Human erythropoietin dimers with markedly enhancedin vivo activity

Abstract

Human erythropoietin, a widely used and important therapeutic glycoprotein, has a relatively short plasma half-life due to clearance by glomerular filtration as well as by other mechanisms. We hypothesized that an erythropoietin species with a larger molecular size would exhibit an increased plasma half-life and, potentially, an enhanced biological activity. We now report the production of biologically active erythropoietin dimers and trimers by chemical crosslinking of the conventional monomeric form. We imparted free sulfhydryl residues to a pool of erythropoietin monomer by chemical modification. A second pool was reacted with another modifying reagent to yield monomer with maleimido groups. Upon mixing these two pools, covalently linked dimers and trimers were formed that were biologically active in vitro. The plasma half-life of erythropoietin dimers in rabbits was >24 h compared with 4 h for the monomers. Importantly, erythropoietin dimers were biologically active in vivo as shown by their ability to increase the hematocrits of mice when injected subcutaneously. In addition, the dimers exhibited >26-fold higher activity in vivo than did the monomers and were very effective after only one dose. Dimeric and other oligomeric forms of Epo may have an important role in therapy.Human erythropoietin (Epo) was first purified from the urine of anemic human donors (1). This important accomplishment was made possible by a salient feature of the protein, namely, that its molecular size of 31-37 kDa allows it to pass through the renal glomerulus and to be excreted in the urine.The advent of recombinant human erythropoietin (2-4), an important therapeutic for correcting certain types of anemia, also made possible more detailed studies of erythropoietin physiology and pharmacokinetics. When administered intravenously to rodents, radiolabeled Epo seemed to localize to several different tissues (5). Pharmacokinetic studies in human volunteers with normal renal function have yielded a plasma half-life of 4-13 h (6, 7). This half-life is much shorter than that of other, larger proteins used therapeutically, for example, serum albumin and Ig.We hypothesized that an Epo species with a larger molecular size would have a reduced rate of clearance, thereby lengthening its plasma survival and increasing its in vivo biological activity. Here, we report the synthesis of Epo dimers and trimers using heterobifunctional crosslinking reagents. These Epo species are biologically active. Moreover, Epo dimers exhibit a markedly enhanced plasma survival and are much more efficacious in vivo than is the conventional monomeric Epo. MATERIALS AND METHODS Preparation of Epo Containing Free Sulfhydryl Groups (SH-Epo) or Maleimido Groups (maleimido-Epo).Recombinant human Epo (1.0 mg͞ml) was incubated in the presence of 10-fold molar excess of succinimidyl 6-[3-(2-pyridyldithio)propionamido] hexanoate (LC-SPDP) (Pierce) or in the presence of 10-fold molar excess of succinimidyl 4-(N-maleimido methyl)cyclohexane-1-carboxylate (SMCC) (Pierce...

Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo

“…39 DAE7 proliferation assay calibrated with human recombinant Epo determined that the Epo dimer induced a 6-fold-higher increase in hematocrit compared with the monomeric form when injected on days 1, 3, and 5 in normal mice at concentrations of 300 U/kg. A similar effect on hematocrit has been observed with chemically linked Epo dimer injected in rabbits 22 and with an Epo-Epo fusion protein injected in mice. 23 It has been postulated that this effect is mainly the result of an enhanced blood lifetime of the dimeric form.…”

“…As with a higher Epo concentration (200 U/kg; Figure 5), no difference between monomer and dimer pharmacokinetics was observed (data not shown). Discrepancies between our data and previously published results 22,23 could be related to (1) the sequence or the length of the linker peptide, which was longer in previous studies (17 amino acid residues vs 9 residues in this study), (2) the producing Epo and Epo-Epo cell line, which may have modified the glycosylation pattern, or (3) the monomeric Epo used as reference for the pharmacokinetic studies. In this study, monomeric and dimeric Epo were produced under identical conditions, by the same cell line, without purification but by concentration of the culture medium using ultrafiltration.…”

“…20,21 The association of 2 Epo molecules obtained by either chemical cross-linking or recombinant DNA-mediated fusion of coding regions resulted in a more stable protein than the native monomer with an increased in vivo life span. 22,23 Based on evidence that the bridging of 2 adjacent Epo receptors triggers a conformational change that initiates signal transduction 24,25 and that high-and low-affinity sites for the Epo receptor are present on Epo, [26][27][28] we hypothesized that the fusing of 2 Epo molecules might confer an increase in intrinsic activity by providing 2 closely associated high-affinity domains.In this study, we report the design and characterization of a recombinant fusion protein made of 2 human Epo molecules linked by a peptide linker of 9 amino acids. We show that this dimer has enhanced erythropoietic activity, both in vitro on primary human erythroid progenitors and in vivo in normal mouse compared with its monomer counterpart.…”

An Erythropoietin Fusion Protein Comprised of Identical Repeating Domains Exhibits Enhanced Biological Properties

“…Previously, we produced chemically linked Epo dimers and showed that we could achieve an increase in potency and a decreased frequency of injection, all leading to enhanced in vivo action (6). These chemically linked dimers are, in all likelihood, a mixed population of molecules, presumably consisting of more highly active isoforms and, possibly, less active ones.…”

“…We speculated that these molecules would exhibit an increased plasma half-life and would also possess enhanced activity due to facilitated binding of the repeating units to their cognate receptors and to amplification of the intracellular signaling pathways. We and others have shown previously that such molecules could be produced by chemical cross-linking (6,7). However, a fusion protein with two human erythropoietin (Epo) 1 domains linked by three to seven amino acids exhibited reduced in vitro activity compared with the wild-type monomer (8).…”