Production and purification of anti-angiogenic recombinant protein drug, DAAP and its preclinical study

Abstract

We have recently developed a fusion protein, which simultaneously blocks VEGF and angiopoietins, namely Double anti-angiogenic protein (DAAP). These molecules are known to serve a crucial role in tumor angiogenesis and vascular leakage. I have assessed the efficacy and safety of DAAP as means of a preclinical study. DAAP was purified from stable CHO cell line manipulated to express DAAP, which was found to show synchronous binding to VEGF-A and Ang2 by ELISA analysis. It also inhibited the migration of HUVECs induced by VEGF-A in vitro. For in vivo evaluation, DAAP was administered subcutaneously at a dose of 25 mg/kg (body weight) in wild-type C57BL/6J mouse every 3 days to assess any potential toxicity. All mice survived and maintained their normal body weight. The white blood cell (WBC) counts and differential leukocytes ratio were within reference range. No abnormalities in the levels of blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were observed. In addition, blood pressure and pulse rate were within reference range. However, DAAP administered mouse increase the hematocrit level that indicates the capability of in vivo VEGF-A neutralization, and the risk of thrombosis. In conclusion, DAAP purified from CHO cells is a promising anti-angiogenic recombinant protein drug with high efficacy and safety except the increasing hematocrit.