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

Abstract

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 SDS-polyacrylamide 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 Epo-Epo 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/μg; 76 IU/pmol) was significantly greater than that of Epo (352 IU/μg; 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 Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages. 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 SDS-polyacrylamide 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 Epo-Epo 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/μg; 76 IU/pmol) was significantly greater than that of Epo (352 IU/μg; 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 Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages. erythropoietin erythropoietin receptor SDS-polyacrylamide gel electrophoresis wild-type erythropoietin enzyme-linked immunosorbent assay 20 mm Tris-HCl, 0.5 m NaCl, 0.5% Tween 20 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”) (1Delgado C. Francis G.E. Fisher D. Crit. Rev. Ther. Drug Carrier Syst. 1992; 9: 249-304PubMed Google Scholar). Although apparent success has been achieved with some proteins (2Tsutsumi Y. Tsunoda S. Kamada H. Kihira T. Kaneda Y. Ohsugi Y. Mayumi T. Thromb. Haemostasis. 1997; 77: 168-173Crossref PubMed Scopus (46) Google Scholar), this method can sometimes alter protein structure, reduce biological activity, or cause unanticipated changes in specificity and function (3Mabrouk P.A. Bioconjugate Chem. 1994; 5: 236-241Crossref PubMed Scopus (28) Google Scholar, 4Pettit D.K. Bonnert T.P. Eisenman J. Srinivasan S. Paxton R. Beers C. Lynch D. Miller B. Yost J. Grabstein K.H. Gombotz W.R. J. Biol. Chem. 1997; 272: 2312-2318Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 5Knusli C. Delgado C. Malik F. Domine M. Tejedor M.C. Irvine A.E. Fisher D. Francis G.E. Br. J. Haematol. 1992; 82: 654-657Crossref PubMed Scopus (44) Google Scholar). 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 (6Sytkowski A.J. Lunn E.D. Davis K.L. Feldman L. Siekman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1184-1188Crossref PubMed Scopus (53) Google Scholar, 7Mockridge J.W. Aston R. Morrell D.J. Holder A.T. Eur. J. Endocrinol. 1998; 138: 449-459Crossref PubMed Scopus (11) Google Scholar). 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 (8Qiu H. Belanger A. Yoon H.-W.P. Bunn H.F. J. Biol. Chem. 1998; 273: 11173-11176Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 efficacy over conventional recombinant Epo in vitro and in vivo and is efficacious after a single subcutaneous injection. Two different Epo cDNA constructs were produced by amplifying pSV2-Epo (9Chern Y. Chung T. Sytkowski A.J. Eur. J. Biochem. 1991; 202: 225-229Crossref PubMed Scopus (15) Google Scholar) and then ligated to form the Epo-Epo fusion protein cDNA. The initial preceding (Domain A) Epo DNA strand (Epo A1) was produced by amplifying pSV2-Epo using primers EpA5′ and EpA3-3 (see below). The 3′ end was sequentially extended by amplifying with EpA3-4 and EpA3-5. Epo A2 was produced by amplifying Epo A1 with primers EpA5′ and EpA3-4, and Epo A3 was produced by amplifying Epo A1 with primers EpA5′ and EpA3-5. The succeeding (Domain B) Epo DNA strand (Epo B) was obtained by amplification of pSV2 Epo using EpB5-1 and EpB3′. Gel-purified Epo A3 and Epo B were ligated into the cloning vector pCR-blunt (InVitrogen) using a vector:insert ratio of 1:10. Kanamycin-resistant colonies (50 μg/ml) were plucked, and positive clones were identified by restriction digest analysis using Bgl I. The resulting constructs were designated Epo A (blunt and Epo B) blunt. The primer sequences were as follows: (a) EpA5′, 5′-AGGCGCGGAGATGGGGGTGCAC; (b) EpA3-3, 3′-CCAGATCCACCACCGCCGGCTCTGTCCCCTGTCCTGCAGG; (c) EpA3-4, 3′-CGCCACCGGATCCACCGCCACCAGATCCACCACCGCCGGC; (d) EpA3-5, 3′-TGGTGGGGCAGTACTGCCGCCGCCACCGGATCCACCGCC; (e) EpB5-1, 5′-GCGGCAGTACTGCCCCACCACGCCTCATCTGTGACAGC; and (f) EpB3′, 3′-CAGGTGGACACACCTGGTCATC. Epo A/blunt