Abstract 3187: Improved anti-tumor immune functions of iPSC-derived NK cells with TGFβR2 knock-out and/or IL-15 knock-in by TALEN® editing for use alone or in combination with GPC3 Flex-NKTM bispecific antibody
Authors: Lin Liang , Gao Peng , Chen An-Ping , Chang Hao-Ming , Ashok Preeti , Zou David , Ma Marshall Chao , Eyquem Justin , Boyne Alex , Juillerat Alexandre , Poirot Laurent , Duchateau Philippe , Rath Armin , Burchi Elisabetta , Teper Daniel , Arulanandam Antonio , Li Wei 
Published: April 2023

Abstract
Background: Induced Pluripotent Stem Cell (iPSC)-derived NK cells (iNK) offer an opportunity to generate unlimited homogenous NK cells as allogeneic off-the-shelf therapies. Interleukin-15 (IL-15) signaling enhances proliferation, persistence, cytotoxicity, and metabolic fitness of NK cells. Activation of TGF-β signaling suppresses anti-tumor functions of immune cells, including NK cells, in the tumor microenvironment (TME). In addition, previously, we have reported that our Flex-NKTM bispecific antibody that engages NK cells through NKp46 can enhance the cytotoxicity of the non-edited iNK cells. Therefore, we hypothesized that iNK cells with IL-15 knock-in (KI) and/or TGFβR2 knock-out (KO) could exhibit improved immune function and overcome the immunosuppressive TME. Furthermore, the activity of these edited universal iNK cells could be enhanced when combined with CYT-303, a Flex-NKTM bispecific antibody NK engager targeting GPC3 expressed on many solid tumors including hepatocellular carcinoma (HCC).
Methods: IL15 was knocked-in and/or TGFβR2 was knocked-out in iPSC using Cellectis TALEN® and the verified edited iPSC clones were differentiated and expanded into NK cells. The functional significance of these edits in iNKs were assessed in IL-15 and TGF-β dependent NK cell assays evaluating survival and proliferation, expression of activating receptors, as well as cytolysis of these iNK cells against HCC tumor cells. Cytotoxic activity of these edited cells was also tested in a serial killing assay with or without CYT-303 in the absence or presence of TGF-β.
Results: Compared to the non-edited iNK cells, iNKs with IL-15 KI can extend persistence in vitro in the absence of exogeneous cytokines. In the presence of TGF-β, the expression level of a number of NK cell activating receptors, such as NKG2D, DNAM-1, and NKp30, was decreased and lower cytotoxicity against HCC tumor cells was observed. However, this TGF-β-mediated immune suppression was reversed in TGFβR2 KO iNKs cells which also presented enhanced cytotoxicity against HCC cells. Furthermore, the anti-HCC cytotoxic activity of either single (IL-15 KI) or double edited (TGFβR2 KO and Il-15 KI) iNK cells were further enhanced by CYT-303, even in the presence of TGF-β. Serial killing assays against HCC tumor cells showed that the iNK cell dysfunction observed in later rounds of killing could still be reversed by the combination of these gene edits and CYT-303.
Conclusions: This work demonstrates that KI of IL-15 and KO of TGFβR2 is a promising strategy for TALEN®-engineered iNK cell therapies to overcome the immunosuppressive TME and mount a potent and persistent anti-tumor immune response. The data also provide a solid foundation for combining these edited iNK cells with CYT-303 to address the immunosuppressive TME towards a cure for HCC.
Citation Format: Liang Lin, Peng Gao, An-Ping Chen, Hao-Ming Chang, Preeti Ashok, David Zou, Marshall Chao Ma, Justin Eyquem, Alex Boyne, Alexandre Juillerat, Laurent Poirot, Philippe Duchateau, Armin Rath, Elisabetta Burchi, Daniel Teper, Antonio Arulanandam, Wei Li. Improved anti-tumor immune functions of iPSC-derived NK cells with TGFβR2 knock-out and/or IL-15 knock-in by TALEN® editing for use alone or in combination with GPC3 Flex-NKTM bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3187.