GMP-compliant, large-scale expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells in vitro and in vivo.

Yuna Lee,Dae Seog Heo,Jung Hyun Her,Mi-Young Jung,Sang Mi Kang,Bokyung Min,Hyejin Shin,Okjae Lim,Yu Kyeong Hwang,Eui-Cheol Shin,Jacques Zimmer,Tae Min Kim,Hyejin Chung

doi:10.1371/journal.pone.0053611

Abstract

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3+ T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3−CD16+CD56+ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.

Highlights

Natural killer (NK) cells are specialized lymphocytes that provide a first line of defense against viral infections and cancer [1]
NK inhibition is conferred by killer cell immunoglobulin-like receptors (KIRs), which bind to MHC class I molecules on target cells [2,4]
There has been recent progresses in dendritic cells (DCs) therapy and cytotoxic T lymphocytes (CTLs) therapy, clinical application is somewhat limited since cancer antigens must first be characterized [23,24]

Summary

Introduction

Natural killer (NK) cells are specialized lymphocytes that provide a first line of defense against viral infections and cancer [1]. NK cell activity is regulated by signals from activating and inhibitory receptors [2]. NK inhibition is conferred by killer cell immunoglobulin-like receptors (KIRs), which bind to MHC class I molecules on target cells [2,4]. MHC class I expression tends to be lost or down-regulated in cancer cells [5] and as a consequence, the NK inhibitory signal is abrogated, allowing NK cells to become activated and kill malignant targets. The graft-versus-tumor (GVT) activity of donor NK cells is significantly improved when KIRs and MHC class I are incompatible between donor and recipient, as inhibitory signals are absent [8,9]. Increased GVT activity of NK cells with KIR-MHC incompatibility is the underlying rationale for the development of allogeneic NK cell therapy

Methods

Results

Conclusion