Engineered-NK Cells Expressing Chimeric Ilt Receptors (CIR™) Expand and Eliminate Bone Marrow-Resident HLA-G+ AML in NSG Mice
Authors: Duong Mylinh , Park Jihyun , Senesac Joseph , Ognar Raphael , Wain-Hobson Simon , Bayle Henri 
Published: December 2024

Background
Human leukocyte antigen (HLA)-G binds to Ig-like transcript (ILT) receptors (ILT2 in lymphoid and ILT4 in myeloid cells) and directs immunosuppression via immunoreceptor tyrosine-based inhibitory motif signaling domains. HLA-G expression is highly repressed in normal tissues except for cytotrophoblasts of the placenta. In contrast, more than 50% of cancers upregulate HLA-G as a mechanism to evade immune surveillance thus making HLA-G an ideal tumor target across indications. Because HLA-G is expressed and active as an immunosuppressant in multiple structural isoforms, an antibody-based CAR approach may not be ideal. To target HLA-G+ tumors, we developed Chimeric ILT Receptors (CIR) by linking the extracellular domains of ILT2 or ILT4 receptor with intracellular activation domains to drive activation of Natural Killer (NK) cells. Further, a screen of NK-relevant activation domains identified several leading candidates that induce robust cytotoxicity and NK expansion when engaged with HLA-G+ target cells relative to 4-1BB.CD3ζ.
Methods
CD56+ NK cells were cultured with IL-15 and IL-2 and activated with a feeder-free cocktail of an immobilized cytokine and activating ligand. Activated NK cells were transduced with g-retroviruses directing multi-cistronic expression of CIR, soluble IL-15, and a DCD19 marker and co-transduced with RFP or ONLRluc for fluorescent detection. Coculture assays were performed with GFPFluc-expressing tumor cell lines and monitored using the IncuCyte live-cell imager while supernatants were analyzed for IFN-γ and TNF-α release by ELISA. A single dose of 7.5 x 106 mock or CIR-NK cells were injected intravenously into Molm13 tumor-bearing NSG mice. Tumor and NK cell bioluminescence (BLI) were tracked via GFPFluc and ONLRluc, respectively. FACS analyses of blood, spleen and bone marrow were performed post-euthanasia.
Results
Both ILT2 and ILT4 CIR constructs were efficiently and stably expressed in NK cells (average % CD19+/ONL+= 93.98 ± 2.12). ILT2 or ILT4 CIRs fused to our novel intracellular activation molecules (4-1BB.DAP10 and 4-1BB.DAP10.(2A-)TLR2) demonstrated equally robust targeting of Molm13 cells at an effector to target ratio of up to 1:40 (Molm13-GFP intensity: ILT2.4-1BB.ζ = 24.3 ± 4.8E6 vs. ILT2.4-1BB.DAP10 = 12.3 ± 4.2E6 vs. ILT2.4-1BB.DAP10.2A-TLR2 = 10.7 ± 0.2E6) and enhanced NK cell expansion (NK-RFP intensity: ILT2.4-1BB.ζ = 3.0 ± 1.4E4 vs. ILT2.4-1BB.DAP10 = 11.8 ± 7.7E4 vs. ILT2.4-1BB.DAP10.2A-TLR2 = 12.8 ± 2.0E4). Notably, a single dose of ILT4.4-1BB.DAP10-NK cells induced 60% greater clearance of Molm13 tumor burden in NSG mice in 10 days relative to ILT4.4-1BB.ζ counterparts (tumor BLI: ILT4.4-1BB.ζ = 5.27 ± 0.53E5 vs. 4-1BB.DAP10 = 2.21 ± 1.1E5) in addition to 3-fold greater NK cell expansion on day 14 (NK BLI: 4-1BB.ζ = 1.06 ± 0.39E7 vs. 4-1BB.DAP10 = 3.16 ± 0.9.1E7). Flow cytometric analysis indicated complete tumor clearance by ILT2 and ILT4.4-1BB.DAP10.TLR2-NK cells by day 17 in blood, spleen and bone marrow (% Molm13-GFP+ in bone marrow: ILT4.ζ = 27.0 ± 14.9 vs. ILT4.4-1BB.DAP10.TLR2 = 0.08 ± 0.06 vs. ILT2.4-1BB.DAP10.TLR2 = 0.02 ± 0.03). Moreover, the expanded CIR-NK cells in the blood, spleen and bone marrow express high transgene levels (average % CD19+/ONL+ in spleens: 89.3 ± 3.4) and are CD57+ (average % CD57+ in blood: 44.3 ± 9.7), NKG2D+ (average % NKG2D+ in bone marrow: 58.1 ± 13.6), CD16+ (average % CD16+ in spleens: 89.0 ± 3.7), and CD25+ (average % CD25+ in spleen: 35.2 ± 18). Analysis of cytokines secreted from plasma indicated elevated production of proinflammatory cytokines (IFN-γ and TNF-α) and granzyme B as early as 7 days post CIR-NK injection.
Conclusions
Utilization of the natural receptors (ILT2/4) to target HLA-G enables recognition of multiple immunosuppressive HLA-G isoforms that may circumvent antigen escape by isotype switching of antibody-based therapy. 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.