( B) Heat map of normalized expression values for 2185 genes that are differentially expressed between basal NK cells and NKAE cell products. ( A) Unsupervised hierarchical clustering of NK cells using the 15,919 genes remaining after filtering. Gene expression profiles of basal NK cells and NKAE cell products. GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.ĬliniMACS Prodigy NK cell activation and expansion NK cell immunotherapy NKAE cells clinical-grade manufacturing. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. Obtaining NKAE cells from CD45RA + cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA + cells. NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. 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. RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. We report the optimization of an NK cell activation-expansion process and its validation on clinical-scale. Natural killer (NK) cells represent promising tools for cancer immunotherapy. 11 Pediatric Hemato-Oncology Department, La Paz University Hospital, 28046 Madrid, Spain.10 Hematology Department 12 de Octubre University Hospital, 28041 Madrid, Spain. 9 Hematology and Hemotherapy Department, La Paz University Hospital, 28046 Madrid, Spain.8 Experimental Therapies and Novel Biomarkers in Cancer, La Paz University Hospital Institute for Health Research-IdiPAZ, 28046 Madrid, Spain.7 Applications Department Miltenyi Biotec, 28223 Madrid, Spain.6 Advanced Therapy Medicinal Products Production Unit, Pediatric Hemato-Oncology Service and Pharmacy Service, La Paz University Hospital, 28046 Madrid, Spain.5 Advanced Therapy Medicinal Products Production Unit Pediatric Hemato-Oncology Department, La Paz University Hospital, 28046 Madrid, Spain.4 Translational Research in Pediatric Oncology, Hematopoietic Transplantation & Cell Therapy, La Paz University Hospital Institute for Health Research-Institute of Medical and Molecular Genetics (INGEMM-IdiPAZ), 28046 Madrid, Spain. 3 Institute of Medical and Molecular Genetics (INGEMM), La Paz University Hospital, 28046 Madrid, Spain.2 Translational Research Group in Paediatric Oncology Haematopoietic Transplantation & Cell Therapy, La Paz University Hospital Institute for Health Research-IdiPAZ, 28046 Madrid, Spain.1 Hematological Malignancies Lab-H12O Clinical Research Unit, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain.
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