# US 2024/0189350 A1 · Cancer Cytotoxic Exosome Formulations and Methods for Use in Treating Cancer > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2024/0189350 A1 - Document ID: US20240189350A1 - Status: Published application - Kind: A1 - Application number: 18/514,019 - Filed: 2023-11-20 - Published: 2024-06-13 - Earliest claimed: 2022-11-23 - Assignees: Jjr&D, LLC - Applicants: Jjr&D, LLC - Inventors: Joaquin J. Jimenez; John P. McCook - Web page: https://patentgrep.com/patent/US20240189350A1 ## CPC - A61K 35/15: Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells (presenting a specific antigen A61K39/00; therapeutic combinations of antibodies, or fragments thereof, and blood-derived cells A61K39/00) - A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)} - A61K 39/0005: {Vertebrate antigens (from snakes A61K39/38)} - A61K 40/17: Monocytes; Macrophages - A61P 35/02: specific for leukemia - C12N 5/0642: {Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells} - C12N 5/0645: {Macrophages, e.g. Kuepfer cells in the liver; Monocytes} - A61K 9/08: Solutions {(composition of solutions A61K47/00)} - A61K 2039/572: {cytotoxic response} - A61K 2039/804: {Blood cells [leukemia, lymphoma]} - A61K 2239/38: characterised by the dose, timing or administration schedule - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Compositions and methods for treating cancer, particularly leukemia, using a cytotoxic composition comprising monocytes activated by β-glucan. The monocytes are preferably incubated with the β-glucan and then processed to extract particles, such as microvesicles and exosomes from the treated monocytes to produce the cytotoxic composition. Preferably the cytotoxic composition comprises at least 50% exosomes having a size of 150 nm or less that are activated with β-glucan. Zymosan is the preferred β-glucan. The cytotoxic composition has an apoptosis effect. When a subject having cancer is treated according to preferred embodiments, the cytotoxic composition preferably induces a cytokine response in the subject's immune system. The combination of the cytotoxic composition and cytokine response are synergistic. ## Claims ### Claim 1 A method of preparing a cytotoxic composition for treating cancer, the method comprising: collecting phagocytic cells, comprising one or more of monocytes, neutrophils, and any type of tissue macrophage; incubating the phagocytic cells with a β-glucan to form an incubated phagocytic composition; ultrafiltering the incubated phagocytic composition to separate out and isolate exosomes in an isolation composition; and centrifuging the isolation composition to produce the cytotoxic composition ### Claim 2 (depends on 1) The method of preparing a cytotoxic composition according to claim 1 wherein incubating the phagocytic cells comprises: adding the β-glucan to the phagocytic cells at a concentration of around 10 to 20 μg/ml of the phagocytic cells 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 time of around 24 to 36 hours to produce an incubated supernatant; centrifuging the incubated supernatant to produce a first centrifuged supernatant; filtering the first centrifuged supernatant to produce the incubated phagocytic composition. ### Claim 3 (depends on 2) The method of preparing a cytotoxic composition according to claim 2 wherein the first set of conditions comprises one or more of: (1) an incubation temperature of around 36.5 to 37° C., (2) a carbon dioxide level in the incubator of around 4.5 to 5% CO2; (3) a humidity level in the incubator of around 99 to 100% humidity; or (4) maintaining constant air flow through the incubator. ### Claim 4 (depends on 2) The method of preparing a cytotoxic composition according to claim 2 wherein the first set of conditions comprises: (1) an incubation temperature of around 36.5 to 37° C., (2) a carbon dioxide level in the incubator of around 4.5 to 5% CO2; (3) a humidity level in the incubator of around 99 to 100% humidity; and (4) maintaining constant air flow through the incubator. ### Claim 5 (depends on 3) The method of preparing a cytotoxic composition according to claim 3 wherein centrifuging the incubated supernatant comprises centrifuging under a second set of conditions comprising one or more of: (1) a temperature of around 3 to 4° C.; (2) a relative centrifugal force of around 500 to 600 g; or (3) for a duration of around 8 to 10 minutes. ### Claim 6 (depends on 5) The method of preparing a cytotoxic composition according to claim 5 wherein ultrafiltering the incubated phagocytic composition comprises: passing the incubated phagocytic composition through a filter with a molecular weight (MW) cutoff of around 200 to 500 kDa at an operating pressure of around 5 to 7.5 psi to produce an effluent. ### Claim 7 (depends on 6) The method of preparing a cytotoxic composition according to claim 6 wherein centrifuging the isolation composition