Sustained Expansion of Human Natural Killer Cells for Leukemia Therapy.
Authors: Fujisaki Hiroyuki , Kakuda Harumi , Imai Chihaya , Campana Dario 
Published: October 2019

Natural killer (NK) cells are a promising tool for cell therapy of hematologic malignancies. They have potential for enhancing graft-versus-leukemia responses in recipients of hematopoietic stem cell transplant, and can also be used in a non-transplant setting, where haploidentical donor NK cells have been shown to expand in vivo.
NK cells represent a small subset of peripheral blood cells. Hence, it can be problematic to obtain them in quantities sufficient to exert significant anti-leukemic activity in patients. We sought to identify culture conditions that would stimulate vigorous, sustained and specific expansion of CD56+ CD3− NK cells. NK cell proliferation was stimulated by contact with the K562 leukemia cell line transfected with two NK stimulatory molecules: membrane-bound interleukin 15 and 4-1BB ligand. Exposure of peripheral blood cells from 23 donors to irradiated K562-mb15-41BBL cells in the presence of 10 IU/mL interleukin-2 resulted in a median expansion of CD56+ CD3− cells of 22-fold (range, 9- to 87-fold) after only 7 days of culture; expansion of CD3+ T cells was negligible. After 14 days of culture, K562-mb15-41BBL cells were completely lysed by the NK cells and no further expansion occurred. However, further NK cell expansion could be achieved by addition of fresh K562-mb15-41BBL cells to the cultures. Using this method, NK cell expansions ranged from 2,000- to 98,000-fold (n = 4) after 65 days of culture. We noted that NK cells eventually became unresponsive to stimulation and underwent senescence after 2–5 months of culture. To determine whether NK cell senescence could be overcome by enforced expression of human telomerase reverse transcriptase (hTERT), we stimulated NK cells for 1 week with K562-mb15-41BBL cells and then transfected them using an MSCV retroviral vector and the hTERT gene (gift of Dr. J. Dome, St. Jude). hTERT expression and telomerase activity was demonstrated by reverse transcriptase-polymerase chain reaction and telomerase repeat amplification protocol assay. The cultures were then stimulated with periodic pulses of K562-mb15-41BBL cells. In 2 donors, enforced expression of hTERT overcame senescence: NK cells transfected with an empty vector died after 85 and 170 days of culture, whereas hTERT-NK cells continue to grow after more than 350 days of culture, while retaining a normal karyotype. hTERT-NK cells maintained their cytotoxicity against the NK-sensitive leukemic cell lines K562, KG1, U937, HL60 and Jurkat. They could be also be genetically modified to express anti-CD19 chimeric signaling receptors, thus becoming cytotoxic against NK-resistant CD19+ B-lineage acute lymphoblastic leukemia cells. Cytotoxicity against CD19+ targets was similar to that of NK cells transfected with the signaling receptor after only one week of culture.
In conclusion, coculture of human peripheral blood mononuclear cells with pulses of irradiated K562-mb15-41BBL cells allows the generation of a large numbers of NK cells which have powerful anti-leukemic capacity and can be redirected to lyse NK-resistant target cells. Although senescence eventually ensues, this can be overcome by hTERT expression. The culture system described here has now been adapted to large scale expansion for clinical use.