Adrián Fernández1, Alfonso Navarro-Zapata2, Adela Escudero3,4, Nerea Matamala4, Beatriz Ruz-Caracuel4, Isabel Mirones5, Alicia Pernas5, Marta Cobo5, Gema Casado5,6, Diego Lanzarot7, Carlos Rodríguez-Antolín8, María Vela2, Cristina Ferreras2, Carmen Mestre2, Aurora Viejo9, Alejandra Leivas1,10, Joaquín Martínez1,10, Lucía Fernández1, Antonio Pérez-Martínez2,11. 1. Hematological Malignancies Lab-H12O Clinical Research Unit, Spanish National Cancer Research Centre (CNIO), 28029 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. 3. Institute of Medical and Molecular Genetics (INGEMM), 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. 5. Advanced Therapy Medicinal Products Production Unit Pediatric Hemato-Oncology Department, La Paz University Hospital, 28046 Madrid, Spain. 6. Advanced Therapy Medicinal Products Production Unit, Pediatric Hemato-Oncology Service and Pharmacy Service, La Paz University Hospital, 28046 Madrid, Spain. 7. Applications Department Miltenyi Biotec, 28223 Madrid, Spain. 8. Experimental Therapies and Novel Biomarkers in Cancer, La Paz University Hospital Institute for Health Research-IdiPAZ, 28046 Madrid, Spain. 9. Hematology and Hemotherapy Department, La Paz University Hospital, 28046 Madrid, Spain. 10. Hematology Department 12 de Octubre University Hospital, 28041 Madrid, Spain. 11. Pediatric Hemato-Oncology Department, La Paz University Hospital, 28046 Madrid, Spain.
Abstract
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.
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.
Entities:
Keywords:
CliniMACS Prodigy; NK cell activation and expansion; NK cell immunotherapy; NKAE cells; clinical-grade manufacturing
Authors: María Victoria Martínez-Sánchez; José Luis Fuster; José Antonio Campillo; Ana María Galera; Mar Bermúdez-Cortés; María Esther Llinares; Eduardo Ramos-Elbal; Juan Francisco Pascual-Gázquez; Ana María Fita; Helios Martínez-Banaclocha; José Antonio Galián; Lourdes Gimeno; Manuel Muro; Alfredo Minguela Journal: Cancers (Basel) Date: 2021-05-11 Impact factor: 6.639