WO1997008200A1 - Proteine de liaison d'erythropoietine utile dans la regulation de l'erythropoiese et dosage permettant de constater sa presence - Google Patents

Proteine de liaison d'erythropoietine utile dans la regulation de l'erythropoiese et dosage permettant de constater sa presence Download PDF

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WO1997008200A1
WO1997008200A1 PCT/US1995/011043 US9511043W WO9708200A1 WO 1997008200 A1 WO1997008200 A1 WO 1997008200A1 US 9511043 W US9511043 W US 9511043W WO 9708200 A1 WO9708200 A1 WO 9708200A1
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erythropoietin
protein
binding protein
complex
binding
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PCT/US1995/011043
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Gisela K. Clemons
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The Board Of Regents Of The University Of California
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Priority to PCT/US1995/011043 priority Critical patent/WO1997008200A1/fr
Priority to AU33752/95A priority patent/AU3375295A/en
Publication of WO1997008200A1 publication Critical patent/WO1997008200A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention concerns erythropoietin binding protein and an assay for its determination. Particularly, this invention concerns the erythropoietin binding protein and its isolation, identification, characterization, and purification, cloning, and expression.
  • the erythropoietin binding protein is useful for regulation of erythropoiesis by regulating levels of erythropoietin via formation of erythropoietin-erythropoietin binding protein complex. Formation of erythropoietin-erythropoietin binding protein complex prolongs the biological activity of erythropoietin and prevents its ultimate depletion by filtration into the urine or by catabolism in liver.
  • Binding proteins for various hormones have been reported in the literature. A highly specific, high affinity, low-capacity binding protein for human growth hormone has been reported in the J. Clin. Endocrinol. Metab. [62:134
  • the growth hormone binding protein was shown to affect growth hormone homeostasis, its kinetics, and metabolism by modulating its interaction with tissue receptors.
  • Erythropoietin is a glycoprotein hormone which is a principal regulator of erythropoiesis, a production of red blood cell. Erythropoietin enhances erythropoiesis by stimulating formation and proliferation of proerythroblasts into reticulocytes and subsequent release of reticulocytes from bone marrow. Ultimately, erythropoietin stimulates the maturation of reticulocytes into morphologically identifiable red blood cells. After- fetal life, erythropoietin is largely produced by kidney in response to tissue hypoxia. Its production, therefore, is principally regulated by the level of renal oxygenation. Approximately 10-15% of erythropoietin is produced by extrarenal sites, including the liver, which seems to be responsible for residual erythropoietin production in anephric patients and in the fetus.
  • Anemia develops whenever there is a deficiency in erythrocyte count. It develops in response to various causes such as sickling disorders, homozygous beta thalassemia, hereditary spherocytosis, red cell enzymopathies, iron, vitamin B 12 , folate deficiencies, aplastic anemia, Fanconi' s anemia, Blackfan Diamond anemia, or leukemia. These disorders are generally accompanied by increased endogenous erythropoietin production. When, however, a patient suffers from acute renal failure or when the chronic renal failure develops, the inadequate renal production of erythropoietin results in hypoplastic anemia. Similarly, anemia in premature infants develops when a progressive fall in hemoglobin concentration, relatively low absolute reticulocyte counts, and bone marrow erythroid hypoplasia develops from low concentration of serum erythropoietin.
  • erythropoietin Treatment with Exogenous Erythropoietin.
  • the presence and availability of erythropoietin for regulation of erythropoiesis is of extreme importance for sustenance of normal physiological state of the human being.
  • the production, release or levels of erythropoietin in the plasma decrease, there are severe consequences affecting the patient's well being. It is therefore, extremely important that erythropoietin is available or made available to control erythropoiesis even when the production and/or release of erythropoietin is blocked by renal disease or by some other pathological conditions.
  • the subject inventor has unexpectedly discovered the existence of a naturally occurring erythropoietin binding protein which has many pharmacological, purification, and immunoassay applications. For the first time the functioning of naturally occurring EP can be optimized, in some cases eliminating the need for further treatment of a patient.
  • the inventive purified erythropoietin binding protein is also useful in assaying erythropoietin and in isolating and purifying this hormone.
  • Another object of the present invention is the isolation and purification of an erythropoietin binding protein which can form a complex erythropoietin, extending the half-life of erythropoietin in plasma.
  • Still another object of the present invention is to treat or prevent anemia by administering to an anemic person an erythropoietin binding protein alone, or in combination with erythropoietin, to stimulate erythropoiesis.
  • Yet another object of the present invention is the isolation, identification, characterization, and purification of erythropoietin binding protein.
  • a further object of the present invention is to provide an immunoassay for determination of concentration in human plasma of erythropoietin binding protein.
  • Figure 1 depicts elution fraction peaks following gel filtration of erythropoietin binding protein complex and free erythropoietin in filtered normal human serum containing labeled and nonlabeled erythropoietin.
  • Figure 2A shows the concentrated binding protein complex (1) and free unbound labeled erythropoietin (2) on non-denaturing 7% polyacrylamide gels.