comprises: (1) adding PBS to the isolation composition to produce a pellet suspension; and (2) centrifuging the pellet suspension in filter units under a third set of conditions; wherein the third set of conditions comprises one or more of: (a) a temperature of around 36.5 to 37 ºC; (b) a relative centrifugal force of around 2800 to 3000 g; or (c) a duration of around 2 to 3 minutes. ### Claim 8 (depends on 7) The method of preparing a cytotoxic composition according to claim 7 wherein ultrafiltering the incubated phagocytic composition further comprises adding PEG to the effluent in an amount of around 2 to 2.8 mL for every 3 mL of the effluent and rocking the PEG and effluent in a shaker to form the isolation composition; and wherein centrifuging the isolation composition further comprises centrifuging the isolation composition under a fourth set of conditions prior to adding PBS to produce a second centrifuged supernatant, discarding the second centrifuged supernatant, and decanting the PEG to produce a pellet; wherein adding the PBS comprises adding PBS to the pellet and further comprises vortexing the PBS and pellet to produce the pellet suspension; and wherein the fourth set of conditions comprises one or more of: (a) a temperature of around 4 to 5° C.; (b) a relative centrifugal force of around 9,000 to 10,000 g; or (c) a duration of around 50 to 60 minutes. ### Claim 9 (depends on 8) The method of preparing a cytotoxic composition according to claim 8 wherein the β-glucan comprises zymosan. ### Claim 10 (depends on 3) The method of preparing a cytotoxic composition according to claim 3 wherein the β-glucan comprises zymosan. ### Claim 11 A method of treating a mammal afflicted with a cancer using a cytotoxic composition, the method comprising: collecting phagocytic cells, comprising 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; treating the phagocytic cells with a phagocytic cell activator at a concentration to produce cancer cytotoxic particles derived from the phagocytic cells; extracting and purifying the cancer cytotoxic particles to produce the cytotoxic composition comprising primarily exosomes; preparing a pharmaceutical delivery formulation comprising the cytotoxic composition; and injecting the pharmaceutical delivery formulation into the mammal afflicted with cancer at a dose and frequency to effectively treat, reduce, or eliminate the cancer or aid in preventing recurrence. ### Claim 12 (depends on 11) The method of claim 11 wherein the mammal afflicted with the cancer is a human, a canine, or a feline. ### Claim 13 (depends on 12) The method of claim 12 wherein the phagocytic cell activator is a β-glucan. ### Claim 14 (depends on 13) The method of claim 13 wherein the β-glucan is Zymosan. ### Claim 15 (depends on 11) The method of claim 11 wherein the phagocytic cell activator is a TRL2 or TLR6 agonist or ligand. ### Claim 16 (depends on 14) The method of claim 14 wherein the cancer is leukemia. ### Claim 17 (depends on 16) The method of claim 16 wherein the Zymosan used in treating the phagocytic cells has a concentration between 10 μg/mL of the phagocytic cells to 20 μg/mL of the phagocytic cells. ### Claim 18 (depends on 11) The method of claim 11 wherein the pharmaceutical delivery formulation is a sterile injectable intravenous infusion. ### Claim 19 (depends on 11) The method of claim 11 wherein the dose comprises an exosome concentration of around 1×109 and 1×1011 and the frequency is once daily for 7 consecutive days. ### Claim 20 A cytotoxic composition comprising exosomes extracted from a phagocyte solution incubated with zymosan in a concentration of around 10 pg/mL of the phagocyte solution to 20 μg/mL of the phagocyte solution; wherein the phagocyte solution comprises phagocytic cells collected from a mammal. ### Claim 21 (depends on 20) The cytotoxic composition according to claim 20 where in the phagocytic cells comprise one or more of monocytes, macrophages, or neutrophils. ### Claim 22 (depends on 21) The cytotoxic composition according to claim 21 where in the phagocytic cells comprise any type of tissue macrophages. ### Claim 23 (depends on 20) The cytotoxic composition according to claim 20 where in the phagocytic cells comprise Kupffer cells. ### Claim 24 (depends on 21) The cytotoxic composition according to claim 21 where in the phagocyte solution is incubated with the zymosan at an incubation temperature of around 36.5 to 37° C., a carbon dioxide level of around 4.5 to 5% CO2, a humidity level of around 99 to 100% humidity, and while maintaining constant air flow for a duration of around 24 to 36 hours. ### Claim 25 (depends on 24) The cytotoxic composition according to claim 24 wherein the phagocyte solution comprises primarily exosomes. ### Claim 26 (depends on 21) The cytotoxic composition according to claim 21 wherein the phagocyte solution comprises primarily exosomes.