Anti-MSLN chimeric antigen receptor-like NK cell therapy with tumor-penetrating capacity (uCAR-like NK) for solid tumors.

Although natural killer (NK) cells are endowed with intrinsic cytotoxicity, their therapeutic application often faces limitations because of their lack of tumor-specific targeting ability and limited ability to infiltrate solid tumors. To overcome these limitations, we developed anti-mesothelin (MSLN) uCAR-like NK cells, which are designed to enhance both the targeting specificity and tumor infiltration capacity, thereby improving the antitumor efficacy of NK cell-based therapies. We constructed, purified, and validated a tetravalent bispecific cell engager (MSLN×CD16A) via the SpyTag/SpyCatcher system. Cytokine-induced memory-like NK cells, induced by IL-12, IL-15, and IL-18, were precomplexed with MSLN×CD16A to generate anti-MSLN CAR-like NK cells. To further enhance tumor penetration, the tumor-penetrating peptide uCendR was integrated into the system to construct anti-MSLN uCAR-like NK cells. In vitro, anti-MSLN CAR-like NK cells demonstrated selective cytotoxicity against MSLN-positive tumor cells through stable binding with MSLN×CD16A while sparing MSLN-negative cells. In xenograft models bearing MSLN-positive tumors, anti-MSLN CAR-like NK cells exhibited significant antitumor activity, with favorable tolerability and no significant body weight loss or toxicity. Notably, anti-MSLN uCAR-like NK cells, which integrate a tumor-penetrating peptide, displayed enhanced intratumor penetration and superior therapeutic efficacy. Overall, this study establishes a modular, nongenetically engineered uCAR-like NK platform that couples targeted recognition with enhanced tissue access. These findings highlight the potential of anti-MSLN CAR-like NK cells, particularly uCAR-like NK cells with enhanced tumor penetration, as promising therapeutic strategies for MSLN-positive solid tumors and lay the foundation for future clinical applications.