Multi-Functional Genetic Engineering of Pluripotent Cell Lines for Universal Off-the-Shelf Natural Killer Cell Cancer Immunotherapy

Since the initial identification of natural killer (NK) cells, considerable effort has been made to harness their inherent anti-tumor capacities for the treatment of cancer. Despite recent clinical advancements utilizing the adoptive transfer of allogeneic NK cells, particularly with haplo-identical NK cells for acute myeloid leukemia, significant opportunities remain to further augment the anti-tumor activity of NK cells. Clinical trials have shown the persistence of adoptively transferred NK cells to be generally correlated with improved patient outcome. Natural killer cell persistence can be enhanced through several strategies, including by the administration of long-lived sub-populations such as adaptive-memory NK cells or of NK cells that are genetically modified to prolong survival or attenuate rejection by the host immune system. However, conventional sources of primary allogeneic NK cells have proven to be highly variable and difficult to genetically modify.

Human induced pluripotent stem cells (hiPSCs) are unique in their capacity to self-renew and differentiate into any specialized cell type of the body. In addition, hiPSCs can be readily genetically-edited, and single hiPSCs can be clonally selected for the creation of engineered master pluripotent cell lines. As such, hiPSCs represent a renewable and reliable source for generating off-the-shelf engineered cell therapy products that are homogenous and display augmented anti-tumor properties.

To enhance the persistence of hiPSC-derived NK cells, we employed a multi-dimensional strategy for hiPSC modification. First, hiPSCs were derived from fibroblast cells through a proprietary cellular reprogramming process that promotes telomere elongation. Second, hiPSCs were genetically-engineered to express cell surface-bound IL-15 to promote NK cell expansion during hiPSC differentiation and survival upon adoptive transfer. Third, additional hiPSC modifications were introduced to disrupt cell surface HLA Class I presentation and to drive cell surface expression of the non-classical HLA molecule HLA-G, to protect against host-mediated rejection by CD8+ T cells and NK cells, respectively. Lastly, we explored the influence of NKG2C to promote the adaptive-memory NK cell phenotype.

We observed that hiPSC-derived NK cells displayed telomere length that was on average ~2-fold longer as compared to peripheral blood NK cells. The introduction of surface-bound IL-15 supported enhanced proliferation during differentiation with minimal requirements for cytokine support. Disruption of HLA-Class I expression decreased the proliferation of allogeneic CD8+ T cells upon challenge with modified hiPSCs as compared to wild-type hiPSCs (64% for wild-type vs. 29% for modified). This modification increased the cytotoxic activity of NK cells against modified hiPSCs (50% killing at 66 hours for modified as compared to 94 hours for wild-type). To this end, we further modified the hiPSCs to express the non-classical HLA molecule HLA-G. HLA-G protected against NK cell cytotoxicity and extended median survival of modified hiPSCs from 35 hours to 58 hours in an in vitro co-culture system. Finally, engineered hiPSC-derived NK cells can be effectively expanded (>1000x in 14 days) to support an efficient central manufacturing and banking method that enables multi-dose strategies to further support product persistence. Combined, the data suggest that our multi-engineered strategy can support the generation of a universal hiPSC backbone for the derivation of NK cells with improved persistence and anti-tumor efficacy. We are currently adding targeting modalities such as enhanced CD16 or chimeric antigen receptor expression to the discussed universal hiPSC backbone to generate master pluripotent cell lines to enable the manufacture of off-the-shelf NK cell cancer immunotherapy products.