Cancer cytotoxic exosome formulations and methods for use in the treatment of cancer

Abstract

The present invention relates to compositions and methods for treating cancer, particularly leukemia, using a cytotoxic composition comprising monocytes activated by β-glucan. The monocytes are preferably incubated with β-glucan and then treated to extract particles, such as microvesicles and exosomes, from the treated monocytes to produce the cytotoxic composition. Preferably, the cytotoxic composition contains at least 50% β-glucan-activated exosomes having a size of 150 nm or less. Zymosan is a preferred β-glucan. The cytotoxic composition has an apoptotic effect. When a subject with cancer is treated according to the preferred embodiment, the cytotoxic composition preferably induces a cytokine response in the subject's immune system. The combination of the cytotoxic composition and the cytokine response is synergistic.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 427,516, filed November 23, 2022.

[0002] FIELD OF THE INVENTION The present disclosure relates to cytotoxic compositions comprising exosomes that are stimulated with phagocytic cell activators to produce cancer cytotoxic exosomes ("CCEs"), methods for preparing such cytotoxic compositions, and methods for using such cytotoxic compositions in treating cancer, particularly leukemia. [Background technology]

[0003] Hematopoiesis is the process of blood cellular components, such as the specific generation of blood and bone marrow cells. Hematopoiesis has been widely studied, and understanding the process can lead to a better understanding of cancer. Specifically, understanding the role of hematopoietic stem cells (HSCs) can improve our understanding of tumorigenesis. HSC progenitors include erythrocytes, platelets, granulocytes, lymphocytes, and monocytes.

[0004] Pluripotent hematopoietic stem cells (HSCs) have the ability to divide into multipotent progenitor cells or to self-renew. HSCs then divide to produce specialized cells such as mature white blood cells, red blood cells, or platelets, increasing their numbers. Multipotent progenitor cells can initially divide into either myeloid progenitor cells or common lymphoid progenitor cells. Ultimately, common myeloid progenitor cells generate megakaryocytes, erythrocytes, basophils, neutrophils, eosinophils, and monocytes. B and T lymphocytes, as well as natural killer (NK) cells, are all produced by the same lymphoid progenitor cells. Multipotent progenitor cells cannot self-renew; therefore, any cell they produce will constantly divide into different cell types. The differentiation and division of HSCs and progenitor cells is largely governed by signaling factors such as erythropoietin, interleukin (IL)-2, IL-3, IL-6, and IL-7. These signaling factors are triggered by stressors such as invading pathogens, environmental changes, and / or other foreign particles and are released by many cells, including monocytes, endothelial cells, and neutrophils.

[0005] The immune system comprises two holistic systems: the adaptive immune system and the innate immune system. A key component of the innate immune system is the monocyte, a type of white blood cell derived from a common myeloid progenitor cell. Invading microbial cells possess PAMPs (pathogen-associated molecular patterns) on their surface that interact with monocyte toll-like receptors. Within 24 hours of responding to such stimuli, monocytes migrate from the bone marrow into the bloodstream and infiltrate tissues and anatomical spaces (e.g., the peritoneal cavity). First, monocytes adhere to the endothelium and then move freely across the arterial surface to infiltrate the affected site. During this process, monocytes ultimately cross the endothelium through a process known as extravasation. Monocytes can then penetrate the endothelial basement membrane and then travel toward the site of inflammation. Monocytes can further specialize into subtypes depending on their location and the signals they receive from the environment. Human monocytes can be classified into three major populations based on their cluster of differentiation (CD), including non-classical (CD14) and inflammatory (CD16) subtypes. dim , CD16 + ), classical (CD14 + , CD16 -), and intermediate (CD14 + CD16 + Monocyte subtypes are determined by cytokines and growth factors. Under certain conditions, such as prolonged infection and / or inflammation, monocytes can evolve into macrophages and dendritic cells, using a pattern recognition system, to control cellular homeostasis.

[0006] While in the peripheral circulation, monocytes act as phagocytes and antigen-presenting cells, consuming and eliminating bacteria, foreign bodies, and dead or damaged cells. Macrophages are an example of such antigen-presenting cells. Macrophages can further differentiate into different types of macrophages depending on their location. Some examples of these include histiocytes in connective tissue, microglia in the brain, osteoclasts in bone, mesangial cells in the kidney, and alveolar macrophages in the lungs. Monocytes also have the ability to produce cytokines, which attract more cells and proteins to the affected area and trigger a strong immune response.

[0007] In unicellular organisms, phagocytosis is an important process for nutrition. However, in multicellular species, it is the function of specialized phagocytes. Monocytes, macrophages, neutrophils, dendritic cells, osteoclasts, and eosinophils are examples of professional phagocytes in multicellular systems. To initiate the phagocytic process, professional phagocytes must first identify a particle. This is accomplished via specific receptors on the cell membrane. Binding of a particle to a particle-specific receptor allows these phagocytes to directly sense foreign substances. Two or more specific receptor types are present in a single phagocyte; therefore, the junction of different receptors cooperates to recognize and consume different extracellular particles within a single phagocyte. TLRs can activate phagocytic integrins from within, priming the cell for phagocytosis. During the phagocytic process, phagocytes are activated, resulting in the host's release of cytokines, hematopoietic factors, and other effector molecules.

[0008] The use of beta-glucans (β-glucans) as phagocyte activators to enhance immune responses as part of treatment programs for certain diseases and cancers is known in the art. The immune system includes two holistic systems: the adaptive immune system and the innate immune system. β-glucans are thought to act primarily through the relatively nonspecific innate immune system. The innate immune system includes complement proteins, macrophages, neutrophils, and natural killer (NK) cells, and serves as a rapid means of combating infection before the adaptive immune system is affected. Beta-glucans are complex carbohydrates generally derived from several sources, including yeast, bacteria, fungi, and plants. Each type of β-glucan has a unique structure, differing in the way glucose is linked together (e.g., linear β-1,3-glucans derived from bacteria and algae, branched β-1,3-glucans with β-1,6-linkages derived from yeast), and differs in physical and chemical properties. These differences translate into different abilities to elicit various cellular responses, such as phagocyte activation, cytokine expression and production, and activity against specific tumors, as well as the ability to administer β-glucans as part of a therapeutic program.

[0009] The structure of β-glucan derived from baker's yeast (Saccharomyces cerevisiae), also known as zymosan (ZM), is branched with 1,3-β and 1,6-β linkages, which enhances its ability to bind and stimulate macrophages, making it a potent anti-infective β-glucan immunomodulator. ZM is an insoluble β-glucan. ZM activates TLR2 and TLR6 on macrophages to generate inflammatory signals. ZM is also known to be a phagocytic cell inducer that enhances the release of proinflammatory cytokines. Particulate β-glucan and high-molecular-weight soluble β-glucans (e.g., lentinan and schizophyllan) have also been shown to induce cytokine release. In contrast, neutral soluble β-glucans do not induce cytokine release.

[0010] The use of several β-glucans to aid in treatment is known in the art. For example, U.S. Patent No. 8,791,252 discloses the oral administration of (1) a high-molecular-weight (40,000-359,000 dalton) β-glucan derived from barley, oats, wheat, or moss, which has a mixed β-1,3 and β-1,4 bond, together with (2) a tumor-specific, complement-activating monoclonal antibody to treat cancer. When administered alone, neither β-glucan nor the monoclonal antibody (such as the 3F8 mouse MoAb used in the experiments) affected tumor size, but when administered together, tumor growth was significantly reduced. Oral administration of β-glucan was found to be more effective than intraperitoneal administration when combined synergistically with intravenous administration of MoAb.

[0011] The '252 patent also discloses that other glucans with a 1,3-β backbone and a comb-like branch structure with 1,6-β-linked branches, such as lentinan, schizophyllan, and ZM derived from baker's yeast (Saccharomyces cerevisiae), are known for use in cytotoxic applications. However, the use of these glucans has several drawbacks. These drawbacks include the fact that they require the use of T cells, are expensive, insoluble, and difficult to administer, complicate the manufacturing and control process, and may contain proteins and non-β-glucan carbohydrates that may pose allergen concerns. They may also trigger cytokine release from macrophages, resulting in undesirable clinical toxicity. While the '252 patent teaches the direct administration of zymosan and monoclonal antibodies to a subject for treatment, it does not teach or suggest the stimulation or activation of macrophages or monocytes by exposure (ex vivo) to β-glucan, specifically zymosan, under certain conditions to produce CCEs and the use of the resulting CCEs in therapeutic compositions.

