Specific ex vivo expansion of NKG2C+/CD25+ "memory-like" natural killer cells from peripheral mononuclear blood cells for immunotherapy of glioblastoma

This study focuses on NKG2C+ natural killer (NK) cells, a small NK cell subpopulation in the blood of human cytomegalovirus (HCMV) seropositive donors. Beside recognition of peptides derived from classical HLA alleles, these NKG2C+ NK cells are able to recognize a processed HLA-G signal peptide and processed peptides from HCMV-UL40 presented by HLA-E molecules on the surface of glioblastoma cells. Consequently, NKG2C+ NK cells have a higher intrinsic capability to kill specifically glioblastoma cells. In our study we investigated, the selective outgrowth of NK cells directly from peripheral mononuclear blood cells (PBMCs) when co-cultivated with a novel feeder cell line and without the need for tedious NK cell sorting in order to simplify translational efforts.

PBMCs were isolated from the blood of HCMV-seropositive donors. Specific NKG2C+/CD25+ NK cell expansion was achieved by co-cultivation with a novel feeder cell line, modified with an HLA-E trimer presenting the HLA-G-derived VMAPRTLFL peptide. Considering clinical regulations, the feeder cells were irradiated and a CD3 depletion of the PBMCs was conducted. Over the course of two weeks, the NK cells activation and exhaustion markers were analyzed via flow cytometry. Subsequently, the cytotoxicity of the ex vivo expanded NKG2C+/CD25+ NK cells was evaluated by cytotoxicity assays employing allogeneic primary glioblastoma cells.

NK cell expansion from PBMCs with irradiated feeder cells showed a significantly higher growth of NKG2C single positive NK cells, compared to expansion with non-irradiated feeder cells (p=0.0056). Proliferation of CD3 positive natural killer T-cells (NKT cells) as a byproduct, made a CD3 depletion necessary, even though single donors achieved high NK cell purity from expansion of non-depleted PBMCs (median 58.9 %; range 10.4 % - 92.2 %). CD3 depletion showed significantly higher purity (p=0.0015; median 91.8 %; range 79.4 % - 99.3 %), whilst not significantly influencing growth as well as NKG2C expression (median 56.6 %; range 15.0 % - 83.4 %). Regarding cytotoxicity no significant difference between the depleted and non-depleted group could be found.

Our results demonstrate selective outgrowth of NKG2C+/CD25+ NK cells when using GMP compliant protocols approved by regulatory bodies. They furthermore, demonstrate a promising strategy for achieving clinically relevant quantities of NKG2C single positive NK cells for treatment of glioblastoma.