was digested with Sca I and Xho I, whereas Epo B/blunt was digested with Sca I and Bam HI. Gel-purified Epo A/blunt and Epo B/blunt digests were ligated in a 1:1 molar ratio. The resulting 1.2- and 1.4-kilobase bands were gel-purified and then ligated into expression vector pCDNA3.1(−) (InVitrogen) that had been digested previously with Bam HI and Xho I and gel-purified. Ampicillin-resistant colonies (10 μg/ml) were plucked, and positive clones were identified by restriction digest analysis using Ngo M. The reactions (50 μl) contained 0.5 m 5′ or 3′ primer; 10 ng of pSV2-Epo; 200 μm dATP, dUTP, dGTP, and dTTP; 20 mmTris-HCl, pH 8; 2 mm MgCl2; 10 mmKCl; 6 mm (NH4)2SO4; 0.1% Triton X-100; 10 g/ml nuclease-free bovine serum albumin; and 2.5 units/plaque-forming unit DNA polymerase (Stratagene). They were subjected to 25 cycles of (a) 1-min denaturation at 95 °C, (b) 1-min annealing at 58 °C, and (c) 1-min extension at 72 °C. Ligation reactions for pCR-blunt contained 25 ng of vector and a 10-fold molar excess of either Epo A3 or Epo B. 10-μl reactions contained 6 mmTris-HCl, pH 7.5, 6 mm MgCl2, 5 mmNaCl, 0.1 mg/ml bovine serum albumin, 7 mm mercaptoethanol, 0.1 mm ATP, 2 mm dithiothreitol, 1 mm spermidine, and 4 Weiss units of T4 DNA ligase (InVitrogen). Incubations were carried out for 1 h at 16 °C. pcDNA 3.1(−) ligations contained a vector:insert ratio of 5:1, and the reaction conditions described above were used. 50 μl of One Shot TOP10 competent cells (InVitrogen) were transformed with 2 μl of ligation reaction mixture according to the manufacturer's procedure. An Epo mutant with arginine 103 replaced by alanine, pSV2-Epo 103 (R103A) (10Grodberg J. Davis K.L. Sytkowski A.J. Eur. J. Biochem. 1993; 218: 597-601Crossref PubMed Scopus (36) Google Scholar) was spliced into Epo A/blunt to produce Epo 103A/blunt. Epo 103B/blunt was produced by amplifying pSV2-Epo103 using primers EpoB-51 and EpoB3. Restriction sites used to insert the mutant strand were Bcg I and Acc I for Epo 103A/blunt. The procedure for ligating Epo A, Epo B, Epo 103A, and Epo 103B was described above. Three mutant fusion constructs were produced: (a) Epo A-Epo 103B/pcDNA 3.1(−), (b) Epo 103A-EpoB/pcDNA 3.1(−), and (c) Epo 103A-Epo 103B/pcDNA 3.1(−), resulting in fusion proteins Epowt/EpoR103A, EpoR103A/Epowt, and EpoR103A/EpoR103A, respectively. The mutant Epo 103 was also spliced into pcDNA3.1(−) in the Eco NI restriction sites. Total RNA was prepared using TRIZOL reagent (Life Technologies, Inc.). The manufacturer's protocol was followed, and the RNA obtained was separated on 1.2% agarose containing 5.5% formaldehyde and transferred to GeneScreen Plus. The DIG-labeled probe was generated according to the procedure described by the manufacturer. COS1 cells (American Type Culture Collection) were grown to 70% confluence, washed twice with phosphate-buffered saline and 6 mm glucose, and resuspended in phosphate-buffered saline/glucose to a concentration of 2 × 106 cells/ml. 10 μg of DNA were added to 0.5 ml of cells, and the cells were electroporated at 0.3 kV, 250 farads (Bio-Rad Electroporator). The cells were plated into 10 ml of Dulbecco's modified Eagle's medium/high glucose and 10% fetal bovine serum and incubated for 72 h at 37 °C, 5% CO2. Supernatant medium was harvested, centrifuged at 10,000 ×g at 4 °C, and then dialyzed against α- minimum Eagle's medium. These samples were assayed for Epo protein and biological activity. Epo and Epo-Epo fusion proteins were measured by Western blot and by enzyme-linked immunosorbent assay (ELISA). Samples were subjected to SDS-PAGE and transferred electrophoretically to 0.45 μm nitrocellulose membranes in 25 mm Tris-HCl, 192 mm glycine, and 10% methanol. Membranes were then rinsed twice with distilled water and incubated at 4 in and 10% pH The membranes were rinsed twice with washed with for and washed twice with for 5 The membranes were then incubated with A.J. Fisher J.