Targeting and neutralizing human epididymis protein 4 by novel nanobodies to suppress ovarian cancer cells and attenuate cisplatin resistance

Human epididymis protein 4 (HE4) is a glycoprotein secreted by epithelial ovarian cancer (EOC) cells and is a novel and specific biomarker for diagnosing and prognosing EOC. Previous studies have shown that overexpression of HE4 is correlated with EOC tumorigenesis and chemoresistance. However, less has been reported regarding the direct effect of the secreted HE4 protein as an autocrine factor in EOC cells. Here, we investigated the molecular mechanism of the secretory form of HE4 on the growth of EOC cells by applying nanobodies with a targeted interaction of free HE4. Three anti-HE4 nanobodies were selected from an immune library by phage display. HE4 secreted by serum-free cultured OVCAR3 cells increased and was effectively neutralized by anti-HE4 nanobodies, which inhibited cell viability. Treatment with the anti-HE4 nanobody 1G8 decreased Bcl-2 expression and increased BAX, cleaved PARP, and p53 levels, resulting in apoptosis of OVCAR3 cells. Moreover, 1G8 significantly improved the cisplatin response of OVCAR3 cells. Our data suggest that secretory HE4 played a novel pro-survival autocrine role and was a target of the anti-HE4 nanobody to improve the therapeutic effects of cisplatin-based chemotherapy.