Background Natural killer (NK) cell therapy is a potential cancer therapeutic approach. To increase anticancer efficacy, it is necessary to obtain high-purity and high-potency NK cells. Most researchers have used CD3+ depleted seed cells to increase NK cell content, and additionally have been used growth-inactivated tumor cell-based feeder cells to increase NK cell productivity. However, these attempts complicate the manufacturing process and require strict control over the feeder cells. We developed the eHuT-78 cell line, which expresses 4-1BBL, mTNF-a, and mIL-21, as a novel feeder cell for ex vivo NK expansion and the NK cell therapy produced by eHuT-78 cells is currently in phase 2 clinical trials. In this study, we present the feasibility to safely expand highly enriched NK cells ex vivo with potent antitumor efficacy without CD3+ depletion using eHuT-78 cells-derived nanovesicles (CDNVs).

Methods Cells-derived nanovesicles (CDNVs) are obtained by serial extruding the eHuT-78 cells through a membrane extruder. NK cells were expanded from healthy donor PBMCs by CDNVs or γ-irradiated eHuT-78 cells in SCGM media with human plasma and IL-2 for 11 days. We assessed the expanded NK cells for identification, purity and activation markers using flowcytometry as well as direct cytotoxicity and antibody dependent cell mediated cytotoxicity (ADCC) by Herceptin or Erbitux against tumor cells.

Results The Fold expansion of NK cells by CDNVs (CDNV-NK) and eHuT-78 (eHuT78-NK) for 11 days was 994-fold and 760-fold, respectively. The percentage of CD3-CD56+ NK cells was 83% for CDNV-NK and 34% for eHuT78-NK respectively. Whereas the percentage of CD3+ T cells were 12% for CDNV-NK and 63% for eHuT78-NK, respectively. The content of CD14+ and CD19+ cells was less than 0.5% in both groups. CDNV-NK showed the higher expression of NK activating receptors/chemokine receptor (CD16 and NKG2D and CXCR3) than eHuT78-NK. NKp46 and DNAM-1 expression was no significant difference between both groups. CDNV-NK exhibited higher direct cytotoxicity against several leukemic cell lines as well as greater ADCC in combination with trastuzumab or cetuximab against breast cancer cell lines compared to eHuT78-NK.

Conclusions eHuT78 CDNVs can simplify the complex manufacturing process resulting from seed cell isolation and feeder cell use, and can be utilized to manufacture NK cells as a safe and useful technology.