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and 5% for 1 h at °C. and were carried out as described by with a in and 5% for 1 h at °C. and were carried out as described two for 5 bands by the were using a The was with recombinant human Epo. and 5% were added to a COS1 cell containing the Epo-Epo fusion and the was at for 2 0.5 of 5% the of on and 1 of mm and 50 mm pH were added to the of were added to of the prepared samples were incubated for at 37 and then prepared for and Western were as described above. The of samples was in vitro as described by Google Scholar). The of recombinant Epo used to the was against the was with medium containing Eagle's fetal bovine and (Life Technologies, Inc.). Three of mice were used. administration of Epo or was and its was using blood obtained by two from the An was on to the of its and The were and an identical of Epo or Epo-Epo fusion protein by subcutaneous injection. The biological of the samples were in by in vitro The frequency of was On the of was was used to the changes the and of were An level of was used. Two of mice were used. the administration of Epo or μl of blood were from the into The were and the plasma was and at °C. 6 of Epo or Epo-Epo by and blood samples were obtained at 5 1 2 4 and h The injections were by a of the The of and the of were by an We constructed an Epo-Epo fusion protein cDNA at the 5′ end with the complete of the acid human Epo C. R. A. J. R. M. T. T. PubMed Scopus Google S. R. F. Proc. Natl. Acad. Sci. U. S. A. 82: PubMed Scopus Google Scholar) with (Domain is by encoding a flexible of the of the is a encoding the acid Epo protein by a (Domain COS1 cells were with either conventional Epo cDNA (8Qiu H. Belanger A. Yoon H.-W.P. Bunn H.F. J. Biol. Chem. 1998; 273: 11173-11176Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, Google Scholar) or Epo-Epo and blot were carried out with Epo cDNA expressed a An of a of was expressed by cells with Epo-Epo blot analysis of RNA from COS1 cells with Epo cDNA or with Epo-Epo cDNA The was with a DIG-labeled of the fusion protein and secretion of Epo-Epo by the COS1 cells were by SDS-PAGE and Western blot analysis using an (10Grodberg J. Davis K.L. Sytkowski A.J. Eur. J. Biochem. 1993; 218: 597-601Crossref PubMed Scopus (36) Google Scholar) Epo migrated as a broad band with an average apparent molecular mass of 37 kDa, with the of the A.J. Feldman L. D.J. Biochem. 1991; PubMed Scopus Google Scholar) at of its sites. In contrast, Epo-Epo migrated with an average apparent molecular mass of 76 kDa. is would be from of the sites in 37 with the of the 17-amino acid or of the and of the fusion protein. The results of N-deglycosylation this of Epo-Epo with an concentration of PubMed Scopus Google Scholar) a species that migrated on SDS-PAGE as a narrow band with an average apparent molecular mass of kDa, with the of The use of of the resulted in a with of The of the in average apparent molecular mass achieved by complete N-deglycosylation of Epo-Epo is twice that for N-deglycosylation of the Epo monomer A.J. Feldman L. D.J. Biochem. 1991; PubMed Scopus Google Scholar, M. S. M. A. J. Biol. Chem. Full Text PDF PubMed Google M. S. R. Eur. J. Biochem. PubMed Scopus Google Scholar). In some the Epo-Epo migrated on SDS-PAGE as a that some molecules the has also been for Epo A.J. Feldman L. D.J. Biochem. 1991; PubMed Scopus Google Scholar). results are with an Epo-Epo to which in molecular mass to for Epo are as with Epo, the of of Epo-Epo some Epo-Epo is biologically active in We COS1 cells with either conventional cDNA or Epo-Epo cDNA. the were and subjected to an in vitro was measured by against recombinant human Epo expressed at μg/ml) and exhibited a specific activity of IU/pmol). specific activity is slightly than that obtained by previously for wild-type Epo expressed in COS1 cells (10Grodberg J. Davis K.L. Sytkowski A.J. Eur. J. Biochem. 