Enhancing Chimeric Antigen Receptor-Extracellular Vesicles (CAR-EV) Technology: The Future of Cancer Therapy

Our research focuses on the development of exosome-based diagnostics and therapeutics. An objective is to develop chimeric antigen receptor exosome-based therapeutics, that are safer and more effective and accessible than cell-based therapies, for the treatment of side tumors. Our EXO-NET and EXO-ACE platforms enable scalable, reproducible and GMP-compliant exosome isolation with high yield and purity, addressing limitations of traditional methods for research and therapeutics.

EXO_NET net and EXO-ACE enable rapid, scalable and GMP-compliant EV isolation, with superior purity and reproducibility, outperforming centrifugation and precipitation for research, diagnostics and therapeutic applications. We have developed a rapid, scalable and validated method to engineer CAR-EVs, and deliver next-generation, cell-free, targeted therapeutics. This provides a robust workflow that can be applied to other disease conditions where there is a clinical need.

Our next goal is to use the methods that we have developed and validated to generate CAR-EVs for the delivery of RNA therapeutics for treating solid tumors and other inflammatory diseases. To begin, bring the ion exchange column to room temperature by allowing it to stand upright for 15 minutes. Using a pipette, add 10 column volumes of regeneration buffer, followed by 10 column volumes of equilibration buffer.

Pass the THOD conditioned medium through a 0.22-micrometer polyethersulfone filter. Now, add up to 31.2 column volumes of the filtered sample into the equilibrated column. Then add 10 column volumes of wash buffer, followed by 2.5 column volumes of elution buffer.

Collect the flow through containing the enriched extracellular vesicles. Using 30 kilodalton ultrafiltration centrifugal filters and a diluent, perform buffer exchange on the extracellular vesicle elute, ensuring a minimum 100-fold dilution. Adjust the final volume of the exchanged solution based on experimental needs.

Pass the enriched extracellular vesicle sample through a 0.22 micrometer filter to sterilize it for in vitro assays. Determine the size distribution, concentration and yield of CAR extracellular vesicles using nanoparticle tracking analysis. For high-throughput pan-exosome extracellular vesicle isolation, switch on and log in to the instrument.

Configure the settings to establish the HT EV protein isolation protocol. Prepare a tip comb plate by placing a tip comb into a 96-well deep-well plate. Add one milliliter of PBS into each well to prepare three wash plates.

Now, prepare an elution plate by pipetting 35 microliters of 1%SDS buffer into each well of a 96-well plate. Then prepare a binding plate by adding one milliliter of the conditioned medium. On the automated isolation system, begin the protocol by selecting the program HT EV Protein Isolation Protocol, and select the play button.

Open the instrument to begin loading the plates. Follow the instrument prompts and load the prepared elution plates, followed by the three-wash deep-well plates into the instrument. Just prior to loading the binding plate, add 30 microliters of pan-exosome capture beads into each well.

Load the binding plate into the instrument, then load the tip comb plate, and close the instrument to initiate binding. The system will mix the conditioned medium sample and magnetic beads continuously at slow speed for 30 minutes, facilitating the efficient capture of extracellular vesicles. Upon completion of the binding step, allow the system to perform three wash steps, mixing at a slow speed for 30 seconds to remove non-specific contaminants.

Allow the automated system to initiate extracellular vesicle lysis for protein recovery by performing a 30-second bottom mix. During incubation, the instrument will pause for seven minutes and 30 seconds, while keeping the pipette tips positioned above each well. Initiate another 30=second mix, followed by another seven-minute-and-30-second pause.

When the protocol concludes, the system will lift the used tip comb, leaving the solution in the 96-well plate and the magnetic beads attached to the tips. Finally, the instrument will proceed to the leave step, returning the tip comb into the tip comb plate. Open the instrument and remove the plates.

GFP fluorescence confirmed CAR expression in transduced T-cells, with localization evident in cell clusters, and colocalization verified by overlaying brightfield and GFP channels. Nanoparticle tracking analysis revealed a significantly higher concentration of extracellular vesicles in the enriched EV fraction compared to conditioned media for both EGFR and HER2 CAR constructs. Western blotting confirmed the presence of granzyme B in CAR-expressing cells and in the isolated extracellular vesicles, with calnexin absent in the extracellular vesicles, indicating lack of cellular contamination.

EGFR-targeting CAR extracellular vesicles significantly reduced MCF-7 breast cancer cell viability by 70%and K-562 blood cancer cell viability by 40%HER2-targeting car extracellular vesicles showed about a 10%reduction in MCF-7 breast cancer cell viability, while having no observable effect on K-562 blood cancer cells.