Abstract
BackgroundThe possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i) the small number of NK cells in peripheral blood, (ii) the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii) the need to activate the NK cells in order to induce NK cell mediated killing and (iv) the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i) if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii) their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii) defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing.MethodsHuman NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated.ResultsCell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors.ConclusionThese data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical evidence necessary to support clinical trials utilizing autologous expanded NK cells, both directly and in combination with monoclonal antibodies in future cell-based immunotherapy in select solid tumors.
Highlights
The possibility that autologous Natural killer cells (NK) cells could serve as an effective treatment modality for solid tumors has long been considered
As a means to facilitate clinical translation, we explored the possibility of these Good Manufacturing Practices (GMP) compliant cell fractions to serve in future NK cell-based immunotherapy studies
While currently elutriated cell fraction 5 is used for monocyte and dendritic cell-based immunotherapy therapy protocols [36,37], we demonstrated that cytolytic NK cells can be expanded from elutriated cell fractions 2, 3 and 4 regardless of the cellular content of these fractions
Summary
The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. Implementation is hampered by (i) the small number of NK cells in peripheral blood, (ii) the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii) the need to activate the NK cells in order to induce NK cell mediated killing and (iv) the constraints imposed by autologous inhibitory receptor-ligand interactions. Clinical effects are demonstrated when inhibitory effects are bypassed by utilizing haplo-identical NK cells and best results are achieved when, in addition, KIR-ligand mismatched NK cells are selected [8,9] This approach requires extensive donor screening and careful depletion of allogeneic T cells from the NK cell product before administration to the host in order to avoid the risk of graft-versus-host disease (GvHD) [10]
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