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US-20260124152-A1 - METHOD OF PRODUCING ENGINEERED EXOSOME-MIMETIC VESICLES

US20260124152A1US 20260124152 A1US20260124152 A1US 20260124152A1US-20260124152-A1

Abstract

A method for producing one or more engineered exosome-mimetic vesicles for targeted drug delivery, including, engineering one or more eukaryotic cells to stably express a Chimeric Antigen Receptor (CAR) protein on their surface; incubating the one or more engineered eukaryotic cells with a therapeutic cargo; and subjecting the one or more incubated cells to a mechanical extrusion process to form nanoscale one or more engineered exosome-mimetic vesicle with the CAR protein displayed on their surface and the therapeutic cargo encapsulated within.

Inventors

  • Peng Shi
  • Xi Zhao

Assignees

  • Hong Kong Centre for Cerebro-Cardiovascular Health Engineering Limited

Dates

Publication Date
20260507
Application Date
20251023

Claims (20)

  1. 1 . A method for producing one or more engineered exosome-mimetic vesicles for targeted drug delivery, comprising: engineering one or more eukaryotic cells to stably express a Chimeric Antigen Receptor (CAR) protein on their surface; incubating the one or more engineered eukaryotic cells with a therapeutic cargo; and subjecting the one or more incubated cells to a mechanical extrusion process to form nanoscale one or more engineered exosome-mimetic vesicle with the CAR protein displayed on their surface and the therapeutic cargo encapsulated within.
  2. 2 . The method of claim 1 , wherein the eukaryotic cells are human embryonic kidney (HEK293) cells.
  3. 3 . The method of claim 1 , wherein the CAR protein is a CD19-targeting CAR.
  4. 4 . The method of claim 1 , wherein the therapeutic cargo is doxorubicin (DOX).
  5. 5 . The method of claim 1 , wherein the mechanical extrusion process comprises sequentially extruding the cells through polycarbonate porous membranes having progressively smaller pore sizes.
  6. 6 . The method of claim 5 , wherein the polycarbonate porous membranes comprise pore sizes of 10 μm, 5 μm, ‘μm, 0.8 μm, 0.4 μm, 0.2 μm or any combination thereof.
  7. 7 . The method of claim 5 , wherein a first extrusion is performed through a polycarbonate porous membrane having a pore size of 5 μm, a second extrusion is performed through a polycarbonate porous membrane having a pore size of 0.8 μm, and a third extrusion is performed through a polycarbonate porous membrane having a pore size of 0.4 μm.
  8. 8 . The method of claim 1 wherein the step of engineering the human embryonic kidney cells (HEK293) comprising the step of performing plasmid transfection on the HEK293 cells to engineer the HK293 cells to express CD19-targeting CAR protein.
  9. 9 . The method of claim 1 , further comprising, the step of isolating the one or more engineered exosome-mimetic vesicles.
  10. 10 . The method of claim 1 , wherein the engineered exosomes comprise a peak size of 145 nm.
  11. 11 . The method of claim 1 , wherein the engineered exosome is configured to achieve a cumulative release concentration of 5.57 μg/ml at 9 hours.
  12. 12 . The method of claim 3 , wherein the cells are incubated with DOX for two hours.
  13. 13 . A method of producing chimeric antigen receptor-presenting exosome-mimetic vesicles loaded with a therapeutic agent, comprising: stably transfecting human embryonic kidney (HEK293) cells with a nucleic acid encoding a CD19-targeting chimeric antigen receptor; incubating the transfected HEK293 cells in a suspension comprising doxorubicin (DOX); sequentially extruding the incubated HEK293 cell suspension through polycarbonate membranes having pore sizes of about 5 μm, about 0.8 μm, and about 0.4 μm to form nanoscale vesicles displaying the chimeric antigen receptor on an outer surface thereof and encapsulating the doxorubicin, and recovering the formed nanoscale vesicles.
  14. 14 . The method of claim 13 , wherein the incubating step comprises incubating the transfected HEK293 cells with doxorubicin at a concentration of about 10 μg/mL for a period of two hours at 37° C.
  15. 15 . The method of claim 14 , wherein the recovered nanoscale vesicles have a mean particle size of from about 120 nm to about 160 nm as measured by nanoparticle tracking analysis.
  16. 16 . The method of claim 14 , further comprising formulating the recovered nanoscale vesicles into a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  17. 17 . An engineered exosome-mimetic vesicle for targeted drug delivery, comprising: a biomimetic vesicle derived from a eukaryotic cell engineered to express a Chimeric Antigen Receptor (CAR) protein on its surface, a therapeutic cargo encapsulated within the vesicle, and wherein the CAR protein is configured to specifically recognize and bind to a target antigen on a target cell, and the vesicle is produced by a mechanical extrusion method.
  18. 18 . The engineered exosome-mimetic vesicle of claim 17 , wherein the eukaryotic cell is a human embryonic kidney (HEK293) cell, wherein the CAR protein is a CD19-targeting CAR, and wherein the therapeutic cargo is doxorubicin (DOX).
  19. 19 . The engineered exosome-mimetic vesicle of claim 17 , wherein the vesicle has a peak particle size distribution of approximately 145 nm.
  20. 20 . The engineered exosome-mimetic vesicle of claim 17 , wherein the mechanical extrusion method involves sequentially extruding cells through polycarbonate porous membranes with progressively smaller pore sizes, and wherein the therapeutic cargo is encapsulated within the vesicle during its formation via the mechanical extrusion method.

