PROJECT SUMMARY: Lung cancer (LC), the most frequent cause of cancer deaths for men and women, is estimated to lead to 228,820 new cases and 135,720 deaths in the United States in 2020. In recent years inhibition of immune checkpoints, such as programmed cell death-1 (PD-1) and programmed cell death ligand- 1 (PD-L1), has been shown to provide survival benefits to patients with LC. However, most patients demonstrate either primary resistance or experience tumor recurrence and die of their disease. Our group has demonstrated that cancer cells upregulate PD-L1 on natural killer (NK) cells, immune cells that can target malignancies without the necessity of chimeric antigen receptors or prior antigen exposure and do not require matching to recipient's human leukocyte antigen for potential activity. Upregulation of PD- L1resulted in enhanced NK-cell function. Furthermore, the PD-L1 inhibitor atezolizumab (AZ) resulted in enhanced leukemic cell killing against myeloid leukemia lacking PD-L1 expression, and mice treated with selective cytokines (IL-12, IL-15, and IL-18) in combination with AZ showed a significant improvement in survival even in the absence of PD-L1 expression in their tumor tissue. We were able to express soluble IL-15 (sIL-15) tagged with a truncated epithelial growth factor receptor in umbilical cord NK cells in vitro, while upregulating endogenous PD-L1 expression on the NK cells. These transduced NK cells maintained greater than 30% antigen-specific tumor lysis compared to mock-transduced NK cells and demonstrated cytotoxicity against A549 Non-Small-Cell LC (NSCLC) cells. Human A549 NSCLC cells were subsequently injected in non-syngeneic mice and followed with treatment with these “enhanced” cordon blood CB NK cells. In comparison to treatment with mock-transduced NK cells, or NK cells expressing sIL-15 (sIL-15-NK) but without ex vivo activation, the enhanced CB NK cells induced substantial reduction in tumor volume. We also performed safety/toxicity in vivo studies of this approach and compared with AZ alone. Our data suggest that anti-PD-L1 mAb therapy has a unique therapeutic role in treating PD-L1 negative cancer, acting through PD-L1(+) NK cells. This activity is achieved independent of PD-1 activity and in the presence of NK-activating cytokines. We hypothesize that cytokine “enhanced” NK cells will provide clinical benefit to NSCLC patients and that the antitumor activity of this approach will be further enhanced by co- administration of AZ. To test this hypothesis and document the safety of this strategy, we propose additional in vivo safety and efficacy studies followed by a phase 1 study in which CB NK cells, genetically modified to express sIL-15, followed by ex-vivo expansion in the presence of IL-2, IL-18, and IL-12 will be administered either by themselves or combined with AZ, following lymphocyte depletion, to NSCLC patients whose tumor has previously progressed on or after treatment with PD-1/PD-L1 inhibitors.