A Nova EPO-Fusion Protein with Prolonged Half-Life in Circulation and Enhanced Activity In Vivo.
Authors: Wang Haitao , Du Yong , Zhang Rui , Xu Jin , Liu Longbin 
Published: April 2020

Abstract
Clinical practice merits the search of long-lasting erythropoietic proteins that reduce the injection requirements. Darbepoietin, the first long-lasting EPO, was created by replacing two amino acids of EPO molecule to increase the sugar contents. Darbepoietin has a half-life of about 24 hours and enhanced functional activities. However, the structural changes of EPO molecules may increase the potential of antibody production in logn-term application. With our NovaFusion technology, we successfully created a nova EPO-fusion protein that contains two identical molecules of natural human EPO sequences and a fragment of another human protein(Nova-EPO). We produced recombinant Nova-EPO protein by CHO cells and then conducted extensive laboratory and anemial studies. Pharmacokinetic studies in primates showed that the half-life of Nova-EPO was about 35 hours comparing to about 8 hours of that of recombinant EPO. In pharmacodynamic studies, we compared, on the same molar basis, the erythropoietic effects of Nova-EPO, recombinant EPO and Darbepoietin in normal mice or rats with experimental renal anemia. The experimental renal anemia was created by the surgical removal of 5/6 renal tissues in rats. Five doses (0.1, 0.5, 2.5, 12.5 or 62.5 ug/kg), two administration methods (subcutaneouly or intravenously) and three administration intervials(three injections per week, one injection per week or one injection per two weeks) were selected in different combinations. With three injections per week, both Nova-EPO and EPO induced dose-dependent elevation of hemoglobulin (HB) levels in normal mice. Lower doses of Nova-EPO increased the HB levels to the extent only achived by the higher doses of EPO, and the highest elevation of HB levels was induced by Nova-EPO. With one injection per week in normal mice, the dose-dependent elevation of HB levels was more significant in the Nova-EPO groups than in the EPO groups, and 12.5ug/kg of Nova-EPO induced the increase of HB levels to the same extent by 62.5ug/kg of EPO. The elevation of HB levels in Nova-EPO groups also remained longer than that in EPO groups after the termination of treatment. These data demonstrated that Nova-EPO has functional activities in vivo and stimulates stronger erythropoiesis. The erythropoietic properties of Nova-EPO were further eveluated in rats with experimental anemia, and compared with EPO and Darbepoietin. When administrated once per week, Nova-EPO, EPO and Darbepoietin all induced the increase of HB levels and corrected the anemia. The levels of HB in Nova-EPO and Darbepoietin groups, however, were higher and decreased more slowly than those in EPO groups. The highest HB levels were observed in Nova-EPO groups. After reducing the injection to once per two weeks, Nova-EPO and Darbepoietin still induced satisfactory elevation of HB levels. Similarly, Nova-EPO induced stronger erythropoietic responses than Darbepoietin. In immunogenisis studies in primates, injection of 5ug/kg of Nova-EPO three times per week for four weeks failed to produce antibodies against Nova-EPO molecules. Acute toxic studies in mice showed that injection of up to 13mg/kg of Nova-EPO intravenously did not result in pathologic changes. Collectively these data indicated that Nova-EPO has significantly prolonged half-life in vivo and enhanced erythropoietic activities. With no immunogenisis in primates, Nova-EPO has the potential to become a better alternative than the long-lasting erythropoietic therapeutics on the market and is justified to be further evaluated in clinical studies.