Development of CAR-NK Cells for Drug Delivery Systems

CAR-modified NK cells can provide an alternative "off-the-shelf" vector for CAR-based therapies and provide more targeted drug delivery to tumor sites through surface engineering. NK cells are identical to the parental cell line, eliminating the issue of donor variability. The use of surface-engineered CAR-NK cells for targeted drug delivery has broad applicability, as both CAR targets and drug payloads may be altered to treat multiple cancer types.

What are CAR-NK Cells?

Functional CAR molecules expressed on NK cells consist of three components: the extracellular structural domain, the transmembrane region, and the intracellular signaling structural domain. The extracellular structural domain consists of a signaling peptide and a single chain antibody fragment (scFv) that recognizes the antigen. A hinge region connects this structure to the transmembrane region, which also connects intracellularly to the intracellular structural domain that contains the activation signal. Successful CAR design is achieved through a combination of careful design and functional testing.

CAR-NK Cells Modification

NK cells are a type of cytotoxic lymphocytes that play an important role in tumor immune surveillance. Genetic engineering of NK cells to express chimeric antigen receptors can enhance the tumor-specific killing effect of NK cells. By using modified NK cells as carriers for nanodrug-targeted transport and directing drug-laden nanoparticles to target sites, the efficacy of chemotherapy can be enhanced in vitro and in vivo while reducing off-target toxicity.

• CAR assay on the surface of NK cells

Three days after transduction, CAR-NK cells were incubated with biotinylated proteins in PBS + 4% FBS. Cells were subsequently incubated with FITC-conjugated streptavidin in PBS + 4% FBS for 10 minutes at 4°C and read using flow cytometry. Non-transduced NK cells were used as a negative control.

• Binding of nanoparticles to cells and in situ polyethylene glycolization

Equal volumes of nanoparticles are suspended in nuclease-free water with NK cells and incubated at 37°C. Cells and nanoparticles were mixed every 10 minutes for 30 minutes. Cells are incubated with thiol-capped polyethylene glycol (PEG) to quench residual moieties on the cell-bound particles. For quantification of cell-bound particles, the particles are fluorescently labeled with lipid-like fluorescent dyes. Particle fluorescence was detected by flow cytometry and luminescence zymography. carboxyfluorescein succinimidyl ester (CFSE) -, which allows easy detection of NK cell binding using confocal microscopy - was used for CAR-NK cells.

• Cytokine release assay

NK cells are co-incubated with target cells in 96-well plates for 6 hours. Brefeldin A is added to each well to prevent protein translocation. At the end of incubation, cells are permeabilized using the kit and stained. Unstimulated cells are used as negative controls. Results are read using flow cytometry.

• Cytotoxicity assay

Cell viability was assessed using the Cell Proliferation Kit II. The percentage of cell viability was determined by subtracting the absorbance value obtained in the wells with only medium from the treated wells, which were then normalized with control wells containing cells without drug.

CD Formulation has been providing fully customized solutions to our partners and supporting the development of new therapies to improve lives. Our CAR-NK cell therapy experts have developed CAR-NK cell formulations and proven preparation processes based on advanced technologies.