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
Hematological Malignancies Lab-H12O Clinical Research Unit, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain.
Translational Research Group in Paediatric Oncology Haematopoietic Transplantation & Cell Therapy, La Paz University Hospital Institute for Health Research-IdiPAZ, 28046 Madrid, Spain.
Cancers (Basel). 2021 Feb 2;13(3):577. doi: 10.3390/cancers13030577.
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.
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.
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.
GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.
自然杀伤（NK）细胞是癌症免疫治疗的有前景的工具。我们报告了NK细胞激活-扩增过程的优化及其在临床规模上的验证。
使用RPMI-1640、干细胞生长培养基（SCGM）、NK MACS和TexMACS作为培养基。通过将全外周血单个核细胞（PBMC）或CD45RA细胞与经辐照的K562mbIL15-41BBL或K562mbIL21-41BBL共培养来获得激活并扩增的NK细胞（NKAE）。分析了扩增倍数、NK细胞纯度、激活状态、细胞毒性和转录组谱。临床级NKAE细胞在CliniMACS Prodigy中制备。
NK MACS和TexMACs实现了最高的NK细胞纯度和最低的T细胞污染。从CD45RA细胞获得NKAE细胞是可行的，尽管PBMC产生的总细胞数和NK细胞纯度高于CD45RA细胞。使用PBMC和K562mbIL21-41BBL细胞实现了最高的扩增倍数和NK纯度。然而，使用任何一种NK细胞来源或激活细胞系时，在激活和细胞毒性方面均未发现差异。转录组谱显示基础NK细胞与用K562mbIL21-41BBL或K562mbIL15-41BBL扩增的NKAE细胞之间存在差异。临床级制备的NKAE细胞符合西班牙监管机构的规范。
使用不同的起始细胞和人工抗原提呈细胞（aAPC）可获得用于临床的GMP级NK细胞。
