Engineering Human Pluripotent Stem Cell Derived Natural Killer Cells to Prevent CD16a Shedding for Enhanced Anti Tumor Killing
Natural killer (NK) cells are lymphocytes able to recognize and lyse transformed cells without HLA restriction and without prior sensitization, mediated through a repertoire of cell surface receptors. Solid tumors are typically more refractory to NK cell mediated killing compared to hematologic malignancies. To improve NK cell mediated killing of solid tumors, we have engineered NK cells to express a more stable form of CD16a. The IgG Fc receptor CD16a represents one of the most potent activators of NK cell anti tumor activity. This receptor binds antibodies attached to target cells to initiate antibody dependent cell mediated cytotoxicity (ADCC) Importantly, CD16a undergoes a rapid down regulation in expression by ADAM17 (A Disintegrin And Metalloprotease 17) mediated proteolytic cleavage upon cell activation. This loss of CD16a expression leads to reduced ADCC activity and decreased anti tumor killing. Previous studies by our group identified cleavage sites in CD16a that are targeted by ADAM17 activity. Substitution of the serine at position 197 for a proline (S197P) in the middle of the cleavage region effectively blocks ADAM17 mediated CD16a cleavage. To evaluate the ability of this cleavage resistant CD16a(S197P) to improve ADCC, we have used a system previously developed by our group to efficiently produce NK cells from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) These hESC/iPSC derived NK cells have a phenotype and function similar to NK cells isolated from peripheral blood (PB NK cells) However, unlike PB NK cells, it is relatively easy to genetically modify hESC/iPSC derived NK cells by engineering at the stem cell level. Here, we use two strategies to generate iPSC derived NK cells with stabilized CD16a expression. First, we used the Sleeping Beautytransposon system to stably express the cleavage resistant CD16a(S197P) in iPSCs. Second, we have used the CRISPR/Cas9 system to delete the ADAM17 gene. This strategy allows us to compare specifically engineering stabilized CD16a expression to the broader effect of ADAM17 deletion which cleaves multiple cell surface proteins. NK cells derived from the CD16a(S197P) iPSCs are resistant to CD16a shedding upon activation with IL 12 and IL 18 as 97.5% of iPSC NK cells are CD16+ before stimulation and 95.2% of cells are CD16+ after stimulation. In contrast, PB NK cells demonstrate a decrease from 85% CD16+ cells before stimulation to 67% CD16+ cells after stimulation. Next, we demonstrate improved ADCC and NK cell degranulation (measured by CD107a expression) in this engineered iPSC derived NK cell compared to non engineered cells upon addition of the anti Her2 antibody (ab) Herceptin to ovarian cancer cells with differing levels of Her2 expression. Specifically, CD16a(S197P) iPSC NK cells stimulated with the Her2 high SKOV3 ovarian carcinoma cells were 26.7% CD107a+ cells with addition of anti Her2 and 10.8% CD107a+ cells without ab. In contrast, unmodified iPSC NK cells were 9.6% vs. 6.9% CD107a+ with and without anti Her2; and PB NK cells were 12.4% vs. 9.4% CD107a+ with and without anti Her2. However, when co cultured with the Her2 low MA 148 ovarian carcinoma none of the three NK cell populations demonstrated increased CD107a+ cells with anti Her2 treatment compared to no ab controls. Similar results were obtained when testing the ADCC of these cells using the Cr 51 release cytotoxicity assay. Killing of SKOV3 cells by CD16a(S197P) iPSC NK cells was markedly increased upon addition of anti Her2 ab from 32.2% cytoxicity with ab vs. 11% cytotoxicity without ab. In contrast, unmodified iPSC NK cells had less killing of the SKOV3 cells (8.1% vs. 3.7% with or without anti Her2 ab) and PB NK cell killing of SKOV3 cells was 20.5% vs. 9.2% with or without anti Her2 ab. Currently, we are comparing anti tumor activity of the CD16a(S197P) iPSC NK cells to the ADAM17 deleted iPSC NK cells, both as resting NK cells and IL12+IL18 stimulated NK cells. Overall, this strategy will allow us to improve the ability of readily available anti tumor antibodies to improve NK cell mediated killing of solid tumors and use engineered iPSC derived NK cells as an "off the shelf" approach for allogeneic cell based immunotherapies. Disclosures Kaufman:Fate Therapeutics: Consultancy.