Preparation of Cytokine-activated NK Cells for Use in Adoptive Cell Therapy in Cancer Patients

Abstract

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.

A Two-Phase Expansion Protocol Combining Interleukin (IL)-15 and IL-21 Improves Natural Killer Cell Proliferation and Cytotoxicity against Rhabdomyosarcoma

“…For repeated NK cell applications, we tested continuously expanded cells and compared their cytotoxicity to that of cryopreserved cells. In line with reports on IL-15-stimulated NK cells (75), in our study, we observed a reduced cytotoxic capacity of cryopreserved cytokine-stimulated compared to continuously expanded NK cells (Figure S3A in Supplementary Material) and therefore performed analysis with the latter if several rounds of NK cell application were necessary. Another advantage of NK cell expansion using a feeder cell free IL-15 + 21 boost protocol will be that it easily fulfils the GMP criteria required for clinical application.…”

HLA‐DR+ NK cells are mostly characterized by less mature phenotype and high functional activity

“…Also, it has been recently shown that combination of IL‐2 and IL‐21 can increase this proportion to even greater extent . IL‐2 and other cytokines, such as IL‐15, IL‐21, are important tools in studying NK cells, as they allow maintaining the viability and growth of the cells in culture up to several weeks . This response to cytokines is widely used to expand NK cells ex vivo for their further applications in adoptive immunotherapy .…”

“…15 IL-2 and other cytokines, such as IL-15, IL-21, are important tools in studying NK cells, as they allow maintaining the viability and growth of the cells in culture up to several weeks. [16][17][18] This response to cytokines is widely used to expand NK cells ex vivo for their further applications in adoptive immunotherapy. [19][20][21] For this purpose, soluble cytokines can be used, but much more impressive results have been acquired using feeder cells (K562, EBV cell lines), modified to express membrane-bound cytokines and other activating molecules, such as 4-1BBL and MICA, 20 mbIL-15 and 4-1BBL.…”

Chimeric antigen receptor (CAR)-modified NK cells against cancer: Opportunities and challenges

“…Nevertheless, compared with T cells and many other human cells, NK cells are more sensitive to freezing and thawing, especially when they have been activated by cytokines. In addition to the variable survival rate during thawing, the cytotoxicity of NK cells decreased significantly after thawing [42]. Also, cytokine-activated NK cells are very sensitive to lower temperatures [43].…”