Synthetic erythropoiesis stimulating proteins

#The invention relates to synthetic erythropoiesis stimulating proteins that are polymer-modified in a defined manner, and methods of production and use.

#Erythropoiesis is the process for the production of red blood cells that occurs to regulate turnover and loss of red blood cells. Erythropoietin (EPO) is a protein hormone that stimulates erythropoiesis. Naturally occurring human EPO is a glycoprotein containing 165 amino acids that is produced primarily in the kidney, secreted into the blood stream, and stimulates production of red blood cells from precursor cells in bone marrow. Although the gene encoding human EPO predicts a molecule with 166 amino acid residues, the carboxy-terminal arginine is removed in a post-translational modification in its mature form.

#Glycosylated human EPO has three N-linked carbohydrate chains attached at positions 24, 38 and 83. It also has one O-linked carbohydrate moiety present at position 126. The effects of glycosylation are complex. Non-glycosylated EPO is active in vitro. However, studies have shown glycosylation to be necessary for full activity in vivo. Studies also have shown that variable glycosylation patterns exhibit variable effects. For example, desialyated EPO exhibits both enhanced in vitro and decreased in vivo activity, an effect attributed to the exposure of galactose residues that are recognized, bound, and cleared by hepatocytes. The branching pattern of fully sialyated EPO also has been shown to make a difference in biological activity. Predominantly tetra-antennary branched EPO shows activity equivalent to “standard” EPO, while predominantly bi-anternnary branched EPO shows three-fold more activity in vitro but only 15% of normal activity in vivo. Moreover, various studies have shown that only N-linked, and not O-linked sugars are important in EPO functioning.

#FIGS. 1A-1E depict schematics of processes for preparing the polymer-modified synthetic erythropoiesis stimulating proteins of the invention.

#FIGS. 2A-2C depict schematics of processes for preparing synthetic erythropoiesis stimulating proteins of the invention.

#FIGS. 3A-3B depict schematics of processes for multi-segment ligations that involve the chemical ligation of three or more non-overlapping peptide segments, i.e., at least one segment is a middle segment corresponding to the final full-length ligation product.

#The present invention relates to synthetic erythropoiesis stimulating proteins, methods of production and use.

#I. Synthetic Erythropoiesis Stimulating Proteins of the Invention

#In a preferred embodiment, the invention is directed to a synthetic erythropoiesis stimulating protein having one or more water-soluble polymers attached thereto. By “erythropoiesis stimulating protein” is intended a protein that has the in vitro biological activity of promoting growth of an erythropoietin-dependent cell line. In a preferred embodiment, the synthetic erythropoiesis stimulating proteins of the invention have the in vivo biological activity of promoting production of red blood cells. Erythropoiesis stimulating activity of a protein may be determined by any of a variety of means, such as by following erythropoiesis after in vivo administration, by assaying the capacity of the protein to mediate the proliferation of EPO-dependent cell lines, etc.