工程化iPSC来源NK细胞对转移性葡萄膜黑色素瘤细胞的抗肿瘤活性

Background: Uveal melanoma (UM) preferentially metastasizes to the liver, leading to a poor prognosis. The immunosuppressive microenvironment of the liver and the low mutation burden of metastatic UM (MUM) contribute to the limited efficacy of immunotherapies, including immune checkpoint inhibitors and adoptive T-cell transfers. Existing evidences highlight the crucial role of Natural Killer (NK) cells in monitoring and targeting circulating MUM cells, suggesting that therapeutic strategies that enhance NK cell activity may offer clinical benefits. Donor variability and limited expansion capacity restricted clinical application of primary NK cells. NK cells derived from induced pluripotent stem cells (iPSCs) present distinct benefits, especially in their ability to be genetically modified to produce cells with improved antitumor characteristics. Therefore, we investigated the antitumor activity of iPSC-NK cells against MUM. Methods: The functions of iPS-NK cells were enhanced by gene editing, resulting in the production of engineered iPS-NK cells (eNK) that express NKG2D, CD16, CCR2, and secrete CCL19 and IL-15. eNK is being developed as AKT-01/HLCN061 through a collaboration between Akatsuki Therapeutics Inc. and Healios K.K.. The functional properties of eNK cells against UM cell lines, UM001 and UM004, were evaluated in comparison with the intact highly cytotoxic NK92 cell line or with NK92 cells genetically modified with CD16 (NK92-CD16). The activities of the NK cells were assessed using (i) cytotoxic assay; (ii) ADCC assay with anti-GD2 antibodies; (iii) CD107a degranulation assay; and (iv) intracellular cytokine staining. The migration of eNK cells toward UM was evaluated using a transwell migration assay. Results: The eNK and NK92 cells exhibit a comparable effectiveness in targeting UM001 cells, killing up to 50% of the tumor cells and showing similar frequency of the cells that produce detectable levels of cytolytic granules and IFNgamma (5-10%). The cytolytic activity of NK92 cells against UM004 cells was negligible, as was the frequency of cells releasing cytolytic granules and expressing IFNgamma. In contrast, eNK cells killed approximately 25% of the UM004 cells, upregulating CD107a membrane expression and intracellular IFNgamma production to a similar extent as in the case of UM001 cells. ADCC against UM004 cell lines in the presence of anti-GD2 antibodies was similar (~15%) for both eNK and NK92-CD16 cells. The TNFalpha intracellular staining in response to UM cells targets was observed only for eNK cells. Importantly, TNFalpha treatment increased CCL2 production by the UM cell lines. In accordance with this, eNK cells exhibited enhanced migration into chambers containing supernatants from TNFalpha-stimulated UM001 or UM004 cells. Conclusions: The data indicated that eNK cells have anti-tumor activity and potential as improved adoptive cell therapeutics in the treatment of MUM.