Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications.
OBJECTIVE
Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available.


METHODS
We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex).


RESULTS
Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation.


CONCLUSIONS
We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.
Center for Cell and Gene Therapy, The Methodist Hospital, Texas Children's Hospital, Houston, TX, USA.
Natalia
N
Lapteva
April G
AG
Durett
Jiali
J
Sun
Lisa A
LA
Rollins
Leslie L
LL
Huye
Jian
J
Fang
Varada
V
Dandekar
Zhuyong
Z
Mei
Kimberley
K
Jackson
Juan
J
Vera
Jun
J
Ando
Minhtran C
MC
Ngo
Elaine
E
Coustan-Smith
Dario
D
Campana
Susann
S
Szmania
Tarun
T
Garg
Amberly
A
Moreno-Bost
Frits
F
Vanrhee
Adrian P
AP
Gee
Cliona M
CM
Rooney
D053330:4-1BB Ligand / Q000378:metabolism; D001781:Blood Component Removal; D018929:Cell Culture Techniques; D002470:Cell Survival; D015925:Cryopreservation; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000378:metabolism; D006801:Humans; D016219:Immunotherapy, Adoptive; D019409:Interleukin-15 / Q000378:metabolism; D020014:K562 Cells / Q000166:cytology*; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism
D053330:4-1BB Ligand; D015395:Histocompatibility Antigens Class I; D019409:Interleukin-15