Chichi Huang

Differential Effects of Long-lived Erythropoietin Receptor Agonists in Rats

Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. Here, we report on experiments designed to study how the pharmacokinetic profiles of EPO receptor agonists, ranging from the short-lived epoetin-? to the long-lived EPO-MIMETIBODYTM constructs CNTO 530 and CNTO 531, influence the pharmacodynamic response in rats. Rats received a single dose of an EPO-R agonist and the effects on reticulocytes, red blood cells and hemoglobin were measured over time. The increase in red blood cells and hemoglobin were negatively correlated with clearance. At doses that cause a similar effect on reticulocytes, very long-lived EPO-R agonists caused prolonged production of red blood cells. In conclusion, we have shown that very long-lived EPO-R agonists cause prolonged production of red blood cells and increase in hemoglobin that is independent of their in vitro potency or the peak release of reticulocytes. These data suggest that EPO may be a survival factor for reticulocytes.

Engineering Peptide Therapeutics Using MIMETIBODY™ Technology

The MIMETIBODY™ platform was developed to expand the opportunities for application of biotherapeutics. While the utility of antibodies as antagonists has been well demonstrated, their application as agonists has been more challenging. For steric reasons, antibodies may be less well suited to perform as agonists or as inhibitors of GPCRs. In contrast, many bioactive peptides function as agonists or by interaction with GPCRs but their development as therapeutics has been challenging due to their small size and metabolic lability. The MIMETIBODY™ platform has been used to develop a variety of stable, long-lived molecules with intrinsic activities similar to that of their parent peptides. This chapter describes methods for construction of expression plasmids, expression and purification strategies, and methods for characterizing the activity of these novel proteins.

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.