<p>Chimeric antigen receptor-natural killer (CAR-NK) cells represent a promising cellular immunotherapy platform for various cancers, offering a favorable clinical safety profile and potential to generate off-the-shelf products. However, broader application of CAR-NK cells is in part restricted by the availability of surface tumor antigens. T-cell receptor (TCR)-like CARs have gained increasing attention due to their ability to recognize peptides derived from intracellular tumor antigens presented by major histocompatibility complex molecules. In the present study, we designed a third-generation CAR targeting WT1(126–134) peptide presented by HLA-A*02:01, harboring a TCR-like scFv linked to CD28, 4-1BB, and CD3ζ signaling domains (CAR-WT1), and introduced it into human NK-92 cells—the only FDA-approved NK cell line for clinical trials. Interestingly, we found that surface expression of CAR-WT1 in NK-92 cells required the presence of human CD3 complex, as observed for full-length TCRs. Quantitative PCR and subsequent pathway and network analyses indicated enhanced immune activation potentially relevant to effector function in CAR-WT1/CD3 NK-92 cells. The established CAR-WT1/CD3 NK-92 cells exhibited significantly greater cytotoxicity against WT1⁺/HLA-A2⁺ target tumor cells than its non-transduced NK-92 cells, consistent with the release of functional cytokines. Once CAR-WT1 was expressed on NK-92 cell surface, surface CD3 appeared dispensable for NK cytotoxicity. Together, our findings provide evidence supporting the feasibility for generating functional TCR-like CAR-WT1 NK-92 cells, thereby broadening the scope of targetable antigens for CAR-NK cell therapy to include intracellular antigens. Other TCR-like CARs with impaired surface expression may be introduced in NK-92 cells using the same strategy, pending further validation.</p>