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Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications
  m, H3 E+ o) v* t3 l# j8 cPosted online on August 17, 2012. (doi:10.3109/14653249.2012.700767): l# L) ~- l! ~; H! E7 ?# y0 V5 [
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http://informahealthcare.com/doi/abs/10.3109/14653249.2012.700767! M# w1 `  \2 A$ h: c

- v, B8 a) _4 @& hNatalia Lapteva1, April G. Durett1, Jiali Sun1, Lisa A. Rollins1, Leslie L. Huye1, Jian Fang1, Varada Dandekar1, Zhuyong Mei1, Kimberley Jackson1, Juan Vera1, Jun Ando1, Minhtran C. Ngo2, Elaine Coustan-Smith3, Dario Campana3, Susann Szmania4, Tarun Garg4, Amberly Moreno-Bost4, Frits Vanrhee4, Adrian P. Gee1,2 & Cliona M. Rooney1,2
1 x1 Z7 V% e$ v+ U. l1Center for Cell and Gene Therapy, The Methodist Hospital, Texas Children's Hospital, Houston, TX) m. T' [8 Y2 w1 K5 G( Q
2Department of Pediatrics, Baylor College of Medicine, Houston, Texas , USA3 r# _  W% u, O4 J" _5 L
3National University of Singapore, Singapore
! ?/ f- Y& R/ w5 B, P* [; n/ e4University of Arkansas Medical Center, Little Rock, Arkansas , USA
/ I- G' @4 E3 _5 r" `% c" LCorrespondence: Cliona M. Rooney, PhD, and Natalia Lapteva, PhD, Center for Cell and Gene Therapy, The Methodist Hospital, Texas Children's Hospital, Baylor College of Medicine, 1102 Bates St, St 1770.10, Houston, TX 77030 , USA. E-mail: cmrooney@txch.org6 H- k: L- s7 ?+ V! D5 ?2 F

5 }+ ~) X+ ]/ H) a5 Q: h5 I: hBackground aims. 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 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 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.- q2 C1 r. \! e# [  K# C, [

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