  • Figure 2B is an autoradiograph showing a clear separation of concentrated erythropoietin binding protein complex and free unbound labeled erythropoietin.
  • Figure 3 shows (A) separation of gel filtration fractions corresponding to erythropoietin binding protein complex on non-denaturing polyacrylamide gel; (B) with respective bands of the gel cut out, eluted, concentrated and incubated with labeled erythropoietin on non-denaturing polyacrylamide gel; and (C) autoradiograph of the complex.
  • Figure 4 depicts elution fraction peak of binding protein eluate, after incubation with labeled erythropoietin, from gel filtration using Sephacryl-S200.
  • Figure 5 is a denaturing SDS polyacrylamide gel of molecular markers (A) and erythropoietin binding protein (B) .
  • Figure 6 depicts elution fraction peaks corresponding to the erythropoietin-erythropoietin binding protein complex and to a labeled free erythropoietin from the normal human serum.
  • Figure 7 depicts elution fraction peaks corresponding to the erythropoietin-erythropoietin binding protein complex and to a labeled free erythropoietin from the human fetal plasma.
  • Figure 8 depicts elution fraction peaks corresponding to the erythropoietin-erythropoietin binding protein complex and to a labeled free erythropoietin in a newborn sheep.
  • Figure 9 depicts elution fraction peaks corresponding to the erythropoietin-erythropoietin binding protein complex and to a labeled free erythropoietin in serum of anephric patient.
  • Figure 10 depicts elution fraction peaks corresponding to the erythropoietin-erythropoietin binding protein complex and to a labeled free erythropoietin in serum of the patient suffering from secondary polycythemia.
  • Figure 11 shows the elution profile erythropoietin-erythropoietin binding protein complex and of free erythropoietin from the normal human plasma containing recombinant human growth hormone.
  • the present invention concerns a newly discovered erythropoietin binding protein in the human serum.
  • Other mammalian species will have the same or closely related proteins. These binding proteins form an erythropoietin-erythropoietin binding protein complex with circulating free erythropoietin. In this way, erythropoietin binding protein extends the half-life of circulating erythropoietin.
  • erythropoietin-erythropoietin binding protein complex prevents rapid excretion by kidney or degradation by liver of free erythropoietin from the blood and, therefore, erythropoietin is able to perform longer its primary function, that is to stimulate formation and maturation of new red blood cells.
  • the binding proteins of the present invention will have important applications in assays and purifications, as well as pharmacology. Erythropoietin. Erythropoietin can be detected in both plasma serum and urine. Under normal condition, erythropoietin is detectable in plasma serum at an average concentration of approximately 10 to 20 mU/ml.
  • the amount of erythropoietin in the serum varies inversely with the level of tissue oxygenation and is dependent on the feedback provided by the lack of excess of oxygen in the tissue.
  • serum erythropoietin level rises when there is tissue hypoxia and stimulates production of red blood cells.
  • Increased levels of erythropoietin were observed to be caused by factors such as anemia, right to left cardiac shunting, chronic obstructive pulmonary disease or exposure to high altitudes.
  • an increased red cell mass induced by erythropoietin corrects tissue hypoxia, serum erythropoietin levels fall and return to their baseline level.
  • erythropoietin stems from its role as erythropoiesis regulator.
  • the cardiovascular and pulmonary systems and circulating erythrocytes function together to deliver oxygen and nutrients throughout the body.
  • the peripheral oxygen delivery in humans is achieved through its binding to hemoglobin of the circulating erythrocytes.
  • the number of circulating erythrocytes is thus decisive for normal health and body function.
  • Erythropoietin in turn, induces proliferation and terminal differentiation of bone marrow erythroid precursors. This ultimately affects the release and maturation of reticulocytes resulting in the larger number of erythrocytes entering the circulation.
  • the kidney cells sense the higher amount of oxygen and cease to further produce or release erythropoietin.
  • EP Binding Proteins in Neonatal and Renal Patients During fetal life, the source of erythropoietin production switches from the liver to the kidney during the third trimester of pregnancy. Significantly greater clearances, short half-lives, shorter residence times, greater distribution volumes and greater erythropoietin production rates have been reported in fetuses and neonates compared to adults. These developmental differences reflect greater distribution of erythropoietin and more rapid metabolism in less mature individuals. The rapid metabolism reduces erythropoietin' s erythropoietic effectiveness and may be the result of lower concentrations of erythropoietin binding protein. In fact, the level of binding protein in fetal and neonatal plasma was found by the inventor to be about two-thirds of that of normal adult plasma.
  • Prematurely born infants are typically afflicted with the anemia of prematurity. These infants not only have low circulating levels of erythropoietin but also inadequate production of erythropoietin. At birth, the liver production ceases to be sufficient to produce all erythropoietin needed. At the same time, kidney is not immediately able to assume the production of erythropoietin. Therefore, the prematurely born infants are often afflicted with anemia. It has been previously reported and confirmed that the level of erythropoietin in prematurely born infants is only about one-half of that of the normal adult.
  • the premature and chronic renal failure anemias can be treated with recombinant erythropoietin in amounts which will be equal to those found in normal adult .