[0012] The use of microvesicles as delivery vehicles for therapeutic agents is also known in the art, and the therapeutic agent may include β-glucan. All blood cells secrete microvesicles (MVs) in vitro and in vivo upon stimulation or stress. Extracellular vesicles (ECVs) include a variety of vesicles, including exosomes, microvesicles, apoptotic bodies, ectosomes, and oncosomes. Increasing evidence suggests that ECVs play an important role in the pathophysiology of disease states. ECVs are important for cell-cell interactions and play a variety of roles, one of the most important of which is acting as signaling mediators. Because ECVs contain a variety of surface chemicals, they interact with various cellular receptors, providing a method for facilitating the exchange of materials between cells. Microvesicles (100-1000 nm diameter range), ectosomes (100-1000 nm diameter range), oncosomes (100-400 nm diameter range), apoptotic bodies (50-5000 nm diameter range), and exosomes (30-150 nm diameter range, average size 113.3 nm) are generated and circulate in the extracellular space near the point of release, where they are either taken up by neighboring cells or slowly degrade. Furthermore, some vesicles travel significant distances by diffusion before appearing in bodily fluids such as blood, urine, and cerebrospinal fluid. Exosomes and microvesicles share structural similarities but differ in size, lipid composition, content, and biological origin. The parent cell, microenvironment, and events leading to their release all influence the contents of ECVs. ECVs are manufactured and excreted through distinct processes. These processes also result in the release of intercellular signaling vesicles.

[0013] Microvesicles are small fragments derived from the plasma membrane that are released into the extracellular space due to pinching and budding of the plasma membrane. Proteins, lipids, and nucleic acids are specifically incorporated into the microvesicles at these sites and released into the environment. Ultimately, this leads to fusion with the target cell and release of the microvesicle components, delivering proteins, lipids, and bioactive chemicals.

[0014] Exosomes are membrane-enclosed vesicles produced intracellularly. Exosomes are initially formed via endocytosis, in contrast to microvesicles, which are formed via exocytosis. Exosomes are often produced by partitioning cargo, such as lipids, proteins, and nucleic acids, within endosomes. Once formed, endosomes associate with structures called multivesicular bodies (MVBs). Exosomes are released via exocytosis when MVBs carrying separated endosomes eventually bind to the plasma membrane. Microvesicles and exosomes are of great importance in research because they are carriers of biomarkers and ideal targets for drug therapy due to their role in many functions of intercellular communication.

[0015] Ectosomes are small vesicles with diameters between 0.1 and 1 μm. They are produced by budding directly from the plasma membrane and released into the extracellular environment. Phospholipids and phosphatidylserine are found on the surface of ectosomes. Ectosome release is currently thought to proceed via an actomyosin-based detachment process, but additional potential mechanisms of ectosome extrusion are emerging. Both resting and activated cells can release ectosomes, while macrophages and microglia are the primary producers. The name ectosomes is clear, emphasizing that these vesicles are released externally, rather than internally. While they are sometimes mistaken for exosomes and microvesicles, what distinguishes ectosomes from other ECVs is their release mechanism. For example, ectosomes are assembled and released from the plasma membrane, whereas exosomes are released by exocytosis of MVBs. Exosomes have the properties of their cell of origin and are usually composed of multiple components (proteins, mRNA, miRNA, etc.), which may differ depending on the state of the cell (e.g., resting vs. stimulated). Ectosomes are necessary for the communication network between cells.

[0016] Another type of ECV is the oncosome. They are membrane-derived secretory vesicles that transport tumorigenic information and protein complexes across cell boundaries. Oncosomes typically range in size from 100 to 400 nm in diameter and contain abnormal oncogenic macromolecules, such as oncogenic proteins. Furthermore, recent studies suggest the existence of "large oncosomes," ranging in size from 1 to 10 μm, but further studies will be required to confirm these suggestions. Oncosomes were first identified as vesicles that transport the oncoprotein EGFRvIII to tumor cells lacking such receptors, allowing the diffusion of tumor-promoting elements and causing mutations.

[0017] Another major type of ECV is apoptotic bodies, which are released during the final stages of apoptosis. Apoptotic cells can produce a variety of membrane-bound vesicles known as apoptotic extracellular vesicles (ApoEVs).

[0018] The use of microvesicle delivery vehicles for therapeutic agents, which may include β-glucan, is disclosed in U.S. Patent Application Publication No. 2020 / 0188311. The '311 application discloses its use for the treatment of inflammatory disorders by introducing a therapeutic agent (such as curcumin) that eliminates activated monocytes and macrophages and reduces the amount of inflammatory cytokines in the subject. Inflammatory disorders treated by the compositions of the '311 application include rheumatoid arthritis and colitis. The '311 application teaches that the compositions can also be used to treat brain tumors, breast cancer, lung cancer, and leukemia and other cancers.

[0019] Although the '311 application teaches microvesicles as equivalent to and interchangeable with exosomes, as discussed above, they are not the same. The '311 application also teaches that microvesicles have not been commercially fully utilized as delivery vehicles due to the difficulty of producing large quantities of microvesicles and the inability to effectively and efficiently utilize microvesicles to deliver therapeutic agents to target cells and tissues while preserving the biological activity of the therapeutic agent. The '311 application teaches encapsulating therapeutic agents in microvesicles specifically derived from edible plants such as grapes, grapefruit, or tomatoes, such that the lipid bilayer of the microvesicles surrounds the therapeutic agent and maintains the biological activity of the therapeutic agent. The encapsulation process includes the steps of: (1) mixing a therapeutic agent with isolated edible plant-derived microvesicles in a buffer solution (e.g., PBS), (2) incubating the mixture for a time to allow encapsulation (e.g., 5 minutes at 22°C), (3) subjecting the mixture to a sucrose gradient (e.g., 8, 30, 45, and 60% sucrose gradient) to separate the free therapeutic agent and free microvesicles from the therapeutic agent encapsulated within the microvesicles, (4) centrifuging to isolate the encapsulated therapeutic agent, and (5) recovering, washing, and dissolving the encapsulated therapeutic agent in an appropriate solution. By using edible plant-derived microvesicles or exosomes, the therapeutic composition of the '311 application is designed to reduce inflammatory cytokine responses in treated subjects. While the '311 application teaches the use of plant-derived microvesicles or exosomes as encapsulation delivery agents for various therapeutic agents, it does not teach or suggest the stimulation or activation of macrophages or monocytes by exposure to β-glucan, particularly zymosan, under specific conditions to produce CCEs (ex vivo) and the use of the resulting CCEs in therapeutic compositions.

[0020] There is a need in the art for more effective treatments of cancer, particularly leukemia, and there is also a need in the art for more effective methods for producing cytotoxic compositions that can stimulate monocytes or phagocytes to achieve apoptosis of cancer cells, preferably increasing the immune response in a treated subject by inducing a cytokine response. Summary of the Invention

[0021] The cytotoxic composition according to a preferred embodiment of the present invention comprises CCEs. Most preferably, exosomes are extracted from human or animal cells (such as phagocytes, monocytes, neutrophils, and / or any type of tissue macrophage) incubated with a phagocyte activator, preferably zymosan, to stimulate, activate, or induce cytotoxic properties, producing CCEs for use in treating diseases or conditions affecting animals (non-humans), humans, or both animals and humans. These cells, preferably from mammals, produce CCEs after ex vivo exposure to zymosan (or other phagocyte activators) under specific conditions, and the CCEs preferably contain normal biomolecular (rthomolecular) or human- or mammal-identical biologically active anti-cancer compounds. According to a preferred embodiment, the anti-cancer activity of the CCEs is further refined or enhanced through separation and enrichment of the CCEs by particle size and molecular weight stratification. Most preferably, the cytotoxic composition according to the preferred embodiment is used to treat cancer, particularly leukemia.

[0022] According to a preferred embodiment, the cytotoxic composition may comprise microvesicles extracted from phagocytes, macrophages, or monocytes. According to another preferred embodiment, the composition may comprise particles extracted from monocytes. According to yet another preferred embodiment, the particles comprise exosomes. According to another preferred embodiment, at least 40% of the particles have a size less than 150 nm, and most preferably at least 50% of the particles have a size less than 150 nm, so that the cytotoxic composition can comprise a majority of CCEs. The remaining particles in a preferred composition may comprise larger ECVs or β-glucan by-products.

[0023] According to other preferred embodiments, the exosomes and ECVs in the cytotoxic compositions disclosed herein are derived from human or animal (preferably mammalian) sources. According to another preferred embodiment, the exosomes and ECVs in the cytotoxic compositions disclosed herein are not derived from plant and / or non-mammalian sources.

[0024] According to a preferred embodiment, the β-glucan is derived from Saccharomyces cerevisiae (or baker's yeast). According to another preferred embodiment, the β-glucan is insoluble. According to another preferred embodiment, the β-glucan is branched at 1,3-β and 1,6-β bonds. According to another preferred embodiment, the β-glucan is zymosan. According to another preferred embodiment, the β-glucan has a MW of about 500 Da to 300 kDa, more preferably about 500 to 296 kDa, and most preferably about 296 to 300 kDa.

[0025] According to another preferred embodiment, the phagocyte activator may comprise zymosan, which may comprise β-glucan.

[0026] According to some preferred embodiments, the β-glucan may include (1) a β-glucan derived from barley, e.g., 1,3-1,4-β-glucan derived from barley, (2) a β-glucan derived from seaweed, e.g., 1,3-1,6-β-glucan (laminarin) derived from seaweed (Laminaria digitata), (3) 1,3-1,6-β-glucan (lentinan), (4) maitake glucan (including 1,3-1,6-β-glucan extracted from Grifola frondosa), (5) schizophyllan, and / or (6) a β-glucan derived from an edible plant (e.g., grapes, grapefruit, tomato, etc.).

