US 12,433,915 B2
Cancer cytotoxic exosome formulations and methods for use in treating cancer
Joaquin J. Jimenez, Miami, FL (US); and John P. McCook, Frisco, TX (US)
Assigned to JJR&D, LLC, Celina, TN (US)
Filed by JJR&D, LLC, Celina, TN (US)
Filed on Nov. 20, 2023, as Appl. No. 18/514,019.
Claims priority of provisional application 63/427,516, filed on Nov. 23, 2022.
Prior Publication US 2024/0189350 A1, Jun. 13, 2024
Int. Cl. A61K 35/15 (2025.01); A61K 9/00 (2006.01); A61K 40/17 (2025.01); A61P 35/00 (2006.01); A61P 35/02 (2006.01); C12N 5/0786 (2010.01); C12N 5/0787 (2010.01)
CPC A61K 35/15 (2013.01) [A61K 9/0019 (2013.01); A61K 40/17 (2025.01); A61P 35/02 (2018.01); C12N 5/0642 (2013.01); C12N 5/0645 (2013.01); A61K 2239/38 (2023.05)] 27 Claims
 
1. A method of preparing a cytotoxic composition for treating cancer, the method comprising steps of:
(A) collecting phagocytic cells, comprising monocytes;
(B) incubating the phagocytic cells with a β-glucan to form an incubated phagocytic composition, wherein incubating the phagocytic cells comprises: (1) 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; (2) 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; (3) centrifuging the incubated supernatant under a second set of conditions to produce a first centrifuged supernatant; and (4) filtering the first centrifuged supernatant to produce the incubated phagocytic composition comprising exosomes that are cytotoxic to cancer cells;
(C) ultrafiltering the incubated phagocytic composition to separate out and isolate the exosomes in an isolation composition, 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, wherein the isolation composition comprises the effluent; and
(D) centrifuging the isolation composition to produce a supernatant and a pellet, wherein the pellet comprises the exosomes;
wherein the first set of conditions in step (B)(2) 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;
wherein the second set of conditions in step (B)(3) comprises: (1) a temperature of around 3 to 4° C.; (2) a relative centrifugal force of around 500 to 600 g; and (3) for a duration of around 8 to 10 minutes; and
wherein the cytotoxic composition comprises the exosomes.