Engineering T cells and natural killer (NK) cells with anti-CD19 chimeric antigen receptors (CAR) has emerged as a powerful new strategy to treat diffuse large B-cell lymphoma (DLBCL). However, CD19 is also expressed on normal B cells, leading to unwanted depletion of B cells, an adverse effect known as “on-target off-tumor” toxicity. The overall objective of our proposed research is to develop an enhanced CAR-NK cell platform that can specifically target DLBCL but spare normal B cells. Previous work finds that the expression of human leukocyte antigen DR (HLA-DR) is lost in 23.9–33.3% of DLBCL patients, presumably to escape anti-tumor immunity. We reason that the specificity of CAR-NK cells can be enhanced by targeting both CD19 overexpression and HLA-DR loss in the above patient population. Our central hypothesis is that a dual CAR-NK cell, which expresses an anti-CD19 CAR and an anti-HLA-DR inhibitory CAR (iCAR), will effectively kill HLA-DR-negative malignant B cells but spare HLA-DR-positive normal B cells. We will test our hypothesis by pursuing two specific aims: 1) Develop and validate an optimal pair of CD19 CAR and HLA-DR iCAR using NK cell-specific activating and inhibitory signaling domains; and 2) Examine the target specificity of dual CAR-NK cells in xenograft mouse models. The proposed research is innovative because HLA loss is an immune escape mechanism frequently used by cancer cells but has not previously been explored to enhance cancer targeting by CAR-T or CAR-NK cells. The successful completion of the proposed study will lead to the development of a dual CAR-NK cell platform for specific targeting of HLA-DR-negative DLBCL. Additionally, our proposed approach can be broadly applied for developing safer CAR-T and NK cell therapy against other hematologic malignancies and solid tumors with HLA loss.