Abstract LB-332: Purified ex vivo expanded and activated human Natural Killer cells efficiently kill glioblastoma cells in a dose- and donor-dependent manner.
Authors: Kmiecik Justyna , Navarro Andrea Gras , Enger Per Ø. , Zelkowski Mateusz , Zimmer Jacques , Enger Martha Chekenya 
Published: August 2014

Glioblastoma (GBM) is the most malignant and frequent brain tumor. Despite combined surgery, chemo- and radiotherapy, GBM patients' survival remains very low, emphasizing the need for further development of novel therapies. The promising strategy is adoptive cellular immunotherapy. Studies investigating purified Natural Killer (NK) cells in adoptive transfer therapies for solid tumors are sparse. Most clinical trials in GBM patients have used Lymphokine Activated Killer (LAK) cells that are composed of T-lymphocytes and NK cells, where the former can contribute to Graft vs Host Disease (GvHD) and inhibit NK cytotoxicity. We hypothesized that purified allogeneic NK cells may be potent effectors for adoptive transfer in GBM patients due to their cytotoxicity, cytokine production and immunomodulatory properties.

We performed in vitro cytotoxicity experiments using patient-derived GBM cells co-cultured with resting or ex vivo expanded and activated NK cells isolated from healthy donors or LAK cells derived from the same donors. We also investigated the role of Killer Immunoglobulin-like Receptor (KIR) versus Human Leucocyte Antigen (HLA) ligand mismatch in determining potency of NK cell-mediated cytotoxicity in vitro and in vivo in human GBM-bearing Nod-Scid mice, treated with activated human NK cells isolated from 2 different donors with or without KIR-HLA ligand mismatch. The primary end points were animals' survival, NK persistence in vivo and mechanisms of therapeutic efficacy. All GBM tumors investigated for cytotoxicity expressed variable levels of MHC class I, MICA/B, ULBP1-3 as evaluated by flow cytometry. High resolution HLA-typing of the GBM cells and KIR-typing of donors was performed at genomic level. Resting and activated NK cells were also characterized for expression levels of NKp46, NKG2D/A and KIRs by flow cytometric phenotyping.

Activated NK cells killed GBM cells more efficiently than resting NK cells and LAK cells in a dose- and donor-dependent manner. By examining tumor-donor pairs for KIR-HLA ligand mismatch we could identify the receptor-ligand combinations that determine greater cytotoxic potential in vitro in suspension cultures vs. in vivo in solid tumors in mice. We demonstrate that KIR-HLA ligand mismatch was important in killing the GBM cells by resting NK cells, however, the cytotoxicity mediated by activated NK cells was less dependent on KIR-HLA mismatch. Activated NK cells upregulated expression of particular KIRs when compared to resting NK cells from the same donor and this partially explains why activated NK cell - mediated cytotoxicity is less dependent on KIR-HLA ligand mismatch.

We conclude that purified and activated NK cells are better effectors against gliomas than LAK cells. However, the KIR-HLA mismatch in solid tumors may not be the primary determinant of activated NK potency in vitro but may have relevance in vivo.

Citation Format: Justyna Kmiecik, Andrea Gras Navarro, Per Ø. Enger, Mateusz Zelkowski, Jacques Zimmer, Martha Chekenya Enger. Purified ex vivo expanded and activated human Natural Killer cells efficiently kill glioblastoma cells in a dose- and donor-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-332. doi:10.1158/1538-7445.AM2013-LB-332

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.