  • Such treatment is currently been successfully utilized, at least in certain cases of chronic renal failure.
  • some patients need higher doses for the same correction of hematocrit, that is for production of sufficient number of red blood cells, than other individuals.
  • prematurely born infants respond to treatment with recombinant human erythropoietin only at 2-3 times the adult dose. In both cases, the lack or low levels of binding protein seems indicated and was in fact found.
  • labeled erythropoietin in the erythropoietin-erythropoietin binding complex When the labeled portion of the erythropoietin in the erythropoietin-erythropoietin binding complex is challenged with nonlabeled cold erythropoietin and the amount of the label is measured, labeled erythropoietin must be displaced with such cold erythropoietin.
  • Human erythropoietin is an acidic glycoprotein with an estimated molecular weight of 34,000.
  • Figure 1 shows the elution profile of erythropoietin-erythropoietin binding protein complex, eluting as fraction 48, and free erythropoietin eluting as fraction 63.
  • the gel filtration on Sephadex was performed as follows: 10 ⁇ l of the 125 I labeled erythropoietin was mixed with 2 ml of normal human serum previously centrifuged and filtered through 0.45 ⁇ m filters to remove particulates and incubated at 37°C for about 1 hour. Samples of the human serum were divided into two groups. One group contained human serum mixed only with 125 I-labeled erythropoietin containing approximately 100,000 cpm (- ⁇ -) ( Figure IA) . The second group contained the same components as the first group but additionally, 1 ⁇ g of cold uniabeled erythropoietin was added (- ⁇ -) ( Figure IB) .
  • the peak corresponding to formed complex has 130,000 molecular weight and that it represents the complex formed between 125 I-erythropoietin and its binding protein.
  • this erythropoietin competed with 125 I-labeled erythropoietin for binding with binding protein. Consequently, the second sample seen in Figure 1(B) showed much smaller peak of the complex than when the 125 I-labeled erythropoietin was used solely.
  • the peak fractions were separated on the non-denaturing 7% polyacrylamide gel by electrophoresis.
  • the appropriate fractions of the peaks containing erythropoietin-erythropoietin binding protein complex were pooled and concentrated to about 75 ⁇ l volume, using Centricon-30 inverse filter which has a molecular weight cut-off of 30,000.
  • the fractions corresponding to free labeled erythropoietin peak were concentrated to 50 ⁇ l.
  • Polyacrylamide gel electrophoresis - nondenaturing and discontinuous (PAGE-ND) method was obtained as a kit from Sigma Chemical Company and is described in detail in Sigma Tech. Bull.. No. EL-100, incorporated herein by reference. After PAGE-ND electrophoresis separation at constant current (50 mAmp) for four hours, the gels were stained with Rapid Coomassie Stain, obtained from Diversified Biotech, dried and autoradiographed on Kodak X-Omat film for about 24 hours, according to method described in Basic Methods in Molecular Biology. 331 (1886) , Eds. L. Davis, M.D. Dibner and, J.F. Battey, Elsevier, N.Y. incorporated herein by reference.
  • Lane 1 corresponds to run 1 in Figure 2(A), that is, it shows the radioactivity of the erythropoietin bound to the binding protein while the run 2 of the Figure 2 (B) corresponds to free 125 I-erythropoietin.
  • Run 1 clearly shows that the radioactivity is concentrated in a region corresponding to the erythropoietin-erythropoietin binding protein peak, while run 2 shows the radioactivity concentrated primarily in the labeled erythropoietin region.
  • such isolation was done by collecting binding protein complex peak obtained by using gel filtration, as described above, from normal human serum, by determining a total protein recovery and separating the concentrated fraction containing erythropoietin- erythropoietin binding protein complex on non-denaturing polyacrylamide gels. After separation, representative samples were stained for identification of the respective bands, regions of interest were cut out from the unstained gels, eluted, concentrated, incubated with labeled erythropoietin and re-electrophoresed on non-denaturing gels. Gels were dried, stained, and autoradiographed as described above.
  • Figure 3 (A) represents separation of nonradioactive binding protein complex separated on non-denaturing polyacrylamide gel.
  • Five ml of normal human serum without labeled erythropoietin in 1 ml aliquots were fractionated on a Sephadex G-100 column (1.5 x 100 cm) .
  • the fractions containing the binding protein were pooled and concentrated to 5 ml.
  • the total protein recovered was 140 mg, i.e., 28 mg/ml.
  • Appropriate aliquot corresponding to 70 ⁇ g/per lane of protein was applied to 7% non-denaturing polyacrylamide gels, 10 lanes per gel.
  • a representative sample of three lanes was stained with Brilliant Commassie Blue Stain for the identification of the respective bands. These three lanes, stained are shown in Figure 3(A), with a region of interest, based on results obtained in Figure 2(A)1, identified and cut from the unstained gels. The cuts were minced with a razor blade, eluted in 2 ml distilled water overnight at 4°C, and concentrated to 200 ⁇ l on AMICON Centricon-30 filter having cut-off molecular weight of 30,000.
