KR100883860B1 - Compositions and methods for preventing erythropoietin-associated hypertension - Google Patents

Abstract

The present invention, when administered to mammals with erythropoietin (Epo), dissolves erythropoietin-binding protein and soluble erythropoietin-binding to prevent or reduce blood pressure elevation usually accompanied by erythropoietin without affecting the erythrocyte volume fraction for Epo treatment. It relates to an antibody against a protein. The present invention is directed to administering erythropoietin (Epo) to a mammal; A method of treating anemia in a mammal, comprising administering to the mammal an agent selected from a soluble Epo binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof. To provide. The present invention also provides a method of reducing hypertension in a mammal receiving Epo, and a pharmaceutical composition containing a recognition protein that binds to the Epo receptor on the lytic Epo-bp and / or extracellular soluble portion of the Epo receptor.

Description

COMPOSITIONS AND METHODS FOR PREVENTING ERYTHROPOIETIN-ASSOCIATED HYPERTENSION}

This application claims priority to US Utility Patent Application No. 10 / 848,689, entitled “Compositions and Methods for Preventing Hypertension Associated with Erythropoietin, filed May 17, 2004.”

Erythropoietin is sold under the names Procret (Ipoietin), Epogen (Ipoietin) and Aranesph (Dabepoetin). Erythropoietin is prescribed for the treatment of anemia, in particular for the treatment of anemia that occurs during chemotherapy of chronic renal failure and cancer.

Erythropoietin (Epo) is a blood vessel proliferating factor that stimulates hematopoiesis to raise red blood cell volume and hemoglobin levels, but also results in blood viscosity (1-5) and blood pressure rise (1-14). In clinical trials, about one third of hemodialysis patients treated with recombinant human Epo had elevated blood pressure. Epo is believed to result in decreased endvascular resistance and cardiac output due to elevated blood viscosity (6). Other scholars have suggested other mechanisms for hypertension caused by Epo, such as excessive blood volume, elevated plasma renin and angiotensin, adrenergic hyperactivity, increased plasma arginine vasopressin, and lowered kinin and prostaglandins (15). Excessive sustained elevation of the daily cycle, excessive blood pressure and blood pressure rise are considered to be based on altered vasomotor time measurements (time structures). Other agents, including those based on hormones and heredity, appear to be contributing factors to changes in blood pressure throughout the day.

Hypertension is one of the most important factors contributing to the involvement of cardiovascular disease. Hypertension is significantly affected by daily rhythms. Fluctuations in the body's hormonal concentrations, which change day and night due to significant automatic changes in the daily cycle (approximately 24 hours), affect blood pressure and heart rate. Extremely important rhythms can make a difference between stimulation and inhibition in malignant disease.

Epo is a glycoprotein hormone with a molecular weight of 34 kilodaltons (kDa) that is produced in the neighboring cells and venous hepatic cells that spread to the mammalian kidney capillaries (3). Epo is secreted in response to oxygen deficiency and acts as an inducer and regulator of erythrocyte differentiation and regulates hematopoietic action by inhibiting progenitor blood cell death and promoting cell division and terminal maturation (16). These effects consist of the binding of Epo to receptors on specific cell surfaces (17). The fundamental structure of human Epo has been known since the mid-1980s (18, 19), but most of the structural aspects of Epo molecules involved in biological activity are unknown. Human Epo contains two disulfide bonds linking cysteines 29 and 33 and cysteines 7 and 161. These two bonds are essential for biological activity (18). Epoetin (recombinant human erythropoietin) was produced by separating human genes and expressing them in Chinese hamster ovary cell lines (4). This recombinant Epo is a 165-amino acid mature protein, which differs from mature natural protein only in that arginine at the carboxyl terminus has been removed from the native protein. Natural human Epo is translated into 193 amino acid peptide from which the 27-amino acid leader sequence has been removed (19, 20). Recombinant Epo has an apparent molecular weight of about 30.4 kilodaltons (kDa) that appears immunologically similar to endocrine hormones and exhibits complete bioactivity (19).

Humans and animals treated with Epo show red blood cell volume (%) and red pigment elevation through hematopoietic stimulation (2-5). Some studies suggest that the increase in red blood cell volume ratio is proportional to the increase in blood pressure in humans (20). In another study involving the treatment of anemia with Epo, blood pressure rose dramatically in 20-30% of patients with an increase in red blood cell volume, requiring blood pressure medication (5).