[0027] According to yet other preferred embodiments, the β-glucan may comprise a combination of two or more of the aforementioned β-glucans.

[0028] According to other preferred embodiments, the β-glucan may not include (1) barley-derived β-glucan, e.g., 1,3-1,4-β-glucan derived from barley, (2) seaweed-derived β-glucan, e.g., 1,3-1,6-β-glucan (laminaran) derived from seaweed (Laminaria digitata), (3) 1,3-1,6-β-glucan (lentinan), (4) maitake glucan (including 1,3-1,6-β-glucan extracted from Grifola frondosa), (5) schizophyllan, and / or (6) β-glucan derived from edible plants (grapes, grapefruit, tomatoes, etc.).

[0029] Zymosan can be classified as a TLR (toll-like receptor) agonist, particularly a TLR-2 and TLR-6 agonist and ligand. TLR-2 and TLR-6 agonists may include heat-killed bacteria and lyophilized preparations of heat-killed bacteria, purified preparations of microbial components such as peptidoglycan, lipoteichoic acid, and zymosan, and synthetic TLR2 / TLR6 agonists such as chemically synthesized lipoproteins (e.g., Pam3CSK4) and small molecules (i.e., CU-T12-9).

[0030] According to other preferred embodiments, cytotoxic compositions comprising exosomes extracted from TLR-2 / TLR-6 agonist-induced phagocytes, monocytes, neutrophils, and / or any type of tissue macrophage are used to treat diseases or conditions that affect only animals (non-humans), conditions that affect only animals (non-humans), conditions that affect only humans, or conditions that affect both animals and humans. Most preferably, the cytotoxic compositions according to the preferred embodiments are used to treat cancer, particularly leukemia.

[0031] Preferred embodiments of the cytotoxic composition are preferably made using one or more of the preferred methods herein. According to certain preferred embodiments, the method of making the cytotoxic composition comprises: (A) collecting phagocytes, preferably phagocytes comprising monocytes, from a pooled mammalian (human or animal) source or directly from the mammal to be treated with the cytotoxic composition; (B) incubating the monocytes with β-glucan; (C) ultrafiltering the incubated solution to separate and isolate small particles from larger particles to produce an isolated solution; and (D) centrifuging the isolated solution to obtain the cytotoxic composition.

[0032] According to another preferred embodiment, step (C) comprises ultrafiltration using a filter medium to separate and isolate exosomes from larger particles such as microvesicles, the exosomes preferably being contained in an isolation solution.

[0033] According to another preferred embodiment, the cytotoxic composition further comprises or is added to a delivery vehicle for use in a method of treating a subject with the cytotoxic composition. The preferred route of administration is parenteral. Other possible routes of administration include intranasal, intraperitoneal, and subcutaneous administration. Most preferably, the cytotoxic composition is not administered orally. According to a preferred embodiment, the vehicle may comprise PBS or normal saline (NS). According to another preferred embodiment, for parenteral administration, the delivery vehicle may also comprise an acid and a base to adjust the pH, and may include one or more of a surfactant, an emulsifier, a suspending agent, a viscosity adjusting agent, a preservative, and an agent to adjust osmolality.

[0034] According to another preferred embodiment, a method of treating diseases and conditions, particularly cancer, with a cytotoxic composition may comprise administering to a subject a dose of a cytotoxic composition according to any preferred embodiment of the composition herein or according to any preferred embodiment of the method of making the composition herein. Most preferably, the cytotoxic composition induces a cytokine response upon administration to a subject that increases the effectiveness of the treatment. Any method of administration may be used according to some preferred embodiments.

[0035] According to one preferred method, the cytotoxic composition (or CCE solution) is injected intraperitoneally or intravenously into a subject (e.g., a human, dog, or cat) at a dose of approximately 4 mL of 10^9 CCEs / 0.1 mL per kg of subject body weight, or 0.04 mL x 10^11 CCEs / 0.1 mL per kg of subject body weight. For example, a human dose for a 70 kg human would be approximately 280 mL of 10^9 CCEs / 0.1 mL, 28 mL of 10^10 CCEs / 0.1 mL, or 2.8 mL of 10^11 CCEs / 0.1 mL. Other doses may also be used. In another preferred embodiment, the cytotoxic composition is sterile CCE (preferably at a concentration of approximately 10^10 to 10^11 per 0.1 mL) diluted with sterile PBS or NS (100-500 mL) and administered intravenously to the subject over several hours. Administration may be repeated daily, weekly, biweekly, triweekly, or monthly until the cancer stops progressing or remission of the cancer is established.

[0036] According to other preferred embodiments, the therapeutic composition is administered only intraperitoneally or intravenously, and not orally.

[0037] According to yet other embodiments, the cytotoxic therapeutic composition may comprise exosomes derived from CCE-producing phagocytes, monocytes, neutrophils, and / or tissue macrophages of any type, which have been stimulated with a phagocyte activator, preferably yeast-derived β-glucan.

[0038] Unlike prior art examples such as the '252 patent and the '311 application, preferred embodiments do not employ direct administration of zymosan to a subject or utilize exosomes (or microvesicles) as a delivery agent for an encapsulated therapeutic agent such as zymosan. Rather, preferred embodiments comprise a CCE solution (or cytotoxic composition) administered to a subject for treatment, the CCE solution comprising CCE produced ex vivo directly by monocytes or macrophages after exposure to an effector antigen (such as zymosan). While the CCE solution may contain modified units of zymosan, the zymosan is not encapsulated in exosomes, and any residual zymosan is likely to be eliminated during the preferred purification steps in the preferred methods for producing the CCE solution described herein. According to certain preferred embodiments, the CCE solution may contain exosomes activated by exposure to zymosan (or other phagocyte activators), but little or no zymosan (or other phagocyte activators) is present in the final CCE solution used for treatment. According to another preferred embodiment, the CCE solution does not contain exosome-encapsulated zymosan (or any phagocyte activator).

[0039] The use of cytotoxic compositions and methods according to preferred embodiments herein has been shown to be effective in having an apoptotic effect on cancer cells. The inventors have found that incubating β-glucan, particularly zymosan, with monocytes, followed by extraction and isolation of microvesicles and / or exosomes from the monocytes, according to preferred embodiments herein, to produce a cytotoxic composition comprising β-glucan-activated or stimulated microvesicles and / or exosomes, is effective in reducing the number of cancer cells and increasing their survival rate. Preferred embodiments of the methods and cytotoxic compositions herein provide an improvement in one or more areas of need in the art. [Brief explanation of the drawings]

[0040] The compositions and methods disclosed herein are further described and explained in connection with the following figures. [Figure 1] FIG. 1 is a flow chart outlining the process steps for the collection of mouse phagocytes, preferably including monocytes, according to one preferred embodiment. [Figure 2] FIG. 2 is a flow chart that outlines the process steps for the collection of human phagocytes, preferably including monocytes, according to a preferred embodiment. [Figure 3] FIG. 3 is a flow chart that outlines process steps for incubating phagocytes, preferably including monocytes, with zymosan, according to a preferred embodiment. [Figure 4] FIG. 4 is a flow chart that outlines the process steps for particle extraction from phagocytes to produce a cytotoxic composition comprising cancer cytotoxic exosomes (“CCE”), according to a preferred embodiment. [Figure 5] 5A and 5B are graphs showing the number and size of exosomes after exosome extraction processing based on FIG. 4 for rat phagocytes / monocytes collected based on FIG. 1. [Figure 6] 6A and 6B are graphs showing the number and size of exosomes after exosome extraction processing based on FIG. 4 for human phagocytes / monocytes collected based on FIG. 2. [Figure 7] FIG. 7 is a chart comparing particle size of extracted exosomes treated with zymosan according to certain preferred embodiments. [Figure 8] FIG. 8 is a graph showing the progression of chloroleukemia and survival time in rats without treatment according to a preferred embodiment. [Figure 9] FIG. 9 is a graph showing the apoptotic effect of a rat CCE composition according to a preferred embodiment on Mia C51 cells compared to a control. [Figure 10] FIG. 10 is a graph showing leukemia progression and survival in rats treated with a CCE composition according to a preferred embodiment compared to controls not treated with CCE. [Figure 11]FIG. 11 is a graph showing leukemia progression and survival in rats treated with a CCE composition according to a preferred embodiment compared to controls not treated with CCE. [Figure 12] FIG. 12 is a graph showing leukemia progression and survival in rats treated with a CCE composition according to a preferred embodiment compared to controls not treated with CCE. [Figure 13] FIG. 13 is a graph showing the apoptotic effect of a human CCE composition on KG1 cells in accordance with a preferred embodiment compared to a control. [Figure 14] FIG. 14 is a graph showing the apoptotic effect of a human CCE composition on HL60 cells in accordance with a preferred embodiment compared to a control. [Figure 15] FIG. 15 is a graph showing the apoptotic effect of a human CCE composition on Mia-PaCa-2 cells compared to a control, according to a preferred embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0041] Cytotoxic compositions according to preferred embodiments disclosed herein comprise microvesicles and / or exosomes derived from phagocytes, including one or more of monocytes, neutrophils, and any type of tissue macrophage, whose cytotoxic properties have been stimulated or activated by incubation with a phagocyte activator. According to a preferred embodiment, the phagocyte activator may comprise a TRL2 or TLR6 agonist or ligand. According to another preferred embodiment, the phagocyte activator may comprise a ZM. Accordingly, disclosed herein are methods for preparing such cytotoxic compositions and using such cytotoxic compositions to treat or prevent a disease or disorder in a subject (preferably a human), comprising administering to the subject a therapeutically effective amount of a composition comprising exosomes derived from monocytes or other phagocytes incubated with zymosan in a vehicle or carrier for intraperitoneal or intravenous administration. Most preferably, the vehicle or carrier is a pharmaceutically acceptable carrier composition suitable for use in humans and / or animals (non-humans).

