An ultra-small bispecific protein augments tumor penetration and treatment for pancreatic cancer

Abstract

Abstract Background and purpose Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths. The once highly anticipated antibody-based pathway-targeted therapies have not achieved promising outcomes, due to drugs' low intrinsic anticancer activity and low penetration across the dense physiological barrier of PDAC tumors. Here, an ultra-small-sized (50 kDa), bispecific protein, called Bi-fp50, that can penetrate deep tumor tissue and effectively inhibit PDAC tumor growth is reported. Methods Bi-fp50 was constructed by a typical synthetic biology method and target both EGFR and VEGF of PDAC cells simultaneously. Characteristics for example binding affinity of Bi-fp50 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and fourier transform infrared spectra (FTIR). Different cell lines (Bxpc3, Aspc1) were used to test the in vitro targeting effect and anticancer ability of Bi-fp50. The orthotopic PDAC tumor model and subcutaneous PDAC tumor model were used to assess in vivo circulation and antitumor effect of Bi-fp50. Results Bi-fp50 with an ultra-small size of 50 kDa (5 ~ 6 nm) had shown a high target binding capacity and in vitro anticancer effect with significant cell killing for Bxpc3 and Aspc1 human PDAC cells. In vivo imaging had shown that Bi-fp50 could vastly enrich deep tumor tissue and had excellent penetration and accumulation when it was injected into orthotopic Bxpc3 xenograft mice. Bi-fp50 also had a high inhibition effect of tumor growth in vivo, accompanied by vascular normalization. No noticeable side effect of Bi-fp50 was found both in vitro and in vivo. Conclusion Compared with scFv2, anti-EGFR scFv, anti-VEGF scFv and Bi-fp50x group, Bi-fp50 with the ultra-small size had the highest binding affinity to both EGFR and VEGF targets. Since Bi-fp50 could penetrate deep pancreatic tumor tissue and had a high antitumor effect in vivo. Our work demonstrates that Bi-fp50 could be a potential candidate as a PDAC tumor suppressor.