Hypertension is the most common and important complication that comes with erythropoietin treatment. Moreover, Epo's therapeutic goal is to increase the red blood cell volume and red soot level, and it has been found that the higher the red blood cell volume, the greater the risk of death and cardiovascular events (PROCRIT warnings, www.rxlist.com/cgi/generic/). epoetin-wcp.htm), which appears to be due to an increase in blood pressure, which is considered to be related to the increase in blood pressure in proportion to the increase in red blood cell volume ratio (20). The epoetin manual warns patients with uncontrolled hypertension not to use epoetin.

There is a need for new methods and compositions for preventing or treating elevated blood pressure in patients treated with Epo. There is a need for new methods and compositions for treating anemia without hypertension.

Summary of the Invention

When the present inventors inject the soluble Epo-binding protein, which is a soluble fragment of the membrane protein Epo receptor, together with Epo, it does not affect the increase of the red blood cell and red blood cell volume, which is the purpose of the Epo treatment, and prevents the blood pressure increase that is normally caused by Epo. It was found. Antibodies made against Epo-binding proteins have also been found to prevent Epo-induced blood pressure rise without affecting Epo-induced elevation of erythrocyte volumes.

Accordingly, the present invention provides a medicament selected from erythropoietin (Epo) for mammals, the soluble Epo-binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof. It provides a method for treating anemia of a mammal, including administering to the mammal.

Another embodiment of the invention comprises administering to a mammal an agent selected from a soluble Epo-binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof. Provided are methods for reducing hypertension in mammals receiving Epo treatment.

Another embodiment of the invention provides a medical therapeutic use of a recognition protein that binds to an Epo receptor on the extracellular lysis portion of the Epo receptor.

Another embodiment of the present invention provides the use of a recognition protein that binds to an Epo receptor on the extracellular soluble part protein of the Epo receptor in the manufacture of a medicament effective for reducing erythropoietin-induced hypertension.

Another embodiment of the invention provides the use of an erythropoietin-binding protein for medical treatment.

Another embodiment of the present invention provides the use of a soluble erythropoietin binding protein for preparing a medicament effective for reducing erythropoietin-induced hypertension.

Another embodiment of the invention is erythropoietin; And a soluble Epo-binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof.

Another embodiment of the invention provides a pharmaceutical composition comprising a recognition protein that binds to an Epo receptor on the extracellular lysis portion of the Epo receptor.

Another embodiment of the invention provides a pharmaceutical composition comprising a soluble Epo-binding protein.

Another embodiment of the present invention provides a medical therapeutic use of a medicament selected from soluble Epo-binding protein (Epo-bp), a recognition protein that binds to an Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof.

Another embodiment of the present invention provides a medicament effective for reducing erythropoietin-induced hypertension of a medicament selected from soluble Epo-binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof. It provides the use for the preparation.

Another embodiment of the invention provides an agent selected from (a) erythropoietin, and (b) soluble Epo-binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof. It provides a medical therapeutic use of the composition comprising.

Another embodiment of the invention provides an agent selected from (a) erythropoietin, and (b) soluble Epo-binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof. Provided are uses for the manufacture of a composition comprising a medicament effective in treating anemia while inducing less hypertension than a comparative composition without component (b).

1 shows a bar graph of an average of about 24 hours of blood pressure when Epo and other drugs were treated in rats of the treatment group, respectively.

2 shows the blood pressure values of each treatment group.

Figure 3 shows the daily cycle red blood cell volume change of the rats of each treatment group.

FIG. 4 lists photographs of spleens cut from rats after Epo (panels A, B and C) or saline (panel D) treatment.

Figure 5 is a light density histogram by immunodetection of Epo, αEpo, Epo-bp and αEpo-bp in the serum and plasma of human volunteers. Error bars represent standard error, SEM.

Justice

As used herein, "erythropoietin" includes erythropoietin isolated from natural resources and recombinant erythropoietin or engineered erythropoietin with erythropoietin bioactivity of erythropoietic stimulation. It contains epoetin and daabepoetin. Preferably, erythropoietin has at least 70%, more preferably at least 90%, of amino acids having sequence homology with human erythropoietin (SEQ ID NO: 3). Sequence homology is calculated using default BLAST parameters for nucleotide sequence control (PubMed website, www.ncbi.nlm.nih.gov/PubMed/).

