Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy. Fernández Adrián,Navarro-Zapata Alfonso,Escudero Adela,Matamala Nerea,Ruz-Caracuel Beatriz,Mirones Isabel,Pernas Alicia,Cobo Marta,Casado Gema,Lanzarot Diego,Rodríguez-Antolín Carlos,Vela María,Ferreras Cristina,Mestre Carmen,Viejo Aurora,Leivas Alejandra,Martínez Joaquín,Fernández Lucía,Pérez-Martínez Antonio Cancers Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation-expansion process and its validation on clinical-scale. METHODS:RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. RESULTS:NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. CONCLUSIONS:GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC. 10.3390/cancers13030577

Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies. Masuyama Jun-ichi,Murakami Takashi,Iwamoto Sanju,Fujita Sanehiko Cytotherapy BACKGROUND AIMS:This study developed a new method to expand CD3(-)CD56(+) natural killer (NK) cells from human peripheral blood mononuclear cells (PBMCs) without feeder cells for clinical trials. METHODS:PBMCs from healthy subjects were co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies and cultured for 14 days in newly developed NKGM-1 medium containing autologous plasma and interleukin-2. Expanded NK cells were examined for cell number, phenotype, in vitro and in vivo cytotoxicity and interferon (IFN)-γ secretion. We also evaluated the proliferative ability of NK cells after cryopreservation. A patient with advanced pancreatic cancer was treated with autologous-expanded NK cells through the use of this method in combination with chemotherapy. RESULTS:Expanded NK cells expressed higher levels of activating molecules compared with resting NK cells and exhibited potent cytotoxicity against K562 cells and IFN-γ secretion by cytokine stimulation. Significant anti-tumor activity was observed in immunodeficient mice injected with the human pancreatic cancer cell line BxPC-3. Large-scale cultures generated a median 5.7 × 10(9) NK cells from 20 mL of peripheral blood (n = 38) after 14 days of culture and 8.4 × 10(9) NK cells after 18 days of culture through the use of a cryopreservation procedure. The number of NK cells and cytotoxic activity in the peripheral blood of the patient with pancreatic cancer greatly increased, and successful clinical responses were observed after multiple infusions of expanded NK cells. CONCLUSIONS:These data demonstrate that this simple and safe methodology for the ex vivo expansion of NK cells can be used for cancer immunotherapy. 10.1016/j.jcyt.2015.09.011