[0042] According to certain preferred embodiments, a method of treating a disease or disorder with a cytotoxic composition may comprise administering to a subject having the disease or disorder (active or in remission), the cytotoxic composition being according to the preferred embodiments described herein. Preferably, the disease or disorder is cancer, such as leukemia. In other preferred embodiments, the disease treated by the cytotoxic composition is a hematopoietic cancer, such as leukemia, lymphoma, and myeloma. In other preferred embodiments, the disease treated by the cytotoxic composition involves or results in a non-hematopoietic solid malignancy.

[0043] According to other preferred embodiments, the cytotoxic composition may further comprise or be administered in combination with one or more additional cancer therapeutic agents, such as a vaccine, an antibody (e.g., a monoclonal antibody), and / or a chemotherapeutic agent. The efficacy of the ZM or TLR-2 / TLR6-induced CCE in the cytotoxic composition may be enhanced against leukemia and other cancers by pre- or post-administration of such additional cancer therapeutic agents to the cytotoxic composition containing the CCE exosomes.

[0044] According to other preferred embodiments, the cytotoxic composition may further comprise or be administered in combination with one or more diagnostic agents, such as bioluminescent tracers and / or radiopharmaceuticals / nuclear medicine agents. The ability of the CCE in the cytotoxic composition to preferentially target cancer cells by pre- or post-administration of such diagnostic agents may improve diagnostic sensitivity and accuracy in detecting cancer cells.

[0045] As used herein, the terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, or ameliorating a disease or condition, symptoms associated therewith, prolonging survival, and / or reducing tumor size or the number of cancer cells. Treating a disease or condition does not necessarily require that the disease, condition, or symptoms associated therewith be completely eliminated.

[0046] As used herein, the terms "treat," "treating," "treatment," and the like can also include "prophylactic treatment." This refers to reducing the probability of a disease or symptom recurring or recurring in a subject who is not experiencing recurrence or recurrence of the disease or symptom but is at risk of or susceptible to recurrence or recurrence, or of a disease or symptom that was previously controlled or a cancer in remission. The term "treat" and its synonyms contemplate administering a therapeutically effective amount of a composition of the present disclosure to an individual in need of such treatment. Within the meaning of the present disclosure, "treatment" also includes prevention of recurrence or phased prevention, as well as treatment of acute or chronic signs, symptoms, and / or dysfunctions. Treatment can be palliative, such as suppressing symptoms. It can be short-term, directed over the medium term, or can be long-term treatment, for example, within the scope of maintenance therapy. As used herein, the terms "prevent," "preventing," and "prevention" are art-recognized and, when used in reference to a condition, include administration of a composition that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to a subject who is not administered the composition. Thus, preventing cancer includes, for example, reducing the number of cancer cells in a subject, stabilizing the number of cancer cells in a subject, slowing the growth of cancer, or slowing a substantial increase in the number of cancer cells in a medical population compared to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0047] As used herein, the terms "derived," "extracted," "isolated," and the like refer to a second substance that has been removed or separated from a first substance, that exists away from its natural environment or form due to human processing, and therefore is not a product of nature.

[0048] As used herein, terms such as "activated," "stimulated," or "induced" with respect to an agent mean that a first agent (e.g., monocytes) is subjected to or treated with a second agent (e.g., β-glucan) to increase the biological activity of the first agent, to increase the effectiveness of the first agent, and / or to produce a new or improved effect of the first agent. As used herein, terms such as "activated," "stimulated," or "induced" with respect to a subject being treated with an agent (e.g., a cytotoxic composition administered to a subject) mean that the subject's biological activity (e.g., an increased immune or cytokine response) is increased, the effectiveness of the agent is increased in treating or preventing a disease or condition in the subject, and / or that the disease or condition is improved or the survival is prolonged.

[0049] A preferred method for preparing a cytotoxic composition includes the steps of: (A) collecting phagocytes, including one or more of monocytes, neutrophils, and any type of tissue macrophage; (B) incubating the phagocytes with a phagocyte activator to stimulate or activate their cytotoxic properties; (C) isolating exosomes by ultrafiltration to separate them from larger particles, such as microvesicles, in an isolation solution; and (D) centrifuging the isolation solution to obtain a cytotoxic composition containing CCE. In step (B), the phagocyte activator preferably includes one or more of β-glucan (preferably ZM) or a TRL2 or TLR6 agonist or ligand.

[0050] Referring to Figure 1, a preferred method for collecting phagocytes (preferably including monocytes) from an animal subject, specifically a rat for purposes of the experimental examples herein, according to step (A) is similar to the method described by Metcalf D et al. (1985) for mouse peritoneal monocyte collection, with some modifications. Preferably, step (A) comprises the steps of: (1) sacrificing 12-16 week old male Sprague-Dawley rats; and (2) flushing the peritoneal cavity of each rat with 15 mL of serum-free (SF) DMEM (Dulbecco's Modified Eagle's Medium) (an average of 12-14 mL is recovered, resulting in a cell concentration of 2.5-4 x 10). 6 / mL, monocytes were 60-70%.) and (3) the cell suspension was added to 75 cm 2 (3) placing the cells in a 37°C tissue culture flask and placing in a humidified incubator with 5% CO2 at 37°C; (4) after 3 hours of incubation, decanting the supernatant; and (5) adding 30 mL of SF DMEM to each flask containing adherent cells. Other known methods of collecting monocytes may also be used in step (A), and variations in specific procedures (temperature, concentration, etc.) may be used as will be understood by those skilled in the art.

[0051] Referring to Figure 2, a preferred method for collecting monocytes from a human subject according to step (A) involves (1) collecting blood in preservative-free anticoagulant; (2) layering the anticoagulated blood over Histopaque-1077 and centrifuging at 400 x g for 45 minutes at room temperature; (3) collecting the interface containing mononuclear cells and platelets, diluting it with culture RPMI 1640 (Sigma) 10% fetal calf serum (FCS), and centrifuging it at 800 RPM for 10 minutes; and (4) collecting the platelet-free pellet, resuspending it in culture medium (RPMI 1640), and centrifuging it at 75 cm. 2and placing the cells in an incubator at 37°C, 5% CO2, 100% humidity, and constant airflow; and (5) after 3 hours of incubation, decanting the supernatant and adding 30 mL of SF RPMI 1640 to each flask containing adherent cells. All steps are preferably performed under sterile conditions. It will be understood by those skilled in the art that other known methods of collecting phagocytes or monocytes may also be used in step (A), and that variations in specific procedures (temperature, concentration, etc.) may be employed.

[0052] 3, a preferred method for incubating collected phagocytes or monocytes with β-glucan according to step (B) includes the steps of: (6) adding β-glucan to each flask containing adherent monocytes at a concentration of about 2-20 μg / ml, more preferably about 10-20 μg / ml, and most preferably about 20 μg / ml; (7) incubating the solution in an incubator for an incubation time of about 24-40 hours, more preferably about 24-36 hours, and most preferably about 24 hours to produce an incubated supernatant; (8) recovering the incubated supernatant; (9) centrifuging the culture supernatant to produce a centrifuged supernatant; and (10) collecting and filtering the centrifuged supernatant to produce a filtered supernatant. Most preferably, step (B) is performed under sterile conditions.

[0053] The incubation in step (B)(7) is preferably carried out under one or more of the following conditions: (i) an incubation temperature of about 36.5-37°C, more preferably about 36.5-37°C, and most preferably about 37°C; (ii) a carbon dioxide level within the incubator maintained at about 4.0-5% CO2, more preferably about 4.5-5% CO2, and most preferably about 5% CO2; (iii) a humidity level within the incubator maintained at about 99-100% humidity, and most preferably 100% humidity; and / or (iv) a constant air flow (preferably ambient air) through the incubator. Most preferably, the incubating step in step (B)(7) may include each of conditions (i)-(iv).

[0054] The centrifugation in step (B)(9) is preferably carried out under one or more of the following conditions: (i) a temperature of about 3-4°C, most preferably about 4°C; (ii) a relative centrifugal force of about 550-600g, most preferably about 600g; and / or (iii) a duration of about 8-10 minutes, most preferably about 10 minutes. Most preferably, the centrifugation in step (B)(9) is carried out under each of conditions (i)-(iii). The centrifuged supernatant is filtered in step (B)(10), preferably using a 0.22-0.45 micron filter, most preferably a 0.22 micron filter (200 nm).

