatidylglycerol is greater than about 33. The composition according to any one of claims 30 to 32, wherein, The phosphatidylglycerol nanovesicles sequester heat shock protein 70 (Hsp70) secreted by the cancer.

34. The composition according to any one of claims 30 to 33, wherein, The composition does not comprise a second therapeutic agent.

35. The composition according to any one of claims 30 to 34, wherein, The phosphatidylglycerol nanovesicles are not conjugated to and / or do not encapsulate a second therapeutic agent.

36. The composition according to any one of claims 30 to 35, wherein, The composition is administered by injection or infusion.

37. The composition according to any one of claims 30 to 36, wherein, The cancer is selected from pancreatic ductal adenocarcinoma, glioblastoma, colorectal cancer, prostate cancer, hepatocellular carcinoma, lung cancer, skin cancer, cervical cancer, ovarian cancer, endometrial cancer, myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, and diffuse large B-cell lymphoma.

38. The composition according to any one of claims 30 to 37, wherein, The composition consists essentially of phosphatidylglycerol nanovesicles.

39. The composition according to any one of claims 30 to 38, wherein, The phosphatidylglycerol is selected from: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG); 1,2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1,2-dielaidoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1,2-dilinoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1,2-dilinolenoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1,2-diarachidonoyl-sn-glycerol-3-[phospho-rac-(1-glycerol)]; 1,2-docosahexaenoyl-sn-glycerol-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol); 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphate-(1'-rac-glycerol); and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate-(1'-rac-glycerol), and combinations thereof.

40. The composition according to any one of claims 30 to 39, wherein, The phosphatidylglycerol is DOPG.

41. The composition according to any one of claims 30 to 40, wherein, The composition comprises phosphatidylglycerol nanovesicles at a concentration of about 50 μM to about 350 μM.

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