Abstract 3186: Functional analysis of chimeric ILT receptor natural killer (CIR™NK) cells that target tumor cells expressing HLA-G <i>in vitro</i> and <i>in vivo</i>

Abstract Background: Human leukocyte antigen (HLA)-G exerts immunosuppression by binding to ILT2 and ILT4 on myeloid and lymphoid cells. While HLA-G expression is repressed in normal tissues except the placenta and cornea, more than 50% of cancers express HLA-G to evade immune surveillance. We developed Chimeric ILT Receptors (CIRs) that link the extracellular domains of ILT2 or ILT4 receptor with novel intracellular costimulatory domains (4-1BB.DAP10 ± TLR2) to drive HLA-G specific targeting and activation of Natural Killer (NK) cells. Methods: CD56+ NK cells were activated and transduced with γ-retroviruses directing multi-cistronic expression of CIR, soluble IL-15, and a ΔCD19 marker. Coculture assays were performed with GFPFluc-expressing tumor cell lines and NK cells transduced with RFP and monitored using the IncuCyte. In vivo, Molm13 tumor-bearing NSG mice were injected i.v. with a single dose of 7.5E6 mock or CIR-NK cells and tumor and NK cell bioluminescence were tracked via GFPFluc and ONLRluc, respectively. Cytokines from cocultures and mouse plasma were analyzed with a 26-panel multiplex kit. Results: CIR-NK cells demonstrated target-specific elimination of HLA-G+ multiple myeloma and AML cancer cells while sparing HLA-G- cancer cells. ILT4.4-1BB.DAP10.TLR2 CIR-NK cells eliminated 2 fold more HLA-G+MM1S tumor cells than mock-transduced NK cells (GFP intensity: mock = 4.21E5 ± 5.14E4 vs. ILT4.4-1BB.DAP10.TLR2 = 2.15E5 ± 2.26E4) but no killing was observed in coculture against HLA-G- Molm14 AML cells. In repeated target coculture where tumor cells were added up to 4 times every 2 days, ILT4.4-1BB.DAP10-2A.TLR2 CIR-NK cells eliminated more than 60% of Molm13 AML cells (GFP intensity: 4-1BB.ζ = 3.96E7 ± 5.37E6 vs. 4-1BB.DAP10.2A-TLR2 = 1.45E7 ± 3.34E6) and expanded more than 2 fold relative to CIR-NK cells containing 4-1BB.ζ (RFP intensity: 4-1BB.ζ = 1.49E5 ± 5.72E3 vs. 4-1BB.DAP10.2A-TLR2 = 3.26E5 ± 5.1E4). Comparison of chemokine and cytokine production with and without Molm13 target revealed that that IL-33 (42 fold), CD40L (45 fold), IP-10 (26 fold), and IL-12 p70 (73 fold) were among the many analytes highly upregulated by ILT4.4-1BB.DAP10 CIR-NK cells that arm NK cells against the cancer. In vivo, a single dose of ILT4.4-1BB.DAP10 CIR-NK cells induced 60% greater clearance of Molm13 tumor burden in NSG mice in 10 days relative to ILT4.4-1BB.ζ counterparts and 3 fold greater in NK expansion on day 14. Proinflammatory cytokine and chemokines secreted into mouse plasma were remarkably higher as early as 7 days post CIR-NK injection (IFN-γ = 4134 ± 127pg/ml; GM-CSF = 594 ± 56pg/ml; granzyme B = 1529 ± 233pg/ml). Conclusions: We demonstrated that engineered CIR-NK cells, coupled with potent intracellular costimulatory domains, can convert HLA-G expression in cancer cells from an inhibitor of immune surveillance into a practical target for anti-cancer therapy. Citation Format: MyLinh Duong, Jihyun Park, Joseph Senesac, Raphael Ognar, Simon Wain-Hobson, Henri Bayle. Functional analysis of chimeric ILT receptor natural killer (CIR™NK) cells that target tumor cells expressing HLA-G in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3186.