1993; 218: 597-601Crossref PubMed Scopus (36) Google Scholar). In contrast, a level of expression of which exhibited a substantially specific activity IU/μg; 76 and protein of Epo and Epo-Epo fusion protein in COS1 cell and Epo-Epo in COS1 cell were by in vitro and by The are the of three activity was by the by the Epo and Epo-Epo in COS1 cell were by in vitro and by The are the of three activity was by the by the in a Both Epo domains of are biologically active. We this by of arginine 103 the arginine 103 equivalent in B, which is arginine of the fusion to results in complete of the protein D. M. Bunn H.F. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, D.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We prepared three different constructs that this (a) EpoR103A/Epowt, (b) Epowt/EpoR103A, and (c) We that of the two domains were then would in a protein that biological activity, with some in specific activity. the mutant be in I, and were expressed and exhibited biological activity. the specific of the two were IU/pmol) and that of the IU/μg; 76 IU/pmol). that of the into Epo resulted in of that the wild-type to the that domains of biologically of receptor and equally The mutant was expressed at the level However, activity or protein was in the COS1 Epo-Epo exhibited enhanced activity in vivo compared with Epo We obtained from three of in the in the form of COS1 cell whereas in the a single of Supernatant medium from COS1 cells with vector was to the as a the of was A increase in was in with In contrast, of the exhibited a significant increase in an in of the half-life of Epo. The of the increased an average of compared with a of in the and a of in the The in of the was significantly different from that of the and that of the We that the pharmacokinetic behavior of Epo-Epo is different from that of Epo We injected two of mice either with 6 of Epo or with 6 blood samples were and the plasma concentration of Epo or Epo-Epo was by The plasma of mice injected with Epo In three of the the Epo concentration to than of the level within 1 Epo was in the plasma of after 4 In contrast, the plasma of injected with Epo-Epo for after 6 Epo-Epo after h in and were above of the in two of the Epo-Epo was in the plasma of two of the after Epo-Epo also exhibited pharmacokinetic In three of the Epo-Epo to after the at 2 h after injection. unanticipated was due to in injections were by and In the have shown that a fusion protein consisting of two complete human Epo domains separated by a flexible has significantly enhanced in vitro and in vivo biological activity compared with the form of recombinant Epo. enhanced activity to be due to an increased specific activity with a different pharmacokinetic The of the Epo-Epo in three of the injected with Epo-Epo We carried out the analysis of the plasma samples twice at and out the of the dose that the increase in with an increasing of samples Biochem. PubMed Scopus Google Scholar). The that Epo-Epo may with or with or more proteins in the at Epo-Epo an could with binding in the resulting in a The pharmacokinetic behavior of Epo-Epo also be by a and with receptors or binding sites on cells in to the Both the of erythropoietin receptors on these cells and a to its binding have been A. M. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, R. T. M. M. T. Y. Eur. J. Biochem. PubMed Scopus Google Scholar, H. C. L. C. Full Text PDF PubMed Scopus Google Scholar, Y. S. M. M. R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, A. H. J. J. 1997; PubMed Scopus Google Scholar). produced linked Epo and that could an increase in potency and a frequency of to enhanced in vivo (6Sytkowski A.J. Lunn E.D. Davis K.L. Feldman L. Siekman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1184-1188Crossref PubMed Scopus (53) Google Scholar). These linked in a of consisting of more active active The use of the fusion protein in the of Although the of the Epo-Epo cDNA was were the success of the that could be to (a) biological activity, (b) (c) the of (d) (e) and (f) in vivo had that the amino of erythropoietin is in its biological activity to the amino acids are Sytkowski A.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, others have shown that arginine may be important in the of Epo D. M. Bunn H.F. