Deletion of CISH and TGFβR2 in iPSC-Derived NK Cells Promotes High Cytotoxicity and Enhances In Vivo Tumor Killing

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. Effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing is the power tool to modify NK cells to potentially overcome these biological limitations. Here, we developed a next-generation iPSC-derived NK cell therapy using CRISPR-AsCas12a gene editing to enhance NK cell function by deleting the CISH and TGFβR2 genes. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene, TGFβR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors, but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR-Cas12a to generate edited iPSC lines that were differentiated into TGFβ R2^-/-/CISH^-/- double knockout (DKO) iNK cells.

Using flow cytometry-based assays we demonstrate that DKO iNK cells phosphorylated less SMAD2/3 relative to unedited control iNK cells in response to IL-15 and TGF-β, while CISH KO NK cells showed enhanced pSTAT3 upon IL-15 stimulation. Additionally, DKO iNKs produced higher levels of cytotoxic cytokines including IFN-γ and TNF-α in response to PMA/ionomycin stimulation. We next explored the ability of these DKO iNKs in controlling 3D SKOV-3 ovarian tumor spheroids in vitro over 5 days of co-culture. Both freshly generated and cryopreserved DKO iNKs demonstrated significantly better tumor killing as compared to unedited control iNKs. Importantly, there was no difference in tumor killing between freshly generated and cryopreserved DKO iNKs, suggesting that the freeze/thaw process does not impact functional capacity.

We utilized the SKOV3-luc IP tumor model to evaluate the in vivo efficacy of cryopreserved iNKs cells. Here, NSG mice with established SKOV3-luc tumors were treated IP with unedited control iNKs or DKO iNKs. DKO iNK cell treatment induced robust anti-tumor efficacy resulting in a significant 7.2- fold and 3.2-fold reduction in tumor burden as compared to vehicle and unedited iNK cell treatment, respectively, at 9 days post-iNK cell dosing.

In summary, we demonstrated that TGFβ R2^-/-/CISH^-/- DKO iPSCs differentiated into iNK cells have potent anti-tumor activity that is maintained after cryopreservation. Together, the increased overall effector function of TGFβ R2^-/-/CISH^-/- DKO human iNK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine is being investigated with other gene edits for future advancement to clinic.