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Background There is an unmet need to develop safe and effective therapies against acute myelogenous leukemia (AML), which is characterized by a rapid and clonal accumulation of myeloid progenitor cells in bone marrow. Interleukin-1 receptor accessory protein (IL-1RAP) is minimally expressed in normal tissues but is consistently overexpressed across multiple genetic subtypes of AML, where it potentiates multiple oncogenic signaling pathways, 1 making it an attractive target for immunotherapies. Natural killer (NK) cells are innate immune cells with significant alloreactive anti-AML effects and ex-vivo expanded NK cells have significantly increased PFS in adults following haplo-HSCT.2 3 We hypothesize that IL-1RAP-targeting chimeric antigen receptor NK (IL-1RAP.CAR NK) cells, when combined with IL-15-based adjuvant immunostimulatory reagents, either a Trispecific Killer Engager (TriKE; cam161533) recognizing CD16 on NK cells and CD33 on AML cells,4 or polymer-conjugated IL15 (NKTR-255),5 would significantly enhance NK cell cytotoxicity against AML.Methods NK cells were isolated from healthy donor peripheral blood mononuclear cells and expanded by coculture with lethally-irradiated K562-mbIL21-41BBL cells. IL-1RAP CAR.NK cells were generated utilizing a Cas9/AAV6 system as previously reported. 6 In vitro cytotoxicity against AML cell lines was assessed in a 24-hour assay at a 1:1 effector-to-target ratio.7 Cam161533 (125 ng/ml) and NKTR-255 (40 ng/ml) were added to selected conditions.Results IL-1RAP CAR.NK cells demonstrated significantly increased cytotoxicity compared to unmodified NK against IL-1RAP high AML cell lines (Molm13, MV4-11 and HEL) (figure 1A), but not against IL-1RAPlow KG1a cells. Both NKTR-255 and cam161533 TriKE significantly enhanced the tumor killing activities of NK cells, and more notably further potentiated the cytotoxicity of IL-1RAP CAR. NK (p<0.001, figure 1B). IL-1RAP CAR. NK cells secreted significantly higher levels of Granzyme B, IFNγ, and perforin compared to unmodified NK cells, and their secretions were further significantly augmented by NKTR-255 or cam161533 (figure 1C).Conclusions IL-1RAP.CAR NK cells, in combination with IL-15- based immunomodulation, exhibited potent in vitro anti-AML cytotoxicity. These results support further evaluation of this combinatorial approach in human AML xenograft models.Acknowledgements Supported by DOD HT94252410682References Mitchell KL Barreyro, Todorova TI, Taylor SJ, Antony-Debre I, Narayanagari SR, Carvajal LA, Leite J, Piperdi Z, Pendurti G, Mantzaris I, Paietta E, Verma A, Gritsman K, Steidl U. IL1RAP potentiates multiple oncogenic signaling pathways in AML. J Exp Med, 2018;215(6):1709-1727.Ciurea SO, Kongtim P, Srour S, Chen J, Soebbing D, Shpall E, Rezvani K, Nakkula R, Thakkar A, Troy EC, Cash AA, Behbehani G, Cao K, Schafer J, Champlin RE, Lee DA. Results of a phase I trial with haploidentical mbIL-21 ex vivo expanded NK cells for patients with multiply relapsed and refractory AML. Am J Hematol 2024:99(5):890-899.Ciurea SO, Schafer JR, Bassett R, Denman CJ, Cao K, Willis D, Rondon G, Chen J, Soebbing D, Kaur I, Gulbis A, Ahmed S, Rezvani K, Shpall EJ, Lee DA, Champlin RE. Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplantation. Blood 2017;130(16):1857-1868.Sarhan D, Brandt L, Felices M, Guldevall K, Lenvik T, Hinderlie P, Curtsinger J, Warlick E, Spellman SR, Blazar BR, Weisdorf DJ, Cooley S, Vallera DA, Onfelt B, Miller JS. 161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS. Blood Adv 2018;2(12):1459-1469.Luo W, Gardenswartz A, Hoang H, Chu Y, Tian M, Liao Y, Ayello J, Rosenblum JM, Mo X, Marcondes AM, Overwijk WW, Cripe TP, Lee DA, Cairo MS. Combinatorial immunotherapy of anti-MCAM CAR-modified expanded natural killer cells and NKTR-255 against neuroblastoma. Mol Ther Oncol 2024;32(4):200894.Naeimi Kararoudi M, Likhite S, Elmas E, Yamamoto K, Schwartz M, Sorathia K, de Souza Fernandes Pereira M, Sezgin Y, Devine RD, Lyberger JM, Behbehani GK, Chakravarti N, Moriarity BS, Meyer K, Lee DA. Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV. Cell Rep Methods 2022;2(6):100236.Chu Y, Nayyar G, Tian M, Lee DA, Ozkaynak MF, Ayala-Cuesta J, Klose K, Foley K, Mendelowitz AS, Luo W, Liao Y, Ayello J, Behbehani GK, Riddell S, Cripe T, Cairo MS. Efficiently targeting neuroblastoma with the combination of anti-ROR1 CAR NK cells and N-803 in vitro and in vivo in NB xenografts. Mol Ther Oncol 2024;32(2):200820.Abstract 649 Figure 1A) In vitro cytotoxicity of NK and IL-1RAP.CAR NK cells against Mol13, MV4-11 and HEL AML cell lines. B) In vitro cytotoxicity and C) Granzyme B, IFNγ and perforin release of NK and IL-1RAP.CAR NK cells, with and without NKTR-255 or cam161533 TriKE