(182w) Arming Engineered Natural Killer Cells with Immunocytokines for Treatment of Acute Myeloid Leukemia

Chimeric antigen receptor (CAR)-expressing natural killer (NK) cells have emerged as a promising immunotherapy candidate for hematological malignances such as acute myeloid leukemia (AML). Our lab previously engineered a panel of CAR-NK cells containing anti-CD123 antibody single-chain variable fragments (scFvs) with a range of target affinities and used this panel to elucidate affinity-based distinctions in CAR-NK cell function across in vitro and in vivo models of AML. In particular, we discovered that CD123-specific CAR-NK cells expressing a low affinity variant of the 7G3 scFv exhibited increased cytokine production and enhanced NK-mediated cytotoxicity upon antigen engagement compared to CAR NK cells expressing the parent 7G3 scFv. These low affinity 7G3 scFv-expressing CAR-NK cells demonstrate strong potential as AML therapeutics, but there remain challenges in effectively priming the NK cells in vivo for sustained antitumor function and persistence. Moreover, the functionality of NK cells is impaired in the context of cancer by increased abundance of immunosuppressive cells, such as regulatory T cells (Tregs).

To address the outstanding need to maintain CAR-NK cell persistence in vivo, supplementation of cytokines can be implemented to augment tumor homing and antitumor activity of NK cells. In particular, interleukin-2 (IL-2) is a key mediator of activated NK cell function, proliferation, and survival. However, due to its pleiotropic nature, IL-2 also signals on Tregs, and in fact the cytokine is more potent on these immunosuppressive cells than it is on NK cells. Our lab has previously engineered a cytokine/antibody fusion protein (immunocytokine, IC) that reverses the Treg bias of IL-2. This molecule, denoted F10 IC, fuses human IL-2 to an engineered anti-IL-2 antibody that modulates the interactions of the cytokine with the IL-2 receptor subunits to enhance its activity on NK cells versus Tregs. We subsequently miniaturized this molecule into a single-chain format, known as Mini F10 IC. Here, we hypothesize that co-delivering Mini F10 IC with low affinity 7G3-expressing CAR-NK cells will lead to improved NK cell persistence and potent antitumor activity in the context of AML therapy. We aim to establish that co-administering Mini F10 IC enhances signaling activity, pro-inflammatory cytokine secretion, and cytotoxicity of CAR-expressing immortalized NK-92 cells or primary human NK cells when co-cultured with AML cells. Building on this, we plan to introduce a bicistronic retroviral vector encoding both the low affinity 7G3 CAR and Mini F10 IC into immortalized NK-92 cells or primary human NK cells. We will assess whether continuous production of the immunocytokine confers enhanced effector functions, both through in vitro co-culture studies with AML cells and through in vivo studies of CAR-NK cell persistence and antitumor efficacy using mouse models of AML.