  • Figure 4 shows an elution pattern of the sample containing 125 I-labeled erythropoietin. The primary peak is eluted in fractions 27 and 28. When the fraction 28 was read against the calibration curve from molecular markers, the molecular weight of the fraction 28 was determined to be around 130,000.
  • the erythropoietin binding protein has a molecular weight of approximately 93,000-96,000.
  • Denaturing SDS polyacrylamide gel electrophoresis separates proteins, protein mixtures or complexes into monomeric proteins and determines their molecular weights. The molecular weight of a given protein is determined by comparing its relative mobility with those of known proteins.
  • Denaturing polyacrylamide gel (PAGE-D) kit has been obtained from Sigma Chemical Company, St. Louis, Missouri, as PAGE-D kit and is described in Technical Bulletin. No. El-2 and in Nature. 227:680 (1970) (LAEMMLI) , incorporated herein by reference.
  • the remaining concentrate (60 ⁇ l) from the experiments described above containing nonlabeled erythropoietin-erythropoietin binding protein complex was subjected to denaturing SDS polyacrylamide analysis using Sigma PAGE-D kit.
  • the concentrate and molecular weight standards were diluted 1:2 in sample denaturing buffer which lyses the protein complex into its protein components, arid boiled for 15 minutes before loading the lanes either with the markers, as seen in Figure 5(A) or with the sample as seen in Figure 5 (B) .
  • phosphorylase (b) (M.W. 97.4) , bovine serum albumin (M.W. 69,000) ; ovalbumin (46,000) ; carbonic anhydrase (M.W. 30,000) ; trypsin inhibitor (M.W. 21.5) and lysozyme (M.W. 14,300) were used.
  • markers When run on PAGE-D gel, markers provided clear separation pattern according to their molecular weights. Since the expected molecular weight of erythropoietin binding protein was in the vicinity of 96,000, phosphorylase having molecular weight 97,400 was used as the standard having highest molecular weight.
  • Figure 5 (A) shows the separation of the molecular standards run alongside of concentrate sample as seen in Figure 5 (B) .
  • Figure 5(B) clearly shows that there is a fraction present which has only slightly lower molecular weight than the molecular marker phosphorylase (M.W. 97,400) .
  • erythropoietin- erythropoietin binding protein complex was determined in normal human serum of healthy adult volunteers, in adult anephric patients, in adult polycythemic patients, and in serum from a fetus and from a newborn sheep. Representative results for each group are shown in Figures 6-10.
  • the endogenous levels of erythropoietin were determined by radioimmunoassay (RIA) , as described in Recent Advances in Nuclear Medicine, 6:19-40 (1983) Ed. J.H. Lawrence and S. Winchell, Grune Shatton, Inc and incorporated herein by reference.
  • the levels of binding protein were determined by gel filtration from the measurement of the labeled erythropoietin bound to the binding protein.
  • the level of the binding protein is expressed in percent of binding of the binding protein to the radiolabeled erythropoietin based on measured cpm.
  • Figure 6 shows the elution pattern of binding protein peak eluting at fraction 48 and the free erythropoietin fraction eluting at fraction 62.
  • the binding protein peak showed 35.1% of binding with the total erythropoietin present in 16.8 mU/ml, as determined by RIA.
  • Figure 7 shows results obtained in human fetal plasma.
  • the binding protein peak showed 23.4% of binding and the total erythropoietin present in amount 18.3 mU/ml, as determined by RIA.
  • the binding protein peak showed 23.3% of binding and the total level of erythropoietin was 9.0 mU/ml, as determined by RIA.
  • the level of erythropoietin in newborn is only about half of that of normal healthy adult and also of the fetus.
  • the kidneys take over and is become fully responsible for the production of erythropoietin.
  • the level of binding protein is almost identical to that of the fetus, confirming that binding protein levels in newborns are similarly as low as in the fetus.
  • Figure 9 shows the results obtained in the serum of anephric patient. Since the anephric patient has no kidney or the function of the kidneys is severely limited or almost completely stopped, there is only very low level (7.7 mU/ml) of erythropoietin present in the anephric patients' serum. At the same time, the level of the binding protein is also very low of only 13.1%, that is about one-third of that of normal adult. The concentrations of erythropoietin binding protein in anephric or uremic patients, although low, may vary among patients and thus account for the fact that some patients require higher doses of human recombinant erythropoietin for the same correction of the hematocrit.
  • Figure 10 shows the results obtained in the serum of polycythemic patients. As seen from the curve, in these patients erythropoietin was present in large quantities (138 mU/ml) while the binding protein level was only 13.4%. Secondary polycythemia is characterized by elevated or high erythropoietin levels as measured by RIA. At higher than normal erythropoietin levels, the bound fraction progressively declines due to partial saturation of the binding protein by endogenous erythropoietin.
  • Table 1 shows that normal human serum has relatively high levels of erythropoietin bound to its binding protein.
  • pathological states particularly in anephric patients, there is a very low level of immunoreactive erythropoietin and the label bound to the binding protein is only one third when compared to normals.
  • the binding protein tends to be higher than in serum from anephric patients but it is still significantly lower than that found in normal human serum.
