Generation and genetic engineering of natural killer cells derived from induced pluripotent stem cells for immunotherapy of solid tumors

Cancer has been well established as a highly morbid disease state both in the United States and worldwide, with cancers such as glioblastoma (GBM), the most aggressive brain tumor, being particularly deadly1,2. In fact, the median survival rate for patients diagnosed with GBM is less than fifteen months3. Despite advances in standard-of-care treatments for GBM, all patients eventually relapse. One promising strategy for treating patients with difficult-to-cure tumors, such as GBM, is through the use of adoptively transferred natural killer (NK) cell-based therapies. NK cells are potent effector cells that are able to specifically target and lyse cancer cells within the body. However, a number of factors limit the efficacy of NK cell immunotherapies, including inadequate sourcing and immunosuppressive mechanisms within the tumor. NK cells have been sourced from blood, as well as from established cell lines, but these cells are either very difficult to obtain or have phenotypic and functional characteristics which are atypical of normal NK cells. Further, GBM-expressed receptors CD73 and CD155 have been shown to suppress NK cell function within the tumor microenvironment (TME)4,5. 

Therefore, to address these drawbacks, we have developed an efficient, chemically defined protocol for generating NK cells from induced pluripotent stem cells (iPSCs), a renewable cell source. These NK cells are representative of functionally mature primary NK cells (PNK). We have, simultaneously, investigated the role of TIGIT/CD155 in immunosuppression of NK cells within GBM, and have found that TIGIT is a functional marker on NK cells, and a novel relationship exists between TIGIT and NK activating receptor 4-1BB. These discoveries have the potential to enhance understanding of NK cell function and drive the development of NK cell-specific engineering for cancer therapies. Lastly, based on these findings, we have developed a novel genetic construct that responsively co-targets CD155 via TIGIT and CD73-induced immunosuppression within the TME of GBM. We have shown that we can genetically engineer iPSCs, and effectively differentiate them into NK cells, retaining genetic modifications in the NK cell state. These engineered NK cells demonstrate superior anti-GBM activity and are effective in controlling tumor growth in an orthotopic patient-derived xenograft GBM model in immunodeficient mice. In summary, our innovative platform represents the first immunotherapy for GBM based on multifunctionally-engineered iPSC-NK cells and a significant technological advancement.