Abstract CD16 consists of two isoforms (CD16a and CD16b) encoded by two highly homologous genes that differ by only 6 amino acids in their extracellular regions. CD16b is expressed by human neutrophils and CD16a by human natural killer (NK) cells. The ectodomain regions of both CD16 isoforms are cleaved proximal to the cell membrane by a proteolytic process referred to as ectodomain shedding. We demonstrate that the membrane metalloprotease ADAM17 cleaves CD16 in isolated leukocytes and in human patients. By mass spectrometry analysis, we determined 3 adjacent cleavage sites in neutrophil CD16b and one cleavage site in NK cell CD16a that occurred at the same location as the predominant cleavage site in CD16b, which is interesting considering CD16b is linked to the plasma membrane via a GPI anchor and CD16a is a transmembrane protein. Antibody-dependent cell cytotoxicity (ADCC) by NK cells is a key mechanism in the anti-cancer effects of therapeutic antibodies, and CD16a exclusively recognizes tumor-bound antibodies. Surface levels of CD16a are rapidly down-regulated upon NK cell activation by cytokines, target cell interaction, and tumor infiltration, which is associated with impaired ADCC. Thus blocking this process has important clinical significance. We contend that maintaining high surface levels of CD16a during NK cell-based immunotherapy will enhance their killing of antibody-bound tumor cells. In ongoing studies, we are examining pharmaceutical and gene-targeting means of preventing CD16a cleavage as a novel therapeutic strategy to enhance the anti-cancer effects of NK cells. Note: This abstract was not presented at the meeting. Citation Format: Hemant K. Mishra, Jianming Wu, Dan S. Kaufman, Bruce Walcheck. Enhancing tumor killing abilities of NK cells by targeting CD16 shedding, and ADAM17 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 800. doi:10.1158/1538-7445.AM2017-800