  • erythropoietin binding protein While the existence of erythropoietin binding protein was determined and its binding to erythropoietin and formation of erythropoietin-erythropoietin binding protein complex shown, the binding specificity was not altogether proven. In order to show that the newly discovered binding protein binds exclusively and selectively to erythropoietin, the same studies as described above for human serum were used. Additionally, however, uniabeled recombinant human growth hormone (5 ⁇ g) obtained from Genentech, S. San Francisco, California, was added to the normal human serum. The serum was mixed with labeled erythropoietin, gel filtrated and the radioactivity of the eluted fractions were determined.
  • Figure 11 illustrates the obtained results.
  • Figure 6 depicting the erythropoietin-erythropoietin binding protein complex peak in normal human serum, under the same conditions, the same results were observed for the serum additionally containing the growth hormone.
  • the binding protein peak of the serum containing the growth hormone was the same and not lower than that observed in normal human serum showing that the binding protein did not bind to the growth hormone but it bound selectively to the labeled erythropoietin.
  • erythropoietin binding protein was purified using generally method described in Biochem J. , 221:617 (1984), incorporated hereby by reference.
  • the method utilizes the affinity chromatography for selective purification on a erythropoietin - Affigel 15 affinity column.
  • the technique is described in detail in Example 4.
  • the purification of binding protein using method described in Example 4 resulted in 200-300 fold purification of the binding protein with no substantial change in binding affinity.
  • erythropoietin binding protein Following the purification of erythropoietin binding protein, protein is sequenced and cloned by methods known in the art. Isolated and purified binding protein is cloned according to methods for cloning and production of recombinant erythropoietin previously described in Proc. Natl. Acad. Sci. , 82:7580 (1985) and in Endocrine Genes, 201-217 (1988) , Ed. Yun-Fai Lau, Oxford University Press, N.Y. incorporated herein by reference.
  • Purified binding protein is used for sequence information, cloning and expression.
  • Mixed oligodeoxyribonucleotide is designed containing all possible coding sequences for selected oligopeptide fragments.
  • a human genomic library is screened for the probes.
  • the isolated human genomic clone containing the binding protein is expressed in a SV 40 promoter-containing plasmid vector.
  • Chinese hamster ovary cells are transfected with the expression vector and direct the production of the erythropoietin binding protein. The binding protein production is monitored by radioimmunoassay.
  • the extent of erythropoietin bound to the binding protein under physiological conditions can be determined. Procedures to demonstrate the presence of erythropoietin binding protein and to separate erythropoietin binding protein free from bound erythropoietin by gel filtration can result in disruption of the binding equilibrium. Therefore, care must be taken to avoid underestimating the bound fraction.
  • the radioimmunoassay offers the highest sensitivity, specificity and accuracy.
  • a radioimmunoassay (RIA) is: 1) specific antibodies to the protein under investigation, 2) the availability of the protein as pure as possible and its ability to accept radioactive label, 3) a standard reference preparation, and 4) a technique to separate the antibody-bound and non-antibody-bound protein.
  • Radioimmunoassay which can be developed for determination of the presence of erythropoietin binding protein is described in Example 5.
  • This invention concerns a newly discovered protein which specifically binds to erythropoietin, forming erythropoietin-erythropoietin binding protein complex.
  • the function of erythropoietin on the erythropoiesis is well known.
  • erythropoietin has relatively a short half-life, and therefore, in the absence of some mechanism to extend it, very large amounts of erythropoietin would have to be synthesized to meet the increased demand. It has now been shown that the half-life of erythropoietin is extended by forming a complex with its binding protein.
  • the formed complex extends the half-life of circulating erythropoietin and in this way, it effects erythropoiesis.
  • the erythropoietin binding protein provides therefore an additional useful treatment for various types of anemias, particularly those which result from inability of the individual to produce its own erythropoietin. In instances where for any reason the synthesis of erythropoietin is decreased or insufficient, the administration of recombinant binding protein would substantially extend its half-life.
  • the dosages which are required in anephric patients or in prematurely born infants may be reduced when administered in combination with the erythropoietin binding protein.
  • the half-life of circulating erythropoietin is substantially extended making the treatment with recombinant human erythropoietin more affordable and practical.
  • the treatment with erythropoietin binding protein is sufficient to extend the half-life of erythropoietin and the erythropoiesis proceeds as if a normal level of erythropoietin is present.
  • the binding protein affects the homeostasis of erythropoietin and presumably the action of erythropoietin by altering its in vivo kinetics and metabolism and by modulating its interaction with tissue receptors. Therefore, changes in binding protein levels or activity may influence the ultimate expression of the biological activity of erythropoietin.
  • the binding protein is administered to a patient intravenously or subcutaneously in therapeutically effective doses which correspond to weight, age, blood circulation volume, affliction and the degree of affliction to be treated.
  • Therapeutically effective doses are doses which are able to extend the half-life of erythropoietin from 6-8 hours to 9 hours and above, and to maintain the level of erythropoietin at such a level that it assures hematocrit above 25% and preferably around 40-47%.