As used herein, "dissolved Epo-binding protein" refers to a protein that has a high affinity to Epo and is a water-soluble protein, not an antibody, that has the effect of reducing Epo-induced hypertension when administered to mammals with Epo. It is preferred that the K _D of the protein binding to Epo is less than 10 μM, more preferably less than 1 μM, even more preferably less than 100 nM, and most preferably less than 20 nM. K _D can be determined by competitive binding analysis as described in Example 6 of US Pat. No. 5,843,726. Preferably, the soluble Epo-binding protein is or comprises a soluble partial sequence of the Epo receptor or a corresponding sequence. The human Epo receptor sequence is SEQ ID NO: 1 (Winkelmann, JC, et al., 1990, Blood 76: 24-30). The soluble portion of the human Epo receptor is the 23-250 residues of SEQ ID NO: 1. In certain embodiments, the residues of the Epo-binding protein involved in Epo-binding are at least 70% identical, more preferably at least 80% identical, even more preferably at least 90% identical to SEQ ID NO: 1 residues. Is the same, and the most ideal is the equivalent.

As used herein, the "extracellular lysis portion of the Epo receptor" refers to the Epo receptor portion exposed to the outer surface of the cell in an aqueous environment. Specifically, it is SEQ ID NO: 2, which is a 25-250 residue of SEQ ID NO: 1 (human Epo receptor) or a homologous lysis residue of another Epo receptor protein.

As used herein, "a recognition protein that binds to the Epo receptor on the extracellular lysis portion of the Epo receptor" binds to the extracellular lysis portion of the Epo receptor and is elevated due to Epo in the mammal when administered to the mammal with Epo. It is a protein that reduces blood pressure. The recognition protein may be a complete antibody against the Epo receptor or an Epo-binding protein and may be an antibody that binds to the Epo receptor soluble portion or fragment binding of the complete antibody. Recognition proteins are also proteins or peptides (eg, proteins or peptides selected by phage display binding) that are not antibodies that bind to the extracellular soluble portion of the human Epo receptor or have a binding affinity of at least 10 ⁵ L / mole, More preferably 10 ⁶ L / mole or even more preferably at least 10 ⁷ L / mole, or most preferably at least 10 ⁸ L / mole to bind to other mammalian Epo receptors.

As used herein, “antibody” includes complete antibodies and antigen-binding fragments of the complete antibody, eg, Fab or F (ab ′) ₂ antibodies. The meaning of “antibody” also includes monoclonal and polyclonal antibodies (eg antisera) and the like.

By "hypertension" by injecting a drug, it means to prevent or reduce the rise of blood pressure in a significant part of the population when the drug is not administered.

Explanation

The method of the present invention comprises administering to a mammal an agent selected from a soluble Epo-binding protein (Epo-bp), a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, and a complex thereof.

In some embodiments of the invention, the medicament is dissolved Epo-bp.

In some embodiments, soluble Epo-bp comprises soluble partial fragments of a mammalian Epo receptor.

In certain embodiments, the soluble Epo-bp contains a fragment of at least 30 residues of SEQ ID NO: 2 (residues 25-250 of the human Epo receptor, SEQ ID NO: 1). SEQ ID NO: 2 is the extracellular lysis portion of the human Epo receptor. In certain embodiments, soluble Epo-bp contains at least 15, at least 50, at least 100, at least 150, or at least 200 residue fragments of SEQ ID NO: 2.

In certain embodiments, the soluble Epo-bp comprises SEQ ID NO: 2 or is SEQ ID NO: 2. The soluble Epo-bp can be represented by SEQ ID NO: 2 as described in US Pat. No. 5,843,726. In general terms, SEQ ID NO: 2 represents a fusion protein having a glutathione-S-transferase (GST) N-terminal leader sequence. SEQ ID NO: 2 is separated from the GST leader sequence by a thrombin cleavage site. The expressed fusion protein is cleaved by thrombin to release SEQ ID NO. Epo-bp of SEQ ID NO: 2 is naturally found in human serum or plasma and may possibly be produced as a cleavage product of the Epo receptor (see Example 2 hereinafter).

In certain embodiments, soluble Epo-bp is at least 70%, at least 80% of the amino acid sequence when calculated using the default BLAST parameter for nucleotide sequence comparison on the PubMed website www.ncbi.nlm.nih.gov / Pubmed / Or at least 90% is identical to SEQ ID NO: 2.

