Anti-CD47 tri-specific killer engager enhances NK cell cytotoxicity against lung cancer.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with immune evasion posing a major therapeutic challenge. One key mechanism involves the 'don't eat me' signal mediated by the interaction between CD47 and signal regulatory protein alpha (SIRPα), which inhibits macrophage phagocytosis and natural killer (NK) cell cytotoxicity, facilitating tumor escape. To overcome this immune evasion, we developed a tri-specific killer engager (TriKE) targeting CD47, termed anti-CD47 TriKE, designed to enhance NK cell-mediated cytotoxicity against lung cancer cells. The activity of anti-CD47 TriKE was evaluated for its ability to induce NK cell proliferation and its binding affinity to NK cells and lung cancer cell lines (A549, NCI-H460, and NCI-H1975). At a concentration of 30 nM, anti-CD47 TriKE effectively promoted NK cell proliferation and exhibited strong binding to both NK cells and lung cancer cells. Functional assays in 2D and 3D co-culture models demonstrated that anti-CD47 TriKE significantly enhanced NK cell specificity and cytotoxicity. Notably, NK cell-mediated cytotoxicity correlated with the basal level of CD47 expression in target cells. In NCI-H1975 cells, which exhibit the highest CD47 expression, target cell viability was reduced by approximately 40%-a significantly greater reduction than in control groups. These findings highlight the potential of anti-CD47 TriKE as a promising immunotherapeutic strategy for lung cancer, particularly in targeting high-CD47-expressing tumor cells and overcoming immune evasion mechanisms.