[0055] The β-glucan used in the incubation step is preferably zymosan (Sigma-Aldrich Z4250-1G). Saccharomyces cerevisiae is a yeast that can produce zymosan (ZM). Zymosan is an insoluble β-1,3-glucan polysaccharide with a molecular weight of approximately 296 kDa that plays an important role in the structure of the yeast host cell wall. Zymosan particles have been used as a model to understand how the innate immune system recognizes microorganisms. Zymosan activates TLR2 and TLR6 on macrophages to generate inflammatory signals. ZM is known to be a phagocytic cell inducer that enhances the release of inflammatory cytokines. ZM is also known to upregulate leukotriene production in monocytes. Macrophages can recognize ZM via pattern recognition receptors (PRRs). Several receptors, including the mannose receptor, complement receptor, and β-glucan receptor, are expressed by phagocytes such as monocytes, macrophages, and dendritic cells; therefore, they are all important in the internalization of zymosan particles. However, the absence of any one of these recognition mechanisms alone has little or no effect on zymosan internalization. In contrast to zymosan phagocytosis, zymosan-induced inflammation is controlled by a single receptor, the heterodimer of toll-like receptors TLR2 and TLR6. Zymosan must activate the TLR2 / TLR6 heterodimer to induce inflammatory responses, such as TNF-α production and NF-kB activation. Zymosan phagocytosis is followed by the recruitment of TLR2 and TLR6 to the phagosome.

[0056] In steps (C) and (D), the filtered supernatant from step (B) is then preferably ultrafiltered and centrifuged to separate CCEs from larger ECVs. Ultrafiltration is a pressure-driven process that uses a semipermeable membrane to separate particles based on molecular size. Only molecules smaller than the pores of the semipermeable membrane can pass through. Ultrafiltration of exosomes is used in the isolation process from the original biological fluid of interest. Ultrafiltration may be performed at about 5°C by slightly modifying the method described previously in "Blatt WF, Robinson SM: Membrane ultrafiltration: The diafiltration technique and its application to microsolute exchange and binding phenomena. Anal Biochem 26:151, 1968." More preferably, Blatt's ultrafiltration is modified according to the following preferred steps:

[0057] 4, a preferred method for ultrafiltration according to step (C) and centrifugation according to step (D) is shown. A preferred method for ultrafiltration according to step (C) may include: (11) ultrafiltering the filtered supernatant from step (B) through a filter having a molecular weight (MW) cutoff of about 100-500 kDa, more preferably about 200-500 kDa, and most preferably about 500 kDa, at an operating pressure of about 5-10 psi, more preferably about 5-7.5 psi, and most preferably about 5 psi, to produce an effluent; (12) collecting the effluent and adding polyethylene glycol 300 (PEG) to the effluent; and (13) mixing them and gently shaking the solution in a shaker to produce a mixed solution.

[0058] A preferred centrifugation method according to step (D) includes the steps of: (14) centrifuging the mixed solution; (15) discarding the supernatant and decanting the remaining PEG (for about 5 minutes) to form a pellet; (16) resuspending the pellet in PBS and vortexing the suspension vigorously for about 1.5 to 3.5 minutes, more preferably for about 2 to 3.5 minutes, and most preferably for about 3 to 3.5 minutes to form a pellet suspension; and (17) transferring the pellet suspension to an appropriate number of filter units with a molecular weight cutoff of about 100 kDa (exosomes have a molecular weight > 100 kDa). (17) centrifuging the filter unit to separate the PBS (bottom) from the semi-fluid pellet solution containing the exosomes (top) and discarding the PBS; (18) recovering the exosome-containing pellet solution and resuspending the exosome-containing semi-fluid pellet solution in PBS to produce an aliquot of a cytotoxic composition comprising CCE; and (19) freezing the aliquot of the cytotoxic composition for future use (if desired, the composition may be used at or near the time of manufacture).

[0059] The PEG added in step (C)(12) is preferably about 2-2.8 mL per 3 mL of effluent, more preferably about 2.5-2.8 mL per 3 mL of effluent, and most preferably about 2.8 mL per 3 mL of effluent. The PEG and effluent are mixed, preferably at a temperature of about 4-5°C, most preferably about 4°C, and shaken on a shaker for about 24-28 hours, most preferably about 24 hours. The centrifugation in step (D)(14) is preferably carried out under one or more of the following conditions: (i) a temperature of about 4-5°C, most preferably about 4°C; (ii) a relative centrifugal force of about 9,000-10,000 g, most preferably about 10,000 g; and / or (iii) a duration of about 50-60 minutes, most preferably about 60 minutes. Most preferably, the centrifugation in step (D)(14) is carried out under each of conditions (i) to (iii).

[0060] The amount of PBS added in step (D)(16) is preferably about 40-50 mL per pellet, more preferably about 45-50 mL per pellet, and most preferably about 50 mL per pellet. Vortexing in step (D)(16) is preferably performed at a temperature of about 4-5°C, most preferably about 4°C.

[0061] Centrifugation in step (D)(17) is preferably carried out under one or more of the following conditions: (i) a temperature of about 4-5°C, most preferably about 4°C; (ii) a relative centrifugal force of about 2,800-3,000 g, most preferably about 3,000 g; and / or (iii) a duration of about 35-40 minutes, most preferably about 40 minutes. Most preferably, centrifugation in step (D)(17) is carried out under each of conditions (i)-(iii). The PBS added in step (D)(18) is preferably added in an amount of about 40-50 mL per semi-fluid pellet, more preferably about 45-50 mL per semi-fluid pellet, most preferably about 50 mL per semi-fluid pellet. Aliquots of the cytotoxic composition containing CCE are preferably frozen at a temperature of about -80°C until ready for their use in step (D)(19). If necessary, thawing is preferably performed in a 37°C water bath for 3-5 minutes.

[0062] One preferred method for treating a mammal suffering from cancer using a cytotoxic composition, preferably a composition according to a preferred embodiment of the present disclosure, comprises the steps of: (1) collecting phagocytes, including one or more of monocytes, neutrophils, and any type of tissue macrophage, from a pooled human or animal source or directly from the mammal to be treated; (2) treating the phagocytes with a phagocyte activator (preferably β-glucan, most preferably zymosan) at a concentration sufficient to produce phagocyte-derived cytotoxic particles; (3) extracting and purifying the cytotoxic particles to produce a cytotoxic composition primarily comprising exosomes; (4) formulating the cytotoxic composition into a suitable sterile, injectable pharmaceutical delivery formulation; and (5) injecting the pharmaceutical delivery formulation into the mammal suffering from cancer at a dose and frequency sufficient to effectively treat, reduce, or eliminate the cancer or help prevent recurrence. Steps (1) through (3) are preferably performed according to steps (A) through (D) described herein for preparing a cytotoxic composition. As will be appreciated by those skilled in the art, any number of acceptable pharmaceutical delivery components, such as PBS or NS, may be added to the cytotoxic composition to prepare a pharmaceutical delivery formulation. Most preferably, the mammal being treated is a human, dog, or cat, although other animals can also be treated. The concentration of the phagocyte activator (preferably β-glucan, most preferably zymosan) for producing the cytotoxic particles is 20 μg / mL to 200 μg / mL.

[0063] According to certain preferred embodiments, the dose of the cytotoxic composition may contain a CCE concentration per subject (e.g., human, dog, or cat) body weight, preferably injected intraperitoneally or intravenously into the subject according to the following ranges and examples: approximately 4 mL at 10^9 CCEs / 0.1 mL per kg of subject body weight, or 0.04 mL at 10^11 CCEs / 0.1 mL per kg of subject body weight. For example, a human dose for a 70 kg human would be approximately 280 mL at 10^9 CCEs / 0.1 mL, 28 mL at 10^10 CCEs / 0.1 mL, or 2.8 mL at 10^11 CCEs / 0.1 mL. Other dosage amounts may also be used. In another preferred embodiment, the cytotoxic composition is sterile CCEs (preferably at a concentration of approximately 10^10 to 10^11 per 0.1 mL) diluted in sterile PBS or NS (100-500 mL) and administered intravenously to the subject over several hours. Administration may be repeated daily, weekly, biweekly, triweekly, or monthly until the cancer stops progressing or remission of the cancer is established.

[0064] For comparative purposes to evaluate the effectiveness of the cytotoxic composition containing CCE prepared and used according to the preferred embodiment, naive exosomes ("NE") were also collected for use as a control. The NE were collected in the same manner as the CCE, including incubation, described above, except that no β-glucan was added in step (2)(a) prior to incubation.

