Ex Vivo Expansion of Highly Cytotoxic Human NK Cells by Cocultivation with Irradiated Tumor Cells for Adoptive Immunotherapy

Abstract

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I-negative or -mismatched tumor cell lines we identified a Jurkat Tlymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1-mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcg receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation.

Phase II Study of Haploidentical Natural Killer Cell Infusion for Treatment of Relapsed or Persistent Myeloid Malignancies Following Allogeneic Hematopoietic Cell Transplantation

“…Several groups are investigating ex vivo NK expansion using platforms both with and without feeder cells. (28–30) Ex vivo expansion may result in a lower activation threshold, but could also decrease mediators of homing necessary to direct NK cells to tumor sites. (31) More importantly, recent evidence suggests that certain ex vivo expansion methods may increase the likelihood of acute GVHD after NK cell infusion.…”

Ex vivo expanded natural killer cells from breast cancer patients and healthy donors are highly cytotoxic against breast cancer cell lines and patient-derived tumours

“…Several studies have shown that NK cells isolated from cancer patients are characterized by decreased cytotoxicity, thus to be used in a clinical setting we first had to investigate the functionality of breast cancer patient NK cells post-expansion [8, 16]. It has been shown by others that expanded NK cells from healthy donors have higher cytotoxic ability than NK cells freshly isolated from peripheral blood [17, 34, 35]. To use autologous NK cells isolated from breast cancer patients as a cancer treatment, it was critical to demonstrate that the expansion process activates NK cells to similar levels as those observed in NK cells expanded from healthy donors.…”

“…Ex-vivo expanded NK cells have enhanced cytotoxicity against K562 cells and other tumour cell lines compared to NK cells freshly isolated from healthy donors and cancer patients [22, 34, 35]. Furthermore, autologous activated NK cells do not have a toxic effect on healthy cells, thus avoiding the toxicity that is often associated with cytokine therapies [36].…”

Selective expansion of human natural killer cells leads to enhanced alloreactivity

“…33 An ex vivo coculture protocol in which NK cell skewing can be induced by the presence of specific KIR ligands (i.e., Skewing NK cell alloreactivity DN Eissens et al HLA molecules) can be an augmentation to these protocols to achieve desired KIR-mediated alloreactivity. Obviously, this should be performed under GMP conditions and the relevant HLA molecules should preferably be expressed in a cell-free system.…”