Before cancer therapies based on extracellular vesicles (EVs) derived from natural killer (NK) cells can be widely used as cancer immunotherapies, commercial-scale manufacturing processes must be developed. Currently, the ability to manufacture NK-EVs is limited by the NK cells’ finite lifespans, production variability, and the use of non-scalable feeder systems.

Researchers at the University of Ottawa are overcoming those limitations by developing a Good Manufacturing Process (GMP)—compliant hollow fiber, closed-loop bioprocessing bioreactor system “to culture and expand NK92 cells in a chemically-defined, serum-free, and Xenofree medium, in a feeder-free environment,” Lisheng Wang, PhD, professor, faculty of medicine, and colleagues wrote in a recent paper.

“This is the first study to show that this culture system can be used to grow and culture NK92 cells while maintaining the NK92 signature and characteristics, such as phenotype, morphology, and cytotoxicity,” they wrote. Their data also suggests it is reliable and cost-effective.

Promising alternative

NK-EVs appear to offer a promising alternative to NK cell-based immunotherapy, which, through the interactions of the tumor microenvironment and tumor-derived extracellular vesicles, degrade cancer cells’ immunogenicity.

The University of Ottawa scientists’ method achieved proof-of-principle, producing large quantities of NK-EVs of consistent quality, high purity, and low toxicity, while retaining essential characteristics of the NK92 cells, “such as expression of surface markers, cytokine payload, and strong cytotoxicity against human K562 leukemia cells.” This process is making the clinical translation of NK-EV-based immunotherapies increasingly feasible.

The team generated three production lots using the same hollow fiber bioreactor cartridge, for a total of 109 clinical-quality NK92 cells and more than 1013 clinical-quality NK-EVs.

As they pointed out, the quantity of NK cells produced is the equivalent of producing approximately 106 or 108 viable cells per kilogram for treatment in less than one week. They say their method yields 40-fold more EVs per milliliter than traditional flask-based methods yield.

Yield appears to be relatively independent from the quantity of starting material, representing only 0.094% of non-vesicular extracellular particles in the final product.

Cell sizes for each batch were well below 200 nm, making filtration a practical purification method for preclinical and clinical quantities. The purification yield from the three lots averaged 2.36 x 1013, which Wang and his team say is “more than enough for preclinical dosing, compared to other large-scale EV production studies.

“Our approach significantly improved the NK92 cell viability and cytotoxicity compared to traditional flask-based culture methods,” they concluded, noting that they plan to fine tune their approach for commercial adoption by exploring other filtration methods to handle large processing volumes.

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