Stabilized CD16a Expression on Pluripotent Stem Cell-Derived Natural Killer Cells

Abstract

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) have been shown to successfully generate functional hematopoietic cells of multiple lineages. One of the cells that these studies have shown is possible to generate are cytotoxic natural killer (NK) cells. These pluripotent stem cell-derived NK cells could potentially be used as a cell-based therapy to treat a wide range of cancers. A potent method through which NK cells kill their targets is antibody-dependent cell-mediated cytotoxicity (ADCC). During this process an Fc gamma receptor, CD16a, recognizes antibody coated target cells and leads to the stimulation of the NK cell’s cytotoxic pathways. However, upon CD16a activation, a metalloprotease known as ADAM17 has been shown to cleave CD16a leading to a decrease in NK cell ADCC. This project seeks to generate genetically modified NK cells that have CD16a expression stabilized to maximize ADCC. One method to accomplish this is the generation of a non-cleavable version of CD16a. This cDNA construct has been transfected into iPSCs using Sleeping Beauty transposase to stably express CD16a. These cell’s CD16a molecules are resistant to cleavage upon cell activation and are also highly expressed on the surface of the NK cells. Preliminary testing has shown that when combined with antibodies specific to their cognate target ligand, these cells have a greater cytolytic response than untransfected peripheral blood NK cells (PBNK). Also, CRISPR-Cas9 has been employed to knock-out ADAM17. Going forward, comparing the cytotoxicity of these two NK cells will show which is best to employ in the treatment of cancers that express ligands for therapeutic antibodies that have proven refractory to other therapies and adjuvants.