Human erythropoietin dimers with markedly enhanced in 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.

Figure 1

SDS/PAGE and Western blot analysis of Epo monomers, chemically modified monomers, and reaction mixture consisting of SH-Epo and maleimido Epo. Monomers (lanes a and c) were treated with 10-fold molar excess of LC-SPDP (lane b) to yield SH-Epo or with 10-fold molar excess of SMCC (lane d) to yield maleimido-Epo. Modified Epo pools (lanes b and d) were mixed and allowed to react (lane b+d). Lane — is untreated monomer. Note new Epo species of ∼70 kDa (dimers) and ∼117 kDa (trimers) in lane b+d.

Figure 2

Size exclusion HPLC chromatogram of mixture after reaction of SH-Epo and maleimido-Epo. The 9.04-min peak corresponds to Epo monomer. Note two peaks eluting at 7.50 and 8.03 min, respectively.

Figure 3

SDS/PAGE and Western blot analyses of fractions collected during SE-HPLC of reaction mixture. Note the higher molecular weight Epo oligomers in earlier fractions. Epo trimers (∼117 kDa) and dimers (∼70 kDa) are well separated from Epo monomers (∼35 kDa). In this preparation, Epo tetramers were also detected in the earliest fractions.

Figure 4

Biological activity profile of fractions collected during SE-HPLC of reaction mixture. Note the peaks of activity corresponding to Epo trimers, dimers, and monomers.

Figure 5

Epo levels in plasma of rabbits injected intravenously with dimers (•) or monomers (▪) at 0 h. Each point represents the mean biological activity determined in triplicate for groups of three to four animals. Standard deviation was equal to or less than ±20% for all values. Note rapid clearance of Epo monomers compared with dimers.

Figure 6

In vivo efficacy of Epo dimers (•) and monomers (▪) in mice injected subcutaneously with 300 IU/kg, days 1, 3, and 5. Hematocrits were obtained pretreatment (Pre) and on day 8 (Post). Points represent the mean Hct of four animals in each group. The vertical bar represents the range of the highest and lowest value.

Figure 7

In vivo efficacy of Epo dimers (•) and monomers (▪) using different dosage regimens. (A) 300 IU/kg, days 1, 3, and 5 (same data as seen in Fig. 6 replotted for comparison). (B) 300 IU/kg, day 1 only. (C) 30 IU/kg, days 1, 3, and 5. (D) 30 IU/kg, day 1 only. Note that for all dosage regimens, Epo dimers are superior to monomers. See legend to Fig. 6.