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). was the Epo (Domain that which is by its amino to the would exhibit biological activity. An greater the Epo (Domain A) with a D. B. 1991; 77: PubMed Google Scholar) that to the of erythropoietin are the of of A to the the with which the and which is for biological activity, would be However, the that Epo domains are active. of the flexible to that used for an factor fusion protein J. T. U. D. R. D. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar) of Epo domains into their The of the of Epo to two binding domains of the C. K.H. B. H. A.J. J. J. S. D.J. J. 1998; PubMed Scopus Google Scholar) that the amino acids and of Epo have with in the 1 whereas and of Epo have in In contrast, of Epo have at the of Epo, and have in These would suggest that the of the the two Epo domains may be in that Epo of can to and of two J. T. U. D. R. D. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar) constructed a fusion protein consisting of factor and linked by an acid of the and fusion protein consisting of and factor linked by a acid of the Both of these fusion proteins could to cell receptors either and exhibited biological activity in vitro with function of In contrast, a by (8Qiu H. Belanger A. Yoon H.-W.P. Bunn H.F. J. Biol. Chem. 1998; 273: 11173-11176Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) used of the to form fusion proteins of the or these fusion proteins were biologically active than the 1 of and the were and flexible to receptor by the fusion protein. However, in to (8Qiu H. Belanger A. Yoon H.-W.P. Bunn H.F. J. Biol. Chem. 1998; 273: 11173-11176Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) increase a in the activity of their fusion proteins compared with into the results of J. T. U. D. R. D. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar) and the results of the with the R. T. M. M. T. Y. Eur. J. Biochem. PubMed Scopus Google Scholar), the used by (8Qiu H. Belanger A. Yoon H.-W.P. Bunn H.F. J. Biol. Chem. 1998; 273: 11173-11176Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) of their fusion proteins to resulting in an activity to that by for and for their the 17-amino acid used in this of Epo-Epo Epo domains to function and resulted in enhanced activity. The potency of Epo-Epo in vitro is significantly than that of conventional recombinant Epo. is and on a are for this may be due to an increased of Epo-Epo or to a in this increase in potency may an increase in receptor Although the that the binding of of Epo-Epo the binding of the positive In this have shown previously that the of by of cells resulted in positive and an enhanced biological Y. S. Sytkowski A.J. PubMed Google Scholar, S. Y. Feldman L. Sytkowski A.J. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). The or binding of Epo domains of the fusion protein also such receptor to increased of that could in enhanced is that the secretion of this was 8-fold that of However, the on were that an increase in was for the in secretion more an increased intracellular and enhanced and and were at greater than that of the that protein may a in the enhanced secretion of the fusion protein. that at arginine 103 of Epo could to a molecule with increased (10Grodberg J. Davis K.L. Sytkowski A.J. Eur. J. Biochem. 1993; 218: 597-601Crossref PubMed Scopus (36) Google Scholar, J. Davis K.L. Sytkowski A.J. Biochem. PubMed Scopus Google Scholar). single in the fusion protein its to increased of that the enhanced of over a in the of secretion of the fusion protein. Although mutant was expressed at the protein by either or is in to that by (8Qiu H. Belanger A. Yoon H.-W.P. Bunn H.F. J. Biol. Chem. 1998; 273: 11173-11176Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) for their fusion proteins with or that were by Although that these results suggest that the and of the may a at the activity and of the They may also and the Epo domains of the fusion protein and the the with and its and secretion We for the of recombinant human erythropoietin and for