Preparation of Cytokine activated NK Cells for Use in Adoptive Cell Therapy in Cancer Patients: Protocol Optimization and Therapeutic Potential
Cell based immunotherapy using donor derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine activated NK cell product. NK cells were isolated either by double depletion (CD3 CD19 or by sequential depletion and enrichment (CD3 CD56+ via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL) 2 or IL 15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T cell contamination. After culture with cytokines, the CD3 CD56+ procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3 CD19 procedure. CD69, NKp44, and NKG2A expression were higher on CD3 CD56+ products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL 2, T cell contamination was lower with IL 15. Therefore, further experiments were performed with CD3 CD56+ products cultured with IL 15. Cryopreservation of IL 15 activated NK cells resulted in a loss of cytotoxicity >92% whereas thawing of isolated, uncultured NK cells followed by culture with IL 15 yielded cells with about 43% of the original lytic activity. Five day IL 15 activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell based immunotherapeutic strategies in a clinical setting.
Journal of immunotherapy