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Background Natural killer cell engagers (NKCEs) offer a versatile platform to enhance NK cell-mediated cytotoxicity. However, conventional NKCEs often fall short in fully activating NK cell due to inhibitory signals such as NKG2A. In this study, we developed a novel NKCE, ES038, that targets LILRB4 on Acute myeloid leukemia (AML) cells while simultaneously blocking inhibitory (NKG2A) and stimulating activating (NKG2C) receptors on NK cells, this dual-targeting strategy enhances NK cell activation and improves their therapeutic potential. AML, particularly in relapsed or refractory cases, remains a significant clinical challenge. Several AML surface markers, such as CD33, CD123, CD47, LILRB4 – have emerged as promising therapeutic targets, with antibody-based therapies demonstrating encouraging results in preclinical and clinical settings. LILRB4, an immunosuppressive checkpoint molecule, is highly expressed in M4/M5 AML subtypes and plays a role in T cell suppression and leukemic cell infiltration. LILRB4, an immunosuppressive checkpoint molecule, is overexpressed in M4/M5 AML and plays a role in T cell suppression and leukemia cells infiltration. 1 Methods ES038 was engineered to bind LILRB4 on AML cells and NKG2A/C on NK cells simultaneously. And the binding activity was assessed by FACS and SPR methods. The blockade of NKG2A and HLA-E interaction was measured by FACS. LILRB4 – dependent NKG2C activation was evaluated in Jurkat-NFAT-NKG2C/CD94/DAP12 reporter system. Functional validation also comprises NK cell activation (CD107a/IFN-γ induction) and cytotoxicity against LILRB4 + AML cell lines (Molm-13/THP-1). Pharmacokinetics profile was evaluated in mouse serum and antibody developability was assessed under stress conditions. In vivo efficacy was evaluated in C1498-LILRB4 syngeneic model monitoring tumor burden and survival.Results ES038 is designed with high-affinity binding domains for LILRB4 and NKG2A and induces NKG2C activation only in LILRB4-dependent manner. ES038 could effectively block the interaction between NKG2A and HLA-E. The dual-targeting NKCE demonstrated superior cytotoxicity compared to NKG2A-only NKCE with enhanced NK cell degranulation (CD107a) and IFN-γ production in double-positive NKG2A + NKG2C + NK population. ES038 exhibited preferable drug developability and mouse PK profile. In vivo, ES038 treatment reduced leukemic tumor growth and extended mice survival.Conclusions This first-in-class NKCE, leveraging dual NKG2A/C engagement, offers a promising strategy in AML and other LILRB4 positive hematologic malignancies.Reference Deng M, Gui X, Kim JE, et al. LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration. Nature. 2018;562(7728):605-609.