BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.
BACKGROUND: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing. RESULTS: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing. DISCUSSION: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.
Authors: Giovanni F Torelli; Nadia Peragine; Sara Raponi; Daria Pagliara; Maria S De Propris; Antonella Vitale; Alice Bertaina; Walter Barberi; Lorenzo Moretta; Giuseppe Basso; Angela Santoni; Anna Guarini; Franco Locatelli; Robin Foà Journal: Haematologica Date: 2014-03-21 Impact factor: 9.941
Authors: Veronika Bachanova; Linda J Burns; David H McKenna; Julie Curtsinger; Angela Panoskaltsis-Mortari; Bruce R Lindgren; Sarah Cooley; Daniel Weisdorf; Jeffrey S Miller Journal: Cancer Immunol Immunother Date: 2010-08-03 Impact factor: 6.968
Authors: Antonio Curti; Loredana Ruggeri; Alessandra D'Addio; Andrea Bontadini; Elisa Dan; Maria Rosa Motta; Sara Trabanelli; Valeria Giudice; Elena Urbani; Giovanni Martinelli; Stefania Paolini; Fiorenza Fruet; Alessandro Isidori; Sarah Parisi; Giuseppe Bandini; Michele Baccarani; Andrea Velardi; Roberto M Lemoli Journal: Blood Date: 2011-07-25 Impact factor: 22.113
Authors: Sudarshawn N Damodharan; Kirsti L Walker; Matthew H Forsberg; Kimberly A McDowell; Myriam N Bouchlaka; Diana A Drier; Paul M Sondel; Kenneth B DeSantes; Christian M Capitini Journal: Cytotherapy Date: 2020-06-11 Impact factor: 5.414
Authors: Jung-Won Choi; Eui Soo Lee; Se Young Kim; Su Il Park; Sena Oh; Jung Hwa Kang; Hyun Aae Ryu; Seahyoung Lee Journal: BMC Cancer Date: 2019-08-19 Impact factor: 4.430