  • Doses of recombinant human erythropoietin needed to maintain such a level of hematocrit in anemic patients vary between 50 U/kg to 1500 U/kg of body weight and are given daily, every second day or biweekly.
  • the therapeutically effective dosages of the recombinant human erythropoietin binding protein are between 10 U/kg to 1000 U/kg of weight in proportion to the degree of anemia and to the dose of erythropoietin needed to correct the hematocrit .
  • Recombinant human erythropoietin binding protein is administered alone or in combination as a complex with recombinant erythropoietin.
  • the complex or the binding protein alone are given therapeutically as well as prophylactically for treatment of anemia, before or after surgery or during any extended bleeding.
  • This example describes the studies which demonstrate the existence of the binding protein for hormone erythropoietin in normal human serum.
  • a normal human serum pool was obtained from Gibco Co. as product no. 200-6150 PG or Y3N 7802.
  • Labeled erythropoietin was prepared by iodination, by chloramine-T method, according to Biochem J.. 89:114 (1963) incorporated herein by reference, of recombinant erythropoietin or erythropoietin extracted from the urine of anemic patients.
  • Each vial for iodination contained approximately 0.7 ⁇ g of lyophilized erythropoietin, or about 57 units (U) .
  • Lyophilized erythropoietin was dissolved in 20 ⁇ l of 0.5 M phosphate buffer at pH 7.5, 20 ⁇ l of distilled water and 1 mCi of Na 125 I in 3 ⁇ l were added to the vial containing dissolved erythropoietin. Then, 10 ⁇ l of chloramine-T (400 ⁇ g/ml) was added and the mixture was submitted to a mild agitation for 1 minute, 100 ⁇ l of metabisulfide (240 ⁇ g/ml) and 300 ml potassium iodine (100 ⁇ g/ml) were added. All three agents were prepared freshly in phosphate buffer (0.05M, pH 7.5) immediately before iodination.
  • the total content of the vial was transferred to a small Sephadex G-25 column having a bed volume 10 ml, which was previously coated with bovine serum albumin (BSA) and eluted in 1 ml aliquots.
  • BSA bovine serum albumin
  • the eluant was carefully monitored and protein bound 125 I labeled protein was eluted as fraction 4, and unreacted free 125 I label as fractions 8-10.
  • the total content of 125 I labeled protein was further fractionated on a 1.5 x 30 cm Sephadex G-150 column and eluted in 20 drops fractions with phosphate buffer (0.05 M, pH 7.5) .
  • the second fractionation yielded two major peaks.
  • the first peak contained damaged label or aggregation of the labeled hormone.
  • the undamaged labeled hormone appeared as a second peak.
  • the average specific activity of iodinated erythropoietin was 200 ⁇ Ci/ ⁇ g which was equivalent to approximately 200,000 cmp/lO ⁇ l/5 mU erythropoietin.
  • Gel chromatography resulted in three peaks separating bound and free hormone from the free iodine, as measured by the radioactivity in individual fractions.
  • the first peak corresponded to a protein or protein complex having -130,000 molecular weight and was identified as erythropoietin-erythropoietin binding protein complex having combined molecular weight of erythropoietin equal 34,000 kD and erythropoietin-erythropoietin binding protein complex equal to 96,000 kDa.
  • Figure 6 shows elution of the complex and free erythropoietin peak. The peak for unbound iodine is not shown.
  • the first and the second peak fractions corresponding to the erythropoietin-erythropoietin binding protein complex and to free erythropoietin, respectively, were pooled and concentrated to about 50-75 ⁇ l in AMICON Centricon-30 filter with a molecular weight cut-off of 30,000.
  • the volume of the second EPO peak concentrate was adjusted to yield approximately the same level of radioactivity.
  • Both preparations were subjected to non-denaturing 7% polyacrylamide gel electrophoresis for 4 hours at 50 mAmp. After electrophoresis, the gel was placed into stain (0.1% Coomassie blue in 40% methanol and 10% acetic acid) for one hour. The gel was dried and autoradiographed for 24 hours and 96 hours using Kodak XAR film and DuPont intensifying screen. The autoradiograph showed a clear separation between specifically bound erythropoietin-erythropoietin binding protein complex and free unbound erythropoietin as seen in Fig. 2, Al and A2.
  • This example describes how the erythropoietin binding protein was identified.
  • Example 2 Five ml of normal human serum without labeled EPO in 1 ml aliquots were placed on the same type of Sephadex G-100 column, as used in Example 1, the same fractions were pooled and concentrated to about 5 ml of total volume, as described in Example 1. The total protein recovered was 140 mg, i.e. 28 mg/ml. Sixty microliters of this concentrate was incubated with 4 ⁇ l of labeled 125 I-erythropoietin (40,000 cpm) in 100 ⁇ l of 0.5% bovine serum albumin for 1 hour at 37°C.
  • the incubation mixture was applied on a column previously calibrated with same markers, as described in Example 1, of Sephacryl S-200 (1.6 x 28 cm) and gel was eluted for 5 hours at room temperature with phosphate buffer. Gel filtration resulted in a single peak of radioactivity at a position of -130,000 M.W.
