Abstract 3603: NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C
Authors: Cho Cynthia , Kimura Nikki , Sood Jessica , Juat Damie , Zhang Max , Chan Carmel , Geng Tao , Hansen Kyle , Trager James 
Published: March 2024

Abstract
Introduction: NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L+ cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic, off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD−). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C.
Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or transduced with empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC50 and IC90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application.
Results: Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines, further validating NKG2D-Ls as therapeutic targets. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells in a synergistic manner, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML.
Citation Format: Cynthia Cho, Nikki Kimura, Jessica Sood, Damie Juat, Max Zhang, Carmel Chan, Tao Geng, Kyle Hansen, James Trager. NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3603.