Automated and closed clinical-grade manufacturing protocol produces potent NK cells against neuroblastoma cells and AML blasts

Abstract

Natural killer (NK) cells have great potential as allogeneic immune cell therapy due to their natural ability to recognize and kill tumor cells, and due to their apparent safety. This study describes the development of an immunotherapy option tailored for high-risk acute myeloid leukemia (AML) in adults and neuroblastoma in children. A GMP-compliant manufacturing protocol for the local production of functionally potent NK cells is detailed in the study, including a comprehensive description of the quality control strategy and considerations for product batch specifications in early clinical development. The protocol is based on the closed, automated CliniMACS Prodigy® platform (Miltenyi Biotec) and a modified Natural Killer Cell Transduction (NKCT) process without transduction and expansion. NK cells are isolated from leukapheresis through CD3 depletion and CD56 enrichment, followed by a 12-hour activation with cytokines (500 IU/ml IL-2, 140 IU/ml IL-15). Three CliniMACS Prodigy® NKCT processes were executed, demonstrating the feasibility and consistency of the modified NKCT process. A three-step process without expansion, however, compromised the NK cell yield. T cells were depleted effectively, indicating excellent safety of the product for allogeneic use. Phenotypic and functional characterization of the NK cells before and after cytokine activation revealed a notable increase in the expression of activation markers, particularly CD69, consistent with enhanced functionality. Intriguingly, even following a brief 12-hour activation period, the NK cells exhibited increased killing efficacy against CD33+ AML blasts isolated from patients and against SH-SY5Y neuroblastoma (NBL) target cells in vitro, suggesting a potential therapeutic benefit for AML and NBL patients.