  • This example illustrates specific binding of the erythropoietin binding protein to erythropoietin.
  • Example 2 The concentrate prepared as in Example 2 was incubated for 1 hour with 4 ⁇ l of labeled EPO having 100,000 cpm and subjected to non-denaturing electrophoresis on 7% polyacrylamide gel electrophoresis for 4 hours at 50 mAmp, as described in Example 1, followed by autoradiography. Autoradiography, under conditions described in Example 1, showed two identifiable bands which had specifically bound labeled EPO. These two bands were cut out and eluted in 50 mM phosphate buffer overnight. The eluate was concentrated and analyzed by electrophoresis on denaturing SDS-PAGE.
  • binding protein (M.W. 21,500); and lysozyme (M.W. 14,500) .
  • One major band of the binding protein was determined to have a molecular weight of approximately 96,000. There were two minor bands with a molecular weight somewhat smaller.
  • This example illustrates purification of erythropoietin binding protein using human erythropoietin - Affigel 15.
  • Human erythropoietin 70 mg was covalently linked to 17 g of Affigel 15 at pH 7.5 (O.lm-HEPES buffer) with gentle agitation for 1 hour at room temperature. Any free, active esters remaining on the gel were blocked with 1.2 ml of 7M-ethanolamine/HCl (pH 8) .
  • the column was then washed with 25 mM Tris/HCl (pH 7.4) containing 40 mM-CaCl 2 , 0.02% (w/v) NaN 3 and Trasylol (lOOOk-i.u./ml) and the bound serum protein(s) were eluted with 4M-urea and 4M-MgCl 2 , either alone or sequentially as described in J. Biol. Chem. , 25:6815 (1979) incorporated herein by reference.
  • the bound fractions were dialyzed against 25 mM-Tris/HCl, pH 7.5, containing 40 mM-CaCl 2 and then assayed as above for 125 I-binding. The yield is about 30-40%.
  • the purified erythropoietin binding protein is confirmed by SDS-PAGE to be a pure protein having a molecular weight 96,000.
  • This example illustrates a radioimmunoassay developed for production of erythropoietin binding protein antibody.
  • the protocol for radioimmunoassay is essentially that which is described in Recent Advances in Nuclear Medicine, 6:19 (1983) Ed. J.H. Lawrence, S. Winchell, Grune Shatton, Inc. incorporated herein by reference.
  • the binding protein for immunization is obtained as above.
  • the binding protein is submitted to two step purification. Normal human serum is fractionated by column chromatography, the appropriate fractions containing the binding protein are pooled, concentrated and subjected to non-denaturing polyacrylamide electrophoresis as described in Example 2.
  • the regions previously identified as containing the binding protein are cut, minced and eluted overnight in distilled water.
  • the eluate (0.1-0.5mg) is emulsified with an equal volume of Freund's complete adjuvant (Sigma, St. Louis, Missouri) and injected subcutaneously at multiple sites into the legs of young New Zealand white rabbits. Additional boosters are given at two week intervals for two months.
  • the rabbits are bled from the central ear artery (50 ml) and the antiserum is evaluated.
  • the antiserum has to have a high titer, i.e., it must be able to be used at a high dilution (1:10,000), it has to be specific, i.e., not cross-react with other proteins, and have a high affinity to the binding protein.
  • the required affinity of the erythropoietin to antiserum has to be higher than the affinity of the binding protein to erythropoietin. This is not difficult to do because antisera in general have higher affinities than binding characteristics under physiological conditions.
  • the eluate from the two step purification procedure above is further purified on an erythropoietin-affinity column as described in Example 4.
  • the binding protein is pure enough for radioiodination using chloramine-T method.
  • 1 ug of protein is needed.
  • the same preparation of purified erythropoietin binding protein as described above is used for the standard preparation in the radioimmunoassay. Based on serum binding protein concentrations of 300 picograms/ml in normal humans, dilutions of the standard is such that normal values fell in the middle part of the standard curve. It is so specifically designed in order to allow the measurement of reduced and increased binding protein concentrations in various erythropoietic states.
  • Serial dilutions of the standard curve is in the range from 2 nanograms/ml to 2 picograms/ml.
  • the separation of the bound binding protein (or antibody) from free binding protein is done by double antiserum technique utilizing the precipitation of the antibody-analyte complex by a second antiserum, an anti-gamma globulin.
  • the first antibody is a rabbit antiserum produced against human erythropoietin binding protein.
  • the antiserum produced in goats against rabbit gamma globulin facilitates the precipitation of the primary antibody binding protein complex.
  • Samples of 100 ⁇ l of undiluted or diluted human serum are pipetted into polystyrene tubes (12 x 75 mm) .
  • the diluent buffer is phosphate buffer (0.05M, pH 7.5) containing 5% of human serum albumin.
  • a volume of 200 ⁇ l of labeled erythropoietin binding protein and 200 ⁇ l of erythropoietin binding protein antiserum are added simultaneously.
  • the labeled erythropoietin binding protein is diluted to contain approximately 10,000 cpm/200 ⁇ l.
  • the antiserum (200 ⁇ l) dilution of 1:20,000, is such that it bound approximately 35-40% of the label.
