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...

The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDSpolyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. Enzymatic N-deglycosylation resulted in an EpoEpo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/g; 76 IU/ pmol) was significantly greater than that of Epo (352 IU/g; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of EpoEpo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.Recombinantly produced proteins are gaining wide use as injectable pharmaceuticals to treat a variety of deficiencies and diseases. A problem encountered in their use is the frequency with which injections must be made in order to maintain a therapeutic level in the circulation. One means of increasing the plasma half-life of injected proteins is chemical conjugation with polyethylene glycol ("pegylation") (1). Although apparent success has been achieved with some proteins (2), this method can sometimes alter protein structure, reduce biological activity, or cause unanticipated changes in specificity and function (3-5). We hypothesized that an alternative approach would be the production of a multivalent molecule consisting of two or more biologically active units of the same protein. 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).We now report the production of a recombinant fusion protein consisting of two complete human Epo molecules in tandem separated by a 17-amino acid linker. Both Epo domains of the fusion protein are equally biologically active. Importantly, the protein has substantially enhanced potency and efficac...

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