“Adherent” versus Other Isolation Strategies for Expanding Purified, Potent, and Activated Human NK Cells for Cancer Immunotherapy

Table 4

Advantages and disadvantages of methods of NK cell isolation and expansion.

NK cell isolation approach			NK cell expansion approach
Method	Advantages	Disadvantages	Method	Advantages	Disadvantages
CliniMACS	(i) Allows a highly purified NK cell product; (ii) Clinical-grade separation method approved for GMP use	(i) Complicated protocol and increased costs; (ii) Two selections may be necessary (CD3 depletion/CD56 positive selection); (iii) Positive selection by anti-CD56 antibody binding could affect NK cell activation	No or brief stimulation	(i) Does not require long-term cell culture; (ii) Minimal costs of cytokines and reagents; (iii) Individual clinical trials have been able to treat a large number of patients due to minimal labor	(i) Require large number of starting PBMCs; (ii) Low percentage and cell number of NK cells; (iii) Minimal reactivation of NK cells that may be preventing antitumor response
OKT-3	(i) Widely used antibody to eliminate CD3^+ cells from culture; (ii) GMP-grade antibody is available; (iii) Eliminates need for expensive MACS procedure and simplifies the process	(i) Percent of NK cells in final culture varies; (ii) Antibody needed for several days, while T cells are present and they could compete with NK cells for cytokines, limiting their effect	Cytokines	(i) GMP-grade IL-2 is widely available and affordable, allowing effective translation; (ii) Capable of generating highly activated NK cells from patients; (iii) Large-scale expansions have been successful and reproducible	(i) Purity of final NK cell product has traditionally been suboptimal; (ii) Low final NK cell number compared to feeder cell expansion; (iii) Cytokines other than IL-2 are more expensive and most are not GMP-grade yet.
Adherent selection	(i) Minimal cost needed; (ii) Simple and reproducible; (iii) Overnight selection, allowing quick elimination of T cells and preventing T cells from taking up cytokines/media; (iv) Upon enrichment IL-2 is used for expansion without any additional feeder cells	(i) Adherent culture flasks are needed instead of culture bags; (ii) Large-scale enrichment and expansion need to be tested using patient PBMCs reproducing similar NK cell purity with healthy donor PBMCs	Feeder cells	(i) Highest number of final NK cells has been achieved using this approach; (ii) Ability to present various activating molecules to NK cells by genetically modifying feeder cells; (iii) Some protocols require no further isolation	(i) GMP-grade feeder cells must be developed and maintained; (ii) Most protocols also require IL-2; (iii) Final product must be ensured to be deficient of all feeder cells before returning to the patient

Note. The table above summarizes the pros and cons of choosing a particular NK cell expansion method in NK cell isolation and NK cell expansion. Considerations for this table include NK cell purity of final product, NK cell viability, reproducibility, cost-effectiveness, GMP protocols, and clinical application.