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PT-401 JUNE 12-2008
13th Congress of the European Hematology Association, June 12-15, 2008
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Title IN VITRO AND IN VIVO PROPERTIES OF PT-401, A NOVEL ERYTHROPOIETIN FUSION PROTEIN
Authors J. Sytkowski,1 J.Y. Jeong,1 K.L. Davis,1 A.L. Socha,1 H.J. Gomez2
Address 1Beth Israel Deaconess Medical Center, Harvard Medical School, BOSTON, MA; 2DNAPrint Pharmaceuticals, Inc., SARASOTA, FL, USA
Reference Haematologica 2008: 93(s1):117 Abs.0288
Text of the Abstract Background and Aims. Recombinant human erythropoietin (rhEpo, epoetin) is widely used for correction of anemia due to chronic kidney disease and other conditions. Modified forms of rhEpo and other erythropoiesis stimulating agents (ESAs) with higher efficacy and longer half-life have been and continue to be developed to overcome rhEpo’s short in vivo half-life. Previously, we described a novel Epo fusion protein, PT-401, comprising identical head-to-tail repeats and a short linker. We now report that PT-401 exhibits unique in vitro characteristics and enhanced in vivo activity in mice compared to rhEpo. Methods. PT-401 was expressed in Chinese hamster ovary (CHO) cells stably transfected with a mammalian expression vector containing the PT-401 cDNA. We used quantitative immunoblotting analysis with the monoclonal anti-Epo antibody AE7A5 to identify and quantify PT-401 protein in the cell culture supernatant. Highly purified PT-401 was obtained by sequential column chromatography. The interaction of PT-401 with the Epo receptor (EpoR) was characterized using radioiodinated PT-401 and BaF3 cells stably expressing the human EpoR (BaF3/huEpoR). Results. The amount of PT-401 secreted into protein-free cell culture medium of cloned CHO cells varied between 4 and 40 mg/L. Clones producing PT-401 with the optimal glycosylation were identified based upon mobility in SDS-PAGE and isoelectric focusing, based upon the observation that the extent of glycosylation of Epo correlates with the in vivo half-life. The specific biological activity of purified PT-401 measure by in vitro bioassay was similar to that of freshly produced PT-401 in the cell culture supernatant, demonstrating that the biological activity of PT-401 was preserved during the purification process. PT-401 exhibited specific, saturable binding to the EpoR. The equilibrium dissociation constant (Kd) of PT-401/EpoR interaction was approximately 4 nM, higher than that for rhEpo (0.14 nM, determined in the same experiment), consistent with longer-acting ESAs. The specific activity of PT-401 determined by in vitro bioassay was lower than that of rhEpo, reflecting the higher Kd, also consistent with other longer-acting ESAs. Importantly, PT-401 increased the mean hematocrit of mice to significantly higher levels compared to rhEpo in all injection schemes tested (three injections with 300 IU/kg or 100 IU/kg, a single injection with 100 IU/kg), demonstrating the superior in vivo activity of PT-401. Conclusions. PT-401 exhibits in vitro characteristics consistent with a very long-acting ESA and an in vivo biological activity superior to that of rhEpo, suggesting important therapeutic advantages.
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