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Anti-CD19 chimeric antigen receptor (CAR)-T cells have improved the outcomes of patients with B cell leukemia and lymphoma. However, their applications and positive outcomes remain limited. CAR-T cells are currently restricted to autologous blood as their source and their use can lead to downregulation of CD19 expression along with complications such as graft-versus-host disease and cytokine release syndrome. The present study aimed to develop anti-CD19/CD22 bispecific CAR structures using an anti-CD22 monoclonal antibody clone from chickens and analyze them in natural killer (NK)-92 cells, a human NK cell line, in vitro and in vivo. Anti-CD19/CD22 CAR-NK-92 cell cytotoxicity was assessed by the survival of target cells and counted using flow cytometry. Anti-CD22/CD19 and loop-structured anti-CD19/CD22 bi-specific CAR-NK-92 cells showed improved efficacy against OCI-Ly7 cells, a human B cell lymphoma cell line, compared with other CAR structures. These results demonstrate the potential of anti-CD19/CD22 bispecific CAR-NK cells and suggested that optimizing CAR structures in NK cells can improve the efficacy of CAR therapy. [ABSTRACT FROM AUTHOR]