Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Abstract

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC). NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials. The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days. Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15), which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL15. Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Activating mutations of STAT5B and STAT3 in lymphomas derived from γδ-T or NK cells

“…Resting NK cells were cultured in the presence of 100 IU of IL2 for 48 h to obtain activated NK cells. Higher levels of NK cell activation were achieved by coculturing freshly isolated peripheral blood lymphocytes with engineered K562 cells, K562-Clone9-mb21, as described in detail before 37,38 .…”

“…The expansion procedure is described briefly as follows: First, primary human NK cells were isolated by negative selection using EasySepHuman NK cell enrichment kit (Stemcell technologies,Vancouver,Canada). Then, primary NK cells were admixed in a 1:2 ratio with 100 Gr irradiated K562-Cl9-mb21cells, which express CD86, 4-1BBL and mIL21 on their surface, and cultured in NK cell expansion medium 38 . Cells were spun down at 400g for 5 min, and the culture medium was renewed every 3 days with fresh culture medium, keeping the cell density at 250,000 cells per ml after every subculture.…”

Umbilical cord blood graft engineering: challenges and opportunities

“…K562-expanded NK cells show potential defects in CCR7-mediated homing to lymph nodes. 129 Modification of K562 feeder cells to transfer CCR7 to NK cells via trogocytosis transiently increases NK cell CCR7 expression and improves homing to LNs of athymic mice. 130 Culture with irradiated EBV LCL reduces CD62L-mediated homing to marrow and secondary lymphoid tissues, which can be improved by adding nicotinamide to the culture.…”

Bringing natural killer cells to the clinic: ex vivo manipulation

“…NK cells expanded with genetically modified K562 cells contain predominantly CD56 ϩ /16 ϩ bright NK cell populations, which do not express CCR7, a chemokine receptor that is known to facilitate NK cell homing to lymph nodes. 67 Although IL-18 can up-regulate CCR7, it does so in only a minority of NK cells. 68 Somanchi et al recently demonstrated that mbIL-21-expressing K562 feeder cells can be further genetically modified to express other transgenes, the products of which can be rapidly and transiently expressed in NK cells via trogocytosis by coculturing with expanded NK cells.…”