Summary: CD19 directed CAR-T cells have transformed the treatment landscape of B-cell lymphoid malignancies. However, despite high initial complete remission rates, relapses occur within the first year of therapy in approximately 50% of patients who receive commercially available autologous CAR19 T-cells. Relapses can be classified into two patterns: CD19-positive relapse related to CAR T-cell exhaustion and senescence, or CD19- negative relapse related to target antigen loss. Patients who relapse after CAR19 T-cell therapy have poor prognosis; hence, there is an urgent need to develop the next-generation of CAR engineered immune effector cells that target tumors with efficacy and with minimal toxicity. There is growing interest in natural killer (NK) cells as a candidate for CAR therapy as they may prevent antigen escape through their innate ability to kill tumor cells and they are safe and well-suited for use in the allogeneic therapy setting. In a first-in-human study, our group showed the safety and promising activity of cord blood (CB) derived CAR-NK cells targeting CD19 in patients with B-lymphoid malignancies (Liu et al NEJM 2020). This proposal aims to build on this platform to develop the next-generation NK cell therapies by enhancing NK cell potency and persistence through optimal costimulatory signaling, cytokine armoring and checkpoint inhibition. We have identified CD70, as a novel therapeutic target in patients with B-NHL after CAR19 T-cell failure and developed a novel strategy to target CD70 by genetically modifying CB-NK cells with a retroviral vector (iC9-CD27-DAP10-CD3ζ-IL-15) that incorporates (i) the gene for a truncated human CD27 (the natural receptor for CD70) to redirect their specificity; (ii) DAP10 as an NK-specific costimulatory domain linked to a CD3ζ signaling endodomain; (iii) IL-15 to support their survival and proliferation, and (iv) inducible caspase-9 (iC9) as a suicide gene. Our preliminary data show the efficacy and safety of this approach in vitro and in vivo and support its translation to the clinic. In addition, we have developed a robust strategy to cryopreserve CAR-NK cells, allowing for the generation of a biobank of off-the-shelf engineered NK cells, thus reducing cost and increasing accessibility. Finally, we have devised a novel strategy to target the immune checkpoint CIS in our CAR-NK cells to modulate their metabolic fitness and potency. We hypothesize that targeting CD70 with iC9/CAR27D10ζ/IL-15 NK cells will greatly improve outcomes in patients with NHL after CAR19 T-cell failure and that by targeting the immune checkpoint CIS we can further enhance the CAR-NK cells’ metabolic fitness and potency. These concepts will be evaluated in three specific aims: In Aim 1 we will conduct a Phase I/II clinical trial to test the safety and efficacy of iC9/CAR27D10ζ/IL-15 NK cells in patients with CD70+ NHL who have failed CAR19 T-cell therapy (FDA approved, IND #27757). In Aim 2 we will apply innovative sing...