Multifunctional-engineered NK cells overcome tumor immunosuppression by combining PD-L1 and HLA-E targeting and endogenous IL15 production.

The immunosuppressive tumor microenvironment (TME) poses a significant challenge to effective cancer immunotherapy, as it enables tumor escape through redundant checkpoint pathways, metabolic constraints, and direct inhibition of effector cells. To overcome these barriers, we developed a next-generation NK cell platform using a tri-cistronic retroviral vector that enhances NK cell activation, recruitment, survival, and metabolic fitness. Pan-cancer transcriptomic analyses reveal consistent co-expression of PD-L1 and HLA-E across tumors, which correlates with immune infiltration accompanied by strong immunosuppression, highlighting these molecules as key targets for immune evasion. To improve NK cell recruitment, activation, cytolytic function, survival, and metabolic fitness in the TME, we developed a next-generation NK cell platform using a tri-cistronic retroviral vector that encodes an extracellular PD-1 domain (exPD1) fused to the intracellular portion of NKG2D with the costimulatory molecule 4-1BB, and expressing soluble IL15 and NKG2A single-chain variable fragments (scFv). Thus, the exPD1 allows the recognition of cells expressing PD-L1, while the intracellular costimulatory signaling transforms the inhibitory interaction PD-1/PD-L1 into an activating one. This strategy effectively targets PD-L1-positive tumor cells and induces de novo PD-L1 expression in otherwise negative tumors. To further enhance anti-tumor activity, we incorporated a module encoding soluble NKG2A-scFv to mask the NKG2A receptor on NK cells and hinder NKG2A/HLA-E inhibitory interaction. Additionally, we improved NK cell survival and expansion by delivering controlled low doses of IL15, which prevents NK cell exhaustion and extends their presence in vivo. This integrated strategy may provide a novel, ready-to-use allogenic mature NK cell therapy that could overcome checkpoint-mediated inhibition, metabolic suppression, and immune escape, offering a promising model for treating high-risk and treatment-resistant tumors.