Description

TECHNICAL FIELD The present disclosure relates to a method of producing one or more engineered exosome-mimetic vesicles, and an engineered exosome-mimetic vesicle. BACKGROUND Immunotherapy represents a significant advancement in modern medicine, utilising the body's natural defence mechanisms to combat disease. These therapeutic approaches have shown considerable promise in treating various conditions, particularly in oncology, where they offer targeted intervention strategies that differ from traditional treatment modalities. In the field of targeted immunotherapies researchers seek to harness the specificity of immune receptors and the biocompatibility of extracellular vesicles. However, current treatment modalities encounter challenges related to safety profiles, manufacturing complexity, and therapeutic accessibility that limit widespread clinical implementation. The ongoing advancement of immunotherapy represents an important area of medical research with potential implications for improving patient outcomes across various disease contexts through enhanced treatment accessibility and improved clinical applications. Methods of improved targeted drug delivery are desired. SUMMARY OF THE INVENTION The present disclosure pertains to the field of biotechnology and nanomedicine, specifically focusing on the development of biomimetic vesicle systems for targeted drug delivery in oncology. In particular, the present disclosure relates to a method of producing an engineered exosome. In a preferred embodiment, the present disclosure relates to a method of producing a Chimeric Antigen Receptor (CAR) exosome. In an example embodiment, the CAR-exosome has targeted drug delivery capabilities. In one example, there is disclosed an apparatus for one step mechanical extrusion of drug loaded CAR-exosomes. The present invention provides a method of producing one or more engineered exosome-mimetic vesicles, and an engineered exosome-mimetic vesicle. In accordance with a first aspect, the present disclosure provides a method for producing one or more engineered exosome-mimetic vesicles for targeted drug delivery, comprising: engineering one or more eukaryotic cells to stably express a Chimeric Antigen Receptor (CAR) protein on a eukaryotic cell surface;incubating the one or more engineered eukaryotic cells with a therapeutic cargo; andsubjecting the one or more incubated cells to a mechanical extrusion process to form nanoscale exosome-mimetic vesicles with the CAR protein displayed on the eukaryotic cell surface and the therapeutic cargo encapsulated within. In one example, the eukaryotic cells are human embryonic kidney (HEK293) cells. In one example, the CAR protein is a CD19-targeting CAR. In one example, the therapeutic cargo is doxorubicin (DOX). In an example embodiment, the mechanical extrusion process comprises sequentially extruding the cells through polycarbonate porous membranes having progressively smaller pore sizes. The claimed method advantageously produces engineered exosome-mimetic vesicles (CAR-exosomes) which combine the targeting specificity of CAR-T cells with the high drug delivery capacity of exosomes. The method provides high production efficiency. The method is also advantageous because the drug e.g., DOX, is loaded into the exosome-mimetic vesicle during vesicle formation, compared to post isolation loading. This is advantageous because it preserves vesicle integrity and improves drug encapsulation efficiency. In one example, the polycarbonate porous membranes comprise pore sizes of 5 μm, 0.8 μm, and 0.4 μm. Alternatively, the pore size may be 10 μm, 5 μm, 1 μm, 0.4 μm or 0.2 μm, or as in some examples, the porous membranes may include pores that are of a single or multiple (combination) of pore sizes, such as (10 μm, 5 μm, 1 μm, 0.4 μm), (5 μm, 1 μm, 0.4 μm, 0.2 μm), (5 μm, 0.8 μm, 0.2 μm) or any combination thereof. Such combination of membrane pores may be determined based on the cell type, operation method and specific application. In one example, a first extrusion is performed through a polycarbonate porous membrane having a pore size of 5 μm, a second extrusion is performed through a polycarbonate porous membrane having a pore size of 0.8 μm, and a third extrusion is performed through a polycarbonate porous membrane having a pore size of 0.4 μm. In one example, the step of engineering the human embryonic kidney cells (HEK293) comprising the step of performing plasmid transfection on the HEK293 cells to engineer the HK293 cells to express CD19-targeting CAR protein. In one example, the cells were prepared as a suspension (3×106 cells/ml) In one example, the method comprising the step of isolating the engineered exosome-mimetic vesicles. The engineered exosome-mimetic vesicles may be isolated by an appropriate isolation process e.g., density gradient ultracentrifugation or tangential flow filtration or ultrafiltration or any other suitable isolation process. In one example, the engineered exo