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Table 4 Summary of NK expansion and activation strategies

From: NK cell-based cancer immunotherapy: from basic biology to clinical development

Stimulation substance Expansion criteria Used clinically Reference Considerations
Cytokines alone: applied separately or in combinations of two
IL-2, IL-15, IL-2/IL-15, IL-2/IL-21
 ~ 5 (two weeks) Yes [7, 100, 120] Generate highly activated NK cells
Possibility of dependence on cytokine
Expansion is facilitated in the presence of autologous PBMC
IL-2/IL-15/IL-21  ~ 8 (two weeks) No [119, 121] Lower rate of NK cell expansion compared to feeder cell
Only IL-2 cytokine is GMP-grade
IL-15/IL-18/IL-27  ~ 17 (two weeks) No [122]  
IL-2, IL-18  ~ 500 (two weeks) No [123]  
Autologous feeder cells
OK432, RN-T cells
 ~ 600 (three weeks) Yes [4] RN-T cells were established by activation PBMC with OKT-3 and RetroNectin FN-CH296
Autologous feeder + Activating Abs
Anti-CD335 (NKp46) and anti-CD2
 ~ 3800 (three weeks) No Patent, 2013, EP2824112B
CD2 and CD335 coated nanomatrices with commercially available cell stimulation beads (Miltenyi Biotec Kit)
OKT-3 (Anti CD3), anti-CD 52 1,537 (18 days) Yes [146] PBMCs are typically irradiated 25 Gy or more
GMP-grade antibody Anti CD3 is available
OKT-3 (Anti CD3)  ~ 1000 (two weeks) Yes [142, 143]  
Anti CD16  > 500 (two weeks) No [147]  
Allogeneic feeder cells PBMC
 + PHA, Ionomycin
 ~ 100 No [139] Without selection final product may contain up to 40% T cell
PBMCs are typically irradiated 25 Gy or more
 + ConA  ~ 100 Yes [138]  
 + anti CD3  ~ 300 Yes [144]  
Allogeneic feeder cells (tumor)
Wilms tumor cell line (HFWT),
 ~ 113 (two weeks) Yes [134] Feeder can be genetically modified to enhance activation
Feeder cells require irradiation and GMP-grade production
Final product needs to be feeder free assured
Jurkat  ~ 100 (two weeks) No [135] Risk of bacterial and viral contamination derived from feeder cells
Transformed feeder cells
Epstein-Barr lymphoblastoid cell line (EBV-LCL),
 ~ 3000 (two weeks) Yes [136] Feeder cells require irradiation
Safety considerations associated with feeder
Engineered feeder
K562 4-1BB + IL15
 ~ 1200 (two weeks) Yes [38, 125,126,127] Increased apoptosis of NK cells noted after extensive expansion
Engineered feeder
K562 4-1BB + IL21
 ~ 30,000 (three weeks) Yes [128,129,130] Greatest rate of expansion reported so far
Lower dose of supportive IL-2 required
Feeder particles
K562 4-1BB + IL21
 ~ 250 (two weeks)   [141] Avoids the safety considerations associated with feeder cells
Laborious to produce
Group A-Streptococcus and zoledronate  ~ 1,560 (three weeks) No [107]  > 90% of NK cells. May not require magnetic cell sorting
Components IL2, streptococcus and zoledronate are FDA approved