[0065] To confirm that the method for producing a cytotoxic composition according to a preferred embodiment primarily collected exosomes rather than larger microvesicle particles, Nanosight software was used to generate Figures 5-7, which show the particle size and count in rat and human cytotoxic composition samples generated according to the preferred method herein. Nanosight is used to characterize nanoparticles in liquid by assessing Brownian motion. The assessments provided in Figures 5A-5B and 6A-6B indicate that the majority of particles in the rat and human cytotoxic compositions were approximately 110 nanometers in size, which is within the exosome size range (30-150 nm) and is close to the average size of exosomes in previous studies. Different lines in these figures represent the results of testing different samples. Serial dilutions of the original samples (before the freezing step) with PBS revealed that the optimal dilution ratio for exosome analysis was 1:40. The original exosome concentrations in both the rat and human cytotoxic compositions based on Nanosight analysis were 5 × 10 10 The Nanosight composite analysis of rat and human exosomes in Figure 7 shows that the majority of particles in the cytotoxic composition are smaller than 150 nm (50% of the particles were calculated to be 141.2 nm). This demonstrates that the preferred method can produce a cytotoxic composition that contains primarily CCEs, rather than other larger ECVs. From a size and function standpoint, it is desirable to have primarily exosomes. Preferably, at least 40% of the particles in the cytotoxic composition produced according to the preferred method herein are smaller than 150 nm in size, indicating that they contain CCEs; more preferably, at least 45% of the particles in the cytotoxic composition produced according to the preferred method herein are smaller than 150 nm in size, and most preferably, at least 50% of the particles in the cytotoxic composition produced according to the preferred method herein are smaller than 150 nm in size. The remaining particles in the cytotoxic composition produced according to the preferred method herein may contain ECVs.

[0066] The following examples illustrate the apoptotic effect of cytotoxic compositions containing CCE according to preferred embodiments disclosed herein. The apoptotic rate was measured using a fluorescent microscope according to the manufacturer's instructions using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method (DeadEnd TM The number of apoptotic cells and viable cells from the cancer cell lines used in the examples was then determined.

[0067] <Example using C51 chloroleukemia in rat Mia>

[0068] The first example model used to test the efficacy of cytotoxic compositions containing CCE prepared according to the preferred method of the present invention was the Mia C51 chloroleukemia model of acute myeloid leukemia in rats. In this model, 1 x 10 5 It has been demonstrated that intraperitoneal injection of Mia C51 results in peritoneal leukemia, with 100% of animals dying from leukemia complications by day 23 post-injection.

[0069] Parental chloroleukemia was first generated by gastric instillation of 20-methylcholanthrene, followed by injection of chloroleukemia cells into neonatal Sprague-Dawley rats (NTac:SD). The resulting cell line (Mia C51, established by Yunis et al. at the University of Miami in 1975) is a well-characterized myeloid leukemia cell line that exhibits characteristics of human chloroleukemia (leukemia, leukemic ascites, and chloroma formation) and human myeloid leukemia. It is maintained in Gibco Dulbecco's Modified Eagle Medium (DMEM)-10% fetal calf serum (FCS) at 37°C in a humidified incubator with 5% CO2.

[0070] For the purposes of this example, MiaC51 cells grown in culture and harvested during the logarithmic phase were collected, centrifuged, and diluted to 1 x 10 6 The cells were resuspended at a concentration of 0.1 mL / mL. Subsequently, 0.1 mL was injected intraperitoneally into randomized 7-day-old rat pups while they were manually restrained. Prior to injection, the area was disinfected with an alcohol swab. The needle, syringe, and cells remained sterile. As shown in Figure 8, 23 days after injection (day 30), all animals succumbed to leukemia.

[0071] Example 1 - The direct effect of a cytotoxic composition (also called a CCE solution) containing CCE prepared according to the preferred method of the present invention was then tested in vitro against Mia C51 cells, as shown in Figure 9. Rat CCE or NE were harvested and resuspended in PBS (in addition to the PBS from step (D)(18) above) to give 25 x 10 6 , 50×10 6 and 1 x 10 7 Three test samples were prepared at exosome concentrations of vesicles / ml. Each concentration of CCE or NE solution was then added directly to Mia C51 cells in a final volume of 0.1 mL CCE (or NE) / mL Mia C51. Control cells were treated with 0.1 mL PBS. As seen in Figure 9, after 48 hours, the PBS control and NE solutions had no apoptotic effect on Mia C51 cell density, and cell density did not decrease. In contrast, the CCE solution caused a dramatic decrease in Mia C51 cell density. The CCE solution exhibited a dose-dependent apoptotic effect on Mia C51 cells (measured by the TUNEL assay described above), with a cell density of 1 x 10 7 When treated with CCE solution at a concentration of 0.05, Mia C51 cells did not survive.

[0072] Example 2 - The effect of the CCE solution prepared according to the preferred method of the present invention was then tested in vivo in rats. Twenty 7-day-old Long-Evans rats were treated with 1 x 10 5Mia C51 were injected intraperitoneally (IP) (day 7) and randomly divided into two groups of 10 mice each. Starting on day 8, group 1 was IP injected daily with 0.1 mL of PBS for 7 days and served as a control. Group 2 received approximately 1 x 10 mice in 0.1 mL of PBS (in addition to the PBS from step (D)(18) above), prepared according to the preferred method herein. 9 CCE solution containing exosomes at a concentration of 0.01 mg / kg / day was administered IP daily for 7 days. As shown in Figure 10, all CCE-treated rats survived and remained leukemia-free at the end of the 30-day study period. In contrast, none of the control rats survived beyond 30 days.

[0073] Example 3 - The effect of the CCE solution prepared according to the preferred method of the present invention was then tested in vivo in rats, with the timing of treatment initiation varied compared to Example 2. Twenty 7-day-old Long-Evans rats were treated with 1 x 10 5 Mia C51 were injected IP (day 7) and randomly divided into two groups of 10 mice each. Starting on day 10 (as opposed to day 8 in Example 2), group 1 was injected IP with 0.1 mL of PBS daily for 7 days to serve as a control, and group 2 received approximately 1 x 10 mice in 0.1 mL of PBS (in addition to the PBS from step (D)(18) above), prepared according to the preferred method herein. 9 A CCE solution containing exosomes at a concentration of 0.01 mg / kg / day was administered IP daily for 7 days. As shown in Figure 11, all CCE-treated rats survived and remained leukemia-free at the end of the 50-day study period. In contrast, none of the control rats survived beyond 30 days. At the end of the 50th day, the CCE-treated rats were sacrificed, and the blood and organs (liver, spleen, and bone marrow) were examined microscopically. No evidence of leukemia was observed.

[0074] Example 4 - The effect of the CCE solution prepared according to the preferred method of the present invention was then tested in vivo in rats, with the timing of treatment initiation varied compared to Example 2. Twenty 7-day-old Long-Evans rats were treated with 1 x 10 5Mia C51 was injected IP (day 7) and randomized into two groups of 10 mice each. Starting on day 22 (15 days after Mia C51 injection, as opposed to starting on day 8 in Example 2 or day 10 in Example 3), group 1 was injected IP with 0.1 mL of PBS daily for 7 days to serve as a control, and group 2 received approximately 1 x 10 mice in 0.1 mL of PBS (in addition to the PBS from step (D)(18) above) prepared according to the preferred method herein. 9 CCE solution containing exosomes at a concentration of 0.01 mg / kg / day was administered IP daily for 7 days. On day 22, leukemia was confirmed in 100% of animals by detection of Mia C51 cells in blood samples. As shown in Figure 12, three rats in Group 2 died of leukemia before the start of CCE treatment on day 22. A rat in Group 1 also died before reaching day 22. After treatment initiation, one rat in Group 2 with progressive disease died of leukemia one day after initiating treatment with CCE solution. However, six of the 10 rats treated with CCE solution survived to day 50 without evidence of leukemia, even when treatment initiation was delayed. All rats in the control group died of leukemia by day 30.

[0075] Example using human CCE

[0076] <Example 5> - Next, as shown in Figure 13, the direct effect of the CCE solution prepared according to the preferred method of the present invention was tested on KG1 cells in vitro.

[0077] The KG1 cell line was isolated from bone marrow aspirate of a 59-year-old male patient with erythroleukemia that progressed to acute myeloid leukemia. It is maintained in Iscove's Modified Dulbecco's Medium (IMDM) with fetal bovine serum at a final concentration of 20% at 37°C in a humidified incubator with 5% CO2. This cell line was specifically selected for its application in research and other immune system disorders.

[0078] Human CCE or NE were harvested and resuspended in PBS (in addition to the PBS from step (D)(18) above) to give 25 × 10 6 , 50×10 6 and 1 x 10 7 Three test samples were prepared at exosome concentrations of vesicles / ml. Each concentration of CCE or NE solution was then added directly to KG1 cells in a final volume of 0.1 mL CCE (or NE) / mL KG1. Control cells were treated with 0.1 mL PBS. As seen in Figure 13, after 48 hours, the PBS control and NE solutions had no apoptotic effect on KG1 cell density, and KG1 cell density did not decrease. In contrast, the CCE solution caused a dramatic decrease in KG1 cell density. The CCE solution exhibited a dose-dependent apoptotic effect on KG1 cells (measured by the TUNEL assay described above), with a cell density of 1 x 10 7 When treated with a CCE solution at a high concentration, almost no cells survived.

[0079] Example 6 Next, as shown in Figure 14, the direct effect of the CCE solution prepared according to the preferred method of the present invention was tested on HL60 cells in vitro.

