Intein-Mediated Production and Anti-tumor Evaluation of DT386-BR2 in Animal Model of Human Breast Cancer

Abstract

PurposeDespite therapeutic advances, breast cancer treatment remains limited by non-selective toxicity, highlighting the need for targeted agents. We previously introduced DT386-BR2 as a selective cytotoxic fusion protein. This study aimed to produce soluble DT386-BR2 using an intein-mediated purification system and to evaluate its cytotoxicity and antitumor efficacy in vitro and in a nude mouse xenograft model. MethodsThe gene encoding DT386-BR2 was cloned into the pTWIN-1 vector and expressed in E. coli BL21 (DE3). Following induction with IPTG (1 mM, 15 degrees C, 24 h), the protein was purified using the IMPACT system. Cytotoxicity against MCF-7 breast cancer cells was determined via MTT assay. In vivo efficacy and safety were assessed in nude mice bearing MCF-7 xenografts. Tumor and vital organs were examined histopathologically using H&E and TUNEL staining. ResultsSoluble DT386-BR2 was successfully expressed and purified. The protein exhibited a potent cytotoxic effect on MCF-7 cells, with an IC50 of 199 nM. In vivo, DT386-BR2 significantly reduced tumor volume compared with controls (P < 0.05), without evidence of metastasis or abnormalities in heart, lungs, kidneys, or spleen. Histological analysis, however, revealed moderate hepatic necrosis in treated mice. ConclusionDT386-BR2 effectively suppressed breast tumor growth in vitro and in vivo, with limited off-target toxicity aside from hepatic effects. These findings highlight DT386-BR2 as a promising targeted therapeutic candidate for breast cancer and warrant further optimization and preclinical safety studies.