Abstract 1532: EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function

Abstract

Abstract We have previously described EDIT-201, a healthy donor derived NK cell therapy with CRISPR-Cas12a mediated CISH and TGFBR2 double gene knockout that demonstrated resistance to TGF-β inhibition and increased tumor control in vitro and in vivo. The tumor microenvironment (TME) is known to be nutrient-deprived due to active tumor cell metabolism leading to competition for essential nutrients with infiltrating effector cells, while at the same time being enriched in immunosuppressive metabolites such as lactic acid due to Warburg Metabolism. To explore whether EDIT-201 is functional in such hostile metabolic conditions, we aimed to more accurately model the metabolic microenvironment in a long-term tumor spheroid model. To model this, we generated SK-OV-3 ovarian tumor spheroids in decreasing concentrations of glucose (10-0.5mM) or glutamine (2-0.1mM), two important fuels for NK cell metabolism, as well as increasing concentrations of inhibitory metabolite lactate (0-50mM), or decreasing pH (7.2-6.5). Each of these metabolic conditions are known to suppress effector cell function; we additionally performed spheroid cells co-cultures in the presence of 10ng/mL TGF-β to increase the potential for NK inhibition. We demonstrated that, relative to control NK cells, EDIT-201 demonstrated rapid and sustained tumor killing in all conditions tested including, the absence of critical nutrients. We next created a multifactorial matrix of metabolic conditions where we combined deprivation of multiple nutrients, in the presence of lactate and/or acidic cell culture media. Remarkably, in all combinations tested, we surprisingly found that EDIT-201 continued to demonstrate increased cytotoxicity against SK-OV-3 spheroids relative to control NK cells, suggesting a clear and robust metabolic advantage of EDIT-201 over control NK cells. We additionally observed a corresponding increase in the concentrations of IFN-γ and TNF-α by EDIT-201 in all of these conditions relative to control NK cells. Given that mitochondrial respiration is key to NK cell persistence and function, we next interrogated the mitochondrial function of EDIT-201. We find that EDIT-201 consistently demonstrated greater spare respiratory capacity (SRC) relative to control NK cells, suggesting enhanced mitochondrial reserve as a result of CISHand TGFBR2 knockout. The increase in SRC likely enables EDIT-201 to meet enhanced energy demands necessary to mediate effector function in metabolically challenging conditions, thus sustaining superior cytotoxic capacity and cytokine production. In summary, our data demonstrate enhanced metabolic function of EDIT-201, resulting in superior cytotoxicity in metabolically unfavorable conditions. These data further support the development of EDIT-201 as a novel cell therapy for cancer. Citation Format: Karrie K. Wong, Steven Sexton, Lincy Prem Antony, Kevin Wasko, Jared Nasser, Kelly Donahue, Amanda Pfautz, Shuqi Zhang, Richard A. Morgan, Christopher M. Borges. EDIT-201: CRISPR-Cas12a gene editing enhances functional metabolism of natural killer cells and enables tumor cell cytolysis in metabolically stressful conditions that inhibit effector cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1532.