Engineering Natural Killer Cells to Target Acute Myeloid Leukemia with HLA-DR Loss - PROJECT SUMMARY/ABSTRACT Engineering T cells and natural killer (NK) cells with chimeric antigen receptors (CAR) has emerged as a powerful new therapeutic approach for cancer, particularly hematologic malignancies. However, most target antigens are not cancer-specific but are also expressed by normal cells (albeit often at lower levels), leading to severe “on- target, off-tumor” toxicity. There is thus a critical need to enhance the anti-cancer specificity of CAR immune cells. While cancer-specific antigens are rarely available, human leukocyte antigens (HLA) are often lost or downregulated in cancer cells, presumably a mechanism to escape immune surveillance. For example, complete or partial loss of HLA-DR, a class II HLA molecule, has been documented in approximately 17% of acute myeloid leukemia (AML) at diagnosis and up to 50% of relapsed cases after transplantation. Our project aims to develop a novel CAR-NK cell that can specifically target AML with HLA-DR loss. Our central hypothesis is that dual CAR- NK cells, which express an activating CAR (aCAR) against CD33 (an AML antigen) and an inhibitory CAR (iCAR) against HLA-DR, will eradicate AML with HLA-DR loss but spare normal myeloid cells, which are HLA-DR positive. In preliminary studies, we engineered NK-92MI cells (a human NK cell line) to express a CD28/CD3ξ- based anti-CD33 aCAR and a PD-1-based anti-HLA-DR iCAR. We found the resulting dual CAR-NK cells could preferentially recognize CD33+HLA-DRneg cells over CD33+HLA-DR+ cells. However, the aCAR/iCAR pair was designed based on T-cell signaling, which may not be optimal in NK cells. Here, we propose to test our hypothesis by pursuing two specific aims: 1) Develop, validate, and optimize a pair of anti-CD33 aCAR and anti- HLA-DR iCAR in NK cells in vitro; 2) Examine the anti-AML potency and specificity of single and dual CAR-NK cells in mouse models. The proposed research is innovative, in our opinion, because HLA-DR loss is an immune escape mechanism frequently observed in AML and other hematologic malignancies, but it has not previously been exploited to enhance cancer targeting by CAR-T or CAR-NK cells. The proposed research is significant because its successful completion will lead to a novel prototype of CAR-NK cells that can specifically target AML with HLA-DR loss, which accounts for a substantial proportion of AML patients at diagnosis and relapse. This approach, if proven successful, can also be broadly used to improve the targeting specificity of CAR-T/NK cells against other types of cancer.