  • the reaction mixture is incubated for 4 days at 4°C.
  • This example describes the diagnostic radioimmunoassay kit for the quantitative measurement of human erythropoietin biding protein in serum.
  • the erythropoietin double antibody radioimmunoassay kit is intended for the quantitative measurement of erythropoietin binding protein in serum as an aid in the diagnosis and treatment manipulation of anemias and polycythemias .
  • This assay is intended for in vitro diagnostic use.
  • the procedure follows the basic principle of radioimmunoassay whereby there is competition between a radioactive and nonradioactive antigen for a fixed number of antibody binding sites.
  • the amount of 125 I labeled erythropoietin binding protein bound to the antibody is inversely proportional to the concentration of erythropoietin binding protein present in the serum.
  • the separation of free and bound antigen is easily and rapidly achieved by using an accelerated double antibody polyethylene glycol system.
  • Lyophilized erythropoietin binding protein standard are prepared containing approximately concentrations of 0, 75, 150, 300, 600, and 1000 pg/ml of purified erythropoietin binding protein from human serum in a protein based buffer.
  • 125 I labeled erythropoietin binding protein is prepared, as described above.
  • Rabbit anti-human serum erythropoietin binding protein is prepared as described in Example 5.
  • Specimen serum is obtained by venipuncture.
  • the serum may be stored at 2-8°C for up to 24 hours and should be frozen at -10°C or lower for longer periods.
  • Test tubes in duplicate, are labeled and arranged for total counts, non-specific binding, standards, controls and specimen samples.
  • Tubes are incubated at room temperature for 16-20 hours .
  • Tubes are vortexed and incubated at room temperature for 30 minutes. 9. Tubes are centrifuged (except Total Count Tubes) for 15-20 minutes at 1500 x g.
  • the supernatant is decanted by inverting each tube or a rack of tubes (when tubes are firmly held) .
  • the tubes are inverted and allowed to drain for 15-20 seconds on absorbent paper. After draining, the mouth of each tube is blotted to remove any droplets adhering to the rim before returning the tubes upright.
  • Results are calculated for the average counts per minute (CPM) for each standard, patient sample, and control.
  • the average of CPM of the NSB are subtracted from all counts to obtain corrected net counts.
  • the % bound is calculated for each standard, patient sample, and control as follows:
  • a curve of radioactivity counts per minute is plotted, % bound for the EPO standards against the EPO concentration on a linear-log graph paper.
  • the EPO-BP concentration of specimen sample is determined from the standard curve.

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Abstract

Cette invention concerne une protéine purifiée de liaison d'érytrhropoiétine mammifère, ainsi que l'isolement, l'identification, la caractérisation, la purification et un dosage immunologique de cette protéine. Cette protéine de liaison de l'érythropoiétine peut être utiliséee dans la régulation de l'érythropoièse par régulation des taux et modification de la demi-vie de l'érythropoiétine. Cette invention concerne également un nécessaire diagnostique permettant de déterminer le taux de protéine de liaison de l'érythropoiétine.
PCT/US1995/011043 1995-08-29 1995-08-29 Proteine de liaison d'erythropoietine utile dans la regulation de l'erythropoiese et dosage permettant de constater sa presence WO1997008200A1 (fr)

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PCT/US1995/011043 WO1997008200A1 (fr) 1995-08-29 1995-08-29 Proteine de liaison d'erythropoietine utile dans la regulation de l'erythropoiese et dosage permettant de constater sa presence
AU33752/95A AU3375295A (en) 1995-08-29 1995-08-29 Erythropoietin binding protein useful for regulation of erythropoiesis and an assay for determination thereof

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PCT/US1995/011043 WO1997008200A1 (fr) 1995-08-29 1995-08-29 Proteine de liaison d'erythropoietine utile dans la regulation de l'erythropoiese et dosage permettant de constater sa presence

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5378808A (en) * 1989-02-03 1995-01-03 Genetics Institute, Inc. Recombinant erythropoietin receptor protein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5378808A (en) * 1989-02-03 1995-01-03 Genetics Institute, Inc. Recombinant erythropoietin receptor protein

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BLOOD, Volume 82, Number 7, issued 01 October 1993, BAYNES et al., "Serum Form of the Erythropoietin Receptor Identified by a Sequence-Specific Peptide Antibody", pages 2088-2095. *
BLOOD, Volume 85, Number 1, issued 01 January 1995, TAKAHASHI et al., "Characterization of Three Erythropoietin (Epo)-Binding Proteins in Various Human Epo-Responsive Cell Lines and in Cells Transfected with Human Epo-Receptor cDNA", pages 106-114. *
GENE, Volume 106, issued 1991, TODOKORO et al., "Isolation of a cDNA Encoding a Potential Soluble Receptor for Human Erythropoietin", pages 283-284. *
JOURNAL OF CELLULAR BIOCHEMISTRY, Supplement 0, Volume 15, Part F, issued 1991, MULCAHY et al., "Generation and Characterization of a Soluble Erythropoietin-Binding Protein", page 146. *

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