Engineering Chimeric Antigen Receptor-Natural Killer Cells Targeting Fungal Infections Using the Non-viral Sleeping Beauty Transposon System

Our research focuses on developing an off-the-shelf CAR cellular therapy for invasive fungal infections. We aim to identify the best targets, CAR designs and immune cells combination to achieve optimal therapeutic outcomes. Recent developments, including our own work, have focused on developing CAR T-cell therapies targeting Aspergillus fumigatus.

These engineered cells have shown promising results in preclinical models by significantly reducing fungal burden, controlling antifungal immunity, and improving overall survival. Currently most research in the field is concentrated on producing CAR T-cells specific to a very limited set of targets using viral vectors or sleeping beauty transposon system. The main challenges involve identifying optimal fungal targets and producing a ready-to-use cellular product that is both effective and scalable for clinical applications.

With this protocol, we are addressing the need for cost effective and scalable methods to generate non-viral CAR-NK cells. It provides an accessible approach for screening new antifungal targets, providing a fundamental step towards off-the-shelf CAR-NK cellular therapy for fungal infections. To begin, check the NK-92 cells under the microscope for clusters in a clear background, add 2 milliliters of cell culture medium per well for each condition into a six-well plate and place the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide.

Transfer 8 times 10 to the power of 6 NK-92 cells to a 15 milliliter conical bottom tube and centrifuge the tube at 200 G for five minutes with an acceleration and deceleration of 3. After discarding the supernatant, fill the tube containing the cell pellet with up to 15 milliliters of pre-warmed PBS and centrifuge again. During centrifugation, pipette 8 micrograms of Af-CAR plasmid DNA, and 4 micrograms of SB100X minicircle into one reaction tube and keep the second tube DNA free to serve as a mock control.

For the transfection system, add 3 milliliters of electrolytic buffer into the first tube and place it into the pipette station. After centrifugation, gently discard the supernatant and resuspend the cell pellet in 200 microliters of resuspension buffer. Then add 100 microliters of the cell suspension into each of the two reaction tubes and mix gently.

Pipette the DNA cell mixture from the first reaction tube using the transfection system pipette, insert the transfection pipette vertically into the tube in the pipette station. Set the first pulse at 1, 650 volts and the pulse time for 20 milliseconds before pressing start to initiate the pulse. After the first pulse, set the second pulse at 500 volts and the pulse time for 100 milliseconds and initiate the second pulse.

Once the second pulse is completed, slowly remove the pipette from the pipette station. Immediately transfer the cells into one well of the prepared culture plate containing 2 milliliters of prewarmed cell culture medium and gently move the plate in a circular motion to evenly distribute the cells. Incubate the plate in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide.

After 30 minutes of incubation, add interleukin-2 to the wells at a final concentration of 150 international units per milliliter. To begin, transfect the NK-92 cells with the desired plasmid. Transfer the cells into a 15 milliliter conical bottom tube.

Then centrifuge the cells and dilute them to a concentration of 1 to 2 times 10 to the power of 6 cells per 100 microliters using the buffer. Add one microliter of anti EGFR-T biotin per 1 times 10 to the power of 6 cells and gently mix to ensure the solution is evenly distributed. Incubate the tube for 25 minutes at 4 degrees celsius.

After incubation, fill the tube with up to 10 milliliters of buffer. Next, centrifuge the tube at 200 G for five minutes with acceleration and deceleration of 3, and discard the supernatant after centrifugation. Then add 8 microliters of cold buffer followed by two microliters of anti-biotin magnetic beads per 1 times 10 to the power of 6 cells.

Incubate the tube for 15 minutes at 4 degrees Celsius. To wash the cells, fill the tube with up to 10 milliliters of buffer and centrifuge. After discarding the supernatant, resuspend the cells in 500 microliters of buffer.

Place an LS column into the max magnet and wash it with 3 milliliters of buffer. Add the cell suspension once the buffer has completely migrated through the column. After washing the column, remove it from the magnet and place it into a clean 15 milliliter conical bottom tube.

Add 5 milliliters of buffer to the column and use the plunger to flush out the EGF-R positive cells as quickly as possible. Transfection efficiency post recovery reached 10 to 20%and max enrichment increased Af-CAR-NK-92 cell purity to over 95%To begin, obtain enriched transgene-expressing NK-92 cells using magnetic-activated cell sorting technique. Then prepare an Aspergillus fumigatus conidia suspension at a concentration of 2.5 times 10 to the power of 5 conidia per milliliter in the medium.

Dispense 100 microliters of the conidia suspension into each well of a 96-well culture plate and incubate the plate at 25 degrees Celsius for 16 hours. On day two, wash the mock and plasmid transfected NK-92 cells twice with the medium and add 5 times 10 to the power of 4 NK-92 cells in 100 microliters of medium per well of the plate containing the fungus to co-culture. Incubate the plate at 37 degrees Celsius in a humidified carbon dioxide incubator for at least 6 hours.

After centrifuging the plate at 300 G for five minutes, harvest 100 microliters of supernatant from each well without touching the bottom. Finally, transfer the supernatants to reaction tubes to perform ELISA. Af-CAR-NK-92 cells showed significantly higher interferon gamma secretion compared to mock NK-92 cells during co-culture with aspergillus fumigatus germ tubes.