[0080] HL60 cells are described as an acute myeloid leukemia cell line and were isolated from the peripheral blood of a 36-year-old female patient with acute promyelocytic leukemia. They are maintained in Iscove's modified Dulbecco's medium (IMDM) with fetal bovine serum at a final concentration of 20% in a humidified incubator at 37°C and 5% CO2. This cell line was selected due to its rapid doubling rate and the ease with which it can be grown consistently in suspension culture with the addition of nutrients and antibiotics.

[0081] Human CCE or NE were harvested and resuspended in PBS (in addition to the PBS from step (D)(18) above) to give 25 × 10 6 , 50×10 6 and 1 x 10 7Three test samples were prepared at exosome concentrations of vesicles / ml. Each concentration of CCE or NE solution was then added directly to HL60 cells in a final volume of 0.1 mL CCE (or NE) / mL HL60. Control cells were treated with 0.1 mL PBS. As seen in Figure 14, after 48 hours, the PBS control and NE solutions had no apoptotic effect on HL60 cell density, and HL60 cell density did not decrease. In contrast, the CCE solution caused a dramatic decrease in HL60 cell density. The CCE solution exhibited a dose-dependent apoptotic effect on HL60 cells (measured by the TUNEL assay described above), with a concentration of 1 x 10 7 When treated with a CCE solution at a concentration of 0.1, no cells survived.

[0082] Example 6 Next, as shown in Figure 15, the direct effect of the CCE solution prepared according to the preferred method of the present invention was tested on Mia-PaCa-2 cells in vitro.

[0083] The Mia-PaCa-2 cell line was isolated from pancreatic tumor tissue of a 65-year-old male patient. This cell line was specifically selected for its application in cancer research. It is maintained in Iscove's Modified Dulbecco's Medium (IMDM), fetal bovine serum (FBS), at a final concentration of 20%, in a humidified incubator at 37°C and 5% CO2. This cell line can double in approximately 40 hours.

[0084] Human CCE or NE were harvested and resuspended in PBS (in addition to the PBS from step (D)(18) above) to give 25 × 10 6 , 50×10 6 and 1 x 10 7Three test samples were prepared at exosome concentrations of 100 vesicles / ml. Each concentration of CCE or NE solution was then added directly to Mia-PaCa-2 cells in a final volume of 0.1 mL CCE (or NE) / mL Mia-PaCa-2. Control cells were treated with 0.1 mL of PBS. As seen in Figure 15, after 48 hours, the PBS control and NE solutions had no apoptotic effect on Mia-PaCa-2 cell density, and Mia-PaCa-2 cell density did not decrease. In contrast, the CCE solution caused a dramatic decrease in Mia-PaCa-2 cell density. The CCE solution exhibited a dose-dependent apoptotic effect on Mia-PaCa-2 cells (measured by the TUNEL assay described above), with a 1x10 7 When treated with a CCE solution at a concentration of 0.1, no cells survived.

[0085] Cytotoxic compositions and methods according to the disclosed embodiments can achieve a reduction in cancer cell (especially leukemic cancer cell) density of at least about 95%, more preferably at least about 100%, compared to before treatment with the cytotoxic composition or CCE solution.

[0086] Other embodiments of cytotoxic compositions and methods of preparing and using same include any combination of the following:

[0087] A. A method for preparing a cytotoxic composition for treating cancer, comprising: (1) collecting phagocytes, including one or more of monocytes, neutrophils, and any type of tissue macrophage; (2) incubating the phagocytes with β-glucan to produce an incubated phagocyte composition; (3) ultrafiltering the incubated phagocyte composition to separate and isolate exosomes into an isolated composition; and (4) centrifuging the isolated composition to produce a cytotoxic composition.

[0088] B. A method for preparing a cytotoxic composition as described in paragraph A, wherein the step of incubating phagocytes comprises: (1) adding β-glucan to the phagocytes at a concentration of about 10-20 μg / ml in the phagocytes to produce a β-glucan composition; (2) placing the β-glucan composition in an incubator under a first set of conditions for an incubation period of about 24-36 hours to produce an incubated supernatant; (3) centrifuging the incubated supernatant to produce a first centrifugation supernatant; and (4) filtering the first centrifugation supernatant to produce an incubated phagocyte composition.

[0089] C. A method of preparing a cytotoxic composition described in paragraph B, wherein the first set of conditions includes one or more of: (1) an incubation temperature of about 36.5-37°C; (2) a carbon dioxide level in the incubator of about 4.5-5% CO2; (3) a humidity level in the incubator of about 99-100% humidity; or (4) maintaining a constant air flow through the incubator.

[0090] D. A method for preparing a cytotoxic composition described in paragraph B, wherein the first set of conditions includes one or more of: (1) an incubation temperature of about 36.5-37°C; (2) a carbon dioxide level in the incubator of about 4.5-5% CO2; (3) a humidity level in the incubator of about 99-100% humidity; and (4) maintaining a constant air flow through the incubator.

[0091] E. A method for preparing a cytotoxic composition described in any of paragraphs A to D, wherein the step of centrifuging the incubated supernatant comprises centrifugation under a second set of conditions including one or more of: (1) a temperature of about 3 to 4°C; (2) a relative centrifugal force of about 500 to 600 g; and (3) a duration of about 8 to 10 minutes.

[0092] F. A method for preparing a cytotoxic composition described in any one of paragraphs A to E, wherein the step of ultrafiltering the incubated phagocyte composition comprises passing the incubated phagocyte composition through a filter having a molecular weight (MW) cutoff of about 200 to 500 kDa at an operating pressure of about 5 to 7.5 psi to produce an effluent.

[0093] G. A method for preparing a cytotoxic composition described in any of paragraphs A-F, wherein centrifugation of the isolated composition comprises: (1) adding PBS to the isolated composition to produce a pellet suspension; and (2) centrifuging the pellet suspension in a filter unit under a third set of conditions, the third set of conditions comprising one or more of: (a) a temperature of about 36.5-37°C; (b) a relative centrifugal force of about 2800-3000 g; and (c) a duration of about 2-3 minutes.

[0094] H. A method for preparing a cytotoxic composition described in any of paragraphs A-G, comprising ultrafiltering the incubated phagocyte composition, further comprising adding PEG to the effluent in an amount of about 2-2.8 mL for every 3 mL of effluent, and agitating the PEG and effluent on a shaker to form an isolated composition; wherein the step of centrifuging the isolated composition further comprises centrifuging the isolated composition under a fourth set of conditions to form a second centrifuged supernatant before the step of adding PBS, discarding the second centrifuged supernatant, and decanting the PEG to form a pellet; wherein the step of adding PBS comprises adding PBS to the pellet and further comprises vortexing the PBS and pellet to form a pellet suspension; and wherein the fourth set of conditions comprises one or more of: (a) a temperature of about 4-5°C; (b) a relative centrifugal force of about 9,000-10,000 g; and (c) a duration of about 50-60 minutes.

[0095] I. A method for preparing a cytotoxic composition according to any of paragraphs AH, wherein the β-glucan comprises zymosan.

[0096] J. A method of treating a mammal suffering from cancer using a cytotoxic composition prepared according to any of paragraphs A-I, or any other cytotoxic composition comprising a β-glucan, preferably zymosan, comprising the steps of: (1) preparing the cytotoxic composition in a suitable sterile injectable pharmaceutical delivery formulation; and (2) injecting the pharmaceutical delivery formulation into a mammal suffering from cancer at a dose and frequency effective to treat, reduce, or eliminate the cancer, or help prevent recurrence.

[0097] K. A method of treating a mammal afflicted with cancer using a cytotoxic composition, comprising: (1) collecting phagocytic cells, including one or more of monocytes, neutrophils, and any type of tissue macrophage, from a pooled human or animal source or directly from the mammal to be treated; (2) treating the phagocytic cells with a phagocytic activator at a concentration that produces phagocytic cell-derived cytotoxic particles; (3) extracting and purifying the cytotoxic particles to produce a cytotoxic composition primarily comprising exosomes; (4) formulating the cytotoxic composition into a suitable sterile injectable pharmaceutical delivery formulation; and (5) injecting the pharmaceutical delivery formulation into the mammal afflicted with cancer at a dose and frequency that effectively treats, reduces, or eliminates the cancer or helps prevent recurrence.

[0098] L. The method of paragraph J or K, wherein the mammal is a human, dog, or cat.

[0099] M. The method of any of paragraphs JL, wherein the phagocyte activator is a β-glucan.

[0100] N. The method of paragraph M, wherein the β-glucan is zymosan.

[0101] O. The method of any of paragraphs JL, wherein the phagocyte activator is a TRL2 or TLR6 agonist or ligand.

[0102] P. The method of any of paragraphs JO, wherein the cancer being treated is leukemia.

[0103] Q. The method of paragraph N, wherein the zymosan concentration used is 10 μg / mL to 20 μg / mL of phagocytes.

[0104] R. The method of any of paragraphs JQ, wherein the pharmaceutical delivery formulation is a sterile injectable intravenous formulation.

