gms | German Medical Science

Objective: Natural killer (NK) cells hold potential as promising effector cells in immunotherapy of human malignancies. Yet, NK cells comprise only 5-15% of PBMCs and various tedious and cost intensive ex vivo expansion protocols are mandatory to expand them to clinically relevant numbers. This study focused on the ex vivo expansion of primary NK cells with novel feeder cell lines and investigation of their cytotoxicity against KIR ligand-matched primary glioblastoma cells. Naturally NK cells are able to kill target cells via antibody-dependent cell-mediated cytotoxicity (ADCC), which is initiated through the recognition of antibody-labelled target cells via crosslinking of their FcγRIII/CD16. Tumour-targeting monoclonal antibodies (mAbs) such as cetuximab can thus be used for treatment of glioma cells with surface expression of EGFR.

Methods: Feeder cell lines were generated by lentiviral gene transfer and used for expansion of allogeneic NK cells. Expanded NK cells from 5 donors were analysed in respect to expression of activating, inhibitory and, immune checkpoint receptors as well as to CD16⁺ subpopulations by flow cytometry. ADCC of expanded matched NK cells was investigated by chromium release assays using cetuximab-labelled KIR/KIR-ligand-matched primary glioblastoma cells. Besides, reactivity against K562 target cells and autologous B cells was tested in CD107a degranulation assays.

Results: Co-cultivation of isolated primary NK cells with feeder cells for 27 days resulted in 40 to 125-fold expansion of donor NK cells. Furthermore, expanded NK cells displayed an up-regulation of activating receptors, and induced expression of high affinity IL-2 receptor. In parallel, an attenuated expression of KIRs and increased expression of inhibitory receptors was monitored. Of note, TIGIT and PD-1 levels were not affected. Although expanded NK cells showed high cytotoxicity against K562 cells, they remained unresponsive to autologous B cells. Furthermore, expanded NK cells exhibited mainly a CD56^bright/CD16⁺ and CD56^dim/CD16⁺ phenotype. These CD16⁺ NK cells showed less alloreactivity yet a stringent ADCC against cetuximab-labelled primary glioblastoma cells.

Conclusion: Our results demonstrate the feasibility of a novel highly efficient ex vivo expansion method to gain functional NK cells with tolerance to KIR/KIR-ligand-matched targets. Further upscaling processes are envisaged to expand allogeneic NK or autologous NK cells to clinical relevant cell numbers for treatment of glioblastoma.