[0105] S. The dose is approximately 1 x 10 9 ~1×10 11 and the frequency is once daily for seven consecutive days.

[0106] T. A cytotoxic composition prepared by the method of any of paragraphs AI.

[0107] U. A cytotoxic composition comprising exosomes extracted from a phagocyte solution incubated with zymosan at a concentration of approximately 10 μg / mL to 20 μg / mL, wherein the phagocyte solution comprises phagocytes collected from a mammal.

[0108] V. The cytotoxic composition of paragraph U, wherein the phagocyte comprises one or more of a monocyte, a macrophage, or a neutrophil.

[0109] W. The cytotoxic composition of paragraph U, wherein the phagocytes comprise any type of tissue macrophage.

[0110] X. The cytotoxic composition of paragraph U, wherein the phagocyte comprises a Kupffer cell.

[0111] Y. The cytotoxic composition of any of paragraphs U-X, wherein the phagocyte solution is incubated with zymosan at an incubation temperature of about 36.5-37°C, a carbon dioxide level of about 4.5-5% CO2, and a humidity level of about 99-100% humidity while maintaining a constant airflow for a duration of about 24-36 hours.

[0112] Z. The cytotoxic composition of any of paragraphs U-Y, wherein the phagocyte solution primarily contains exosomes.

[0113] All numerical values, ratios, or percentages given herein as ranges include each individual amount, value, or ratio within that range, and any subset combination within that range (including subsets that overlap from one preferred range to a more preferred range). Unless specifically excluded, any preferred feature and any component of any composition embodiment and / or method step described herein can be used with any other embodiment even if not specifically described herein with that particular embodiment.

[0114] References to "about" or "approximately" in reference to a numerical value typically mean + / - 1 or + / - 0.5 for a value expressed as an integer (no decimal point) or to one decimal place (e.g., approximately 36.5°C means 35.5 to 37.5°C or 36 to 37°C).

[0115] Additionally, the terms "a" or "an" are used to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one, and the singular also includes the plural unless it is clear that it is meant otherwise.

[0116] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of features is not necessarily limited to only those features and may include features not expressly listed or other features inherent to such process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, "or" refers to an "inclusive-or" and not an "exclusive-or." For example, condition A or B is satisfied by any one of the following: (1) A is true (or present) and B is false (or absent), (2) A is false (or absent) and B is true (or present), and (3) both A and B are true (or present).

[0117] While embodiments of the present invention and their advantages have been described in detail herein, it should be understood that various changes, substitutions, and alterations could be made therein without departing from the spirit and scope of the present disclosure, as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the compositions and methods and processes described herein, but rather the scope of the inventions disclosed herein is intended to be limited only by the broadest interpretation of the appended claims to which the inventors are legally entitled.

Claims

1. 1. A method for preparing a cytotoxic composition for treating cancer, comprising: collecting phagocytes, including one or more of monocytes, neutrophils, and any type of tissue macrophages; incubating phagocytes with β-glucan to form an incubated phagocyte composition; ultrafiltering the incubated phagocyte composition to separate and isolate exosomes into an isolated composition; centrifuging the isolated composition to produce a cytotoxic composition; A method comprising:

2. Incubating phagocytes adding β-glucan to the phagocytes at a concentration of about 10-20 μg / ml of phagocytes to form a β-glucan composition; placing the β-glucan composition in an incubator under a first set of conditions for an incubation period of about 24 to 36 hours to produce an incubated supernatant; centrifuging the incubated supernatant to produce a first centrifugation supernatant; filtering the first centrifugation supernatant to produce an incubated phagocyte composition; 2. A method for preparing the cytotoxic composition of claim 1, comprising:

3. The first set of conditions was: (1) an incubation temperature of about 36.5-37°C; (2) about 4.5-5% CO ２ 3. The method of preparing the cytotoxic composition of claim 2, comprising one or more of: (1) maintaining a constant carbon dioxide level in the incubator at about 99-100% humidity; (2) maintaining a constant humidity level in the incubator at about 99-100% humidity; and (3) maintaining a constant air flow through the incubator.

4. The first set of conditions was: (1) an incubation temperature of about 36.5-37°C; (2) about 4.5-5% CO ２ 3. A method for preparing the cytotoxic composition of claim 2, comprising: (1) maintaining a constant carbon dioxide level in the incubator at about 99-100% humidity; (2) a constant humidity level in the incubator at about 99-100% humidity; and (3) a constant air flow through the incubator.

5. 4. The method of preparing a cytotoxic composition of claim 3, wherein centrifuging the incubated supernatant comprises centrifugation under a second set of conditions comprising one or more of: (1) a temperature of about 3-4°C; (2) a relative centrifugal force of about 500-600 g; and (3) a duration of about 8-10 minutes.

6. 6. The method for preparing a cytotoxic composition of claim 5, wherein ultrafiltering the incubated phagocyte composition comprises passing the incubated phagocyte composition through a filter having a molecular weight (MW) cutoff of about 200 to 500 kDa at an operating pressure of about 5 to 7.5 psi to generate an effluent.

7. Centrifuging the isolated composition includes (1) adding PBS to the isolated composition to form a pellet suspension; and (2) centrifuging the pellet suspension in a filter unit under a third set of conditions; 7. The method of preparing a cytotoxic composition of claim 6, wherein the third set of conditions comprises one or more of: (a) a temperature of about 36.5-37°C; (b) a relative centrifugal force of about 2800-3000 g; and (c) a duration of about 2-3 minutes.

8. Ultrafiltering the incubated phagocyte composition further comprises adding PEG to the effluent in an amount of about 2-2.8 mL for every 3 mL of effluent, and agitating the PEG and effluent on a shaker to form the isolated composition; Centrifuging the isolated composition further includes centrifuging the isolated composition under a fourth set of conditions to produce a second centrifuged supernatant before adding PBS, discarding the second centrifuged supernatant, and decanting the PEG to produce a pellet; adding PBS can include adding PBS to the pellet, and can further include vortexing the PBS and pellet to form a pellet suspension; 8. The method of preparing a cytotoxic composition of claim 7, wherein the fourth set of conditions comprises one or more of: (a) a temperature of about 4-5°C; (b) a relative centrifugal force of about 9,000-10,000 g; and (c) a duration of about 50-60 minutes.

9. The method for preparing a cytotoxic composition according to claim 8, wherein the β-glucan comprises zymosan.

10. The method for preparing a cytotoxic composition according to claim 3, wherein the β-glucan comprises zymosan.

11. 1. A method of treating a mammal suffering from cancer using a cytotoxic composition, comprising: collecting phagocytes, including one or more of monocytes, neutrophils, and any type of tissue macrophages, from a pooled human or animal source or directly from the mammal to be treated; treating phagocytes with a phagocyte activator at a concentration that produces phagocyte-derived cancer cell cytotoxic particles; Extracting and purifying the cancer cytotoxic particles to produce a cytotoxic composition primarily comprising exosomes; preparing a pharmaceutical delivery formulation comprising a cytotoxic composition; injecting a pharmaceutical delivery formulation into a mammal suffering from cancer at a dose and frequency that effectively treats, reduces, or eliminates the cancer or helps prevent recurrence; A method comprising:

12. 12. The method of claim 11, wherein the mammal suffering from cancer is a human, dog, or cat.

13. The method of claim 12, wherein the phagocyte activator is a β-glucan.

14. The method of claim 13, wherein the β-glucan is zymosan.

15. 12. The method of claim 11, wherein the phagocyte activator is a TRL2 or TLR6 agonist or ligand.

16. 15. The method of claim 14, wherein the cancer is leukemia.

17. 17. The method of claim 16, wherein the zymosan used in treating the phagocytes has a concentration of 10 μg / mL to 20 μg / mL of the phagocytes.

18. 12. The method of claim 11, wherein the pharmaceutical delivery formulation is a sterile injectable intravenous infusion.

19. The dose is approximately 1 x 10 ９ ~1 x 10 １１ and the frequency is once daily for seven consecutive days.

20. The exosomes are extracted from a phagocyte solution incubated with zymosan at a concentration of about 10 μg / mL to 20 μg / mL. The cytotoxic composition, wherein the phagocyte solution comprises phagocytes obtained from a mammal.

21. 21. The cytotoxic composition of claim 20, wherein the phagocytes comprise one or more of monocytes, macrophages, and neutrophils.

22. 22. The cytotoxic composition of claim 21, wherein the phagocyte comprises any type of tissue macrophage.

23. 21. The cytotoxic composition of claim 20, wherein the phagocyte comprises a Kupffer cell.

24. The phagocyte solution is incubated at an incubation temperature of about 36.5-37°C, about 4.5-5% CO ２ 22. The cytotoxic composition of claim 21, wherein the cytotoxic composition is incubated with zymosan at a carbon dioxide level of about 1000 psi, a humidity level of about 99-100% humidity, while maintaining a constant airflow for a duration of about 24-36 hours.

25. The cytotoxic composition of claim 24, wherein the phagocyte solution primarily contains exosomes.

26. The cytotoxic composition of claim 21 , wherein the phagocyte solution primarily contains exosomes.

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