In some embodiments of the invention, the soluble Epo-bp is SEQ ID NO: 8, which is SEQ ID NO: 2 with two additional residues of Gly-ser at the amino terminus.

In one embodiment of the invention, soluble Epo-bp is the product of a process including expression of the fusion protein, cleavage with thrombin, and the like. The fusion protein comprises a first polypeptide segment essentially having a thrombin cleavage site at the carboxyl terminus and a second polypeptide segment essentially having SEQ ID NO: 2. The amino terminus of the second segment is covalently attached to the carboxyl terminus of the first segment. The soluble Epo-bp is produced by cleaving the fusion protein with thrombin.

In one embodiment of the invention, the soluble Epo-bp is a fusion protein expression and thrombin comprising SEQ ID NO: 2 linking the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 7) to the amino-terminus It is the product of processes, including cleavage of fusion proteins.

In one embodiment of the invention, the soluble Epo-bp comprises (a) a first polypeptide segment containing amino and carboxyl termini and at the carboxyl terminus having a SEQ ID NO: 7 and (b) SEQ ID NO: 2 and a first poly Expression of a fusion protein consisting of a second polypeptide segment covalently linked to the carboxyl terminus of the peptide segment, cleaving the fusion protein with thrombin, and the like.

In certain embodiments methods of the invention, the medicament is a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor.

Recognition proteins may have the effect of reducing Epo-induced blood pressure rise by binding to the extracellular soluble portion of the intact Epo receptor on the cell membrane, or by circulating soluble Epo-binding protein (a degradation product of the receptor that is naturally circulating in the blood) Binding to the extracellular lysing portion of the Epo receptor, which is thought to be exerted, may exert its effect, or are likely to be effected by both or an unknown mechanism. Describing an embodiment of a medicament as "a recognition protein that binds to the Epo receptor on the extracellular lysis portion of the Epo receptor" is intended to describe the properties of the recognition protein and does not necessarily explain the mechanism of action of the recognition protein.

In certain embodiments, the recognition protein binds to SEQ ID NO: 2. That is, the recognition protein recognizes SEQ ID NO: 2 and binds to some sequence therein. Recognition proteins can be, for example, antibodies made against SEQ ID NO: 2, or antibodies made against the Epo receptor bind to SEQ ID NO: 2, or antibodies made against the peptide fragment of SEQ ID NO: 2.

In certain embodiments, the recognition protein is an antibody. In certain embodiments, the antibody is a complete antibody. In certain embodiments, the antibody is an antibody fragment. For example, the antibody fragment is Fab, Fab 'or F (ab') ₂ or Fv.

In certain embodiments, the antibody is an antibody against SEQ ID NO: 2.

In other specific embodiments, the recognition protein is a protein or peptide that is not an antibody. For example, it may be a protein selected by recognition peptide or phage display. Methods of selecting binding peptides using phage display are described in Sidhu SS, Lowman HB, Cunningham BC, and Wells JA: Phage display for selection of novel binding peptides, Methods in Enzymology 2000; 328: 333-363.

In certain embodiments, the agent is a combination of soluble Epo-binding protein and a recognition protein that binds to an Epo receptor on the extracellular soluble portion of the receptor.

Epo and the medicament may be administered separately or together.

In certain embodiments, the amount of medicament administered is at least equimolar to the Epo dose.

In certain embodiments, the amount of medicament administered is about equimolar to the Epo dose. For example, the moles of drug administered will be between 70% and 125% of the moles of Epo administered.

In certain embodiments of the method of treating anemia, the medicament reduces the blood pressure rise induced by mammalian Epo. That is, when Epo and the drug are applied to a mammal, the blood pressure rise of the mammal is reduced rather than receiving Epo alone.

Preferably, when Epo is administered to a mammal together with an equimolar amount of the drug, 75% or less of the blood pressure increase rate when Epo alone is administered, and more preferably 50% when Epo is only administered to the mammal. I see a rise in blood pressure below that.

One embodiment of the invention is a pharmaceutical composition comprising a recognition protein that binds to an Epo receptor on the extracellular lysis portion of the Epo receptor.

Other pharmaceutical compositions of the present invention include erythropoietin; And a medicament selected from soluble Epo-binding proteins, recognition proteins that bind to Epo receptors on the lytic portion of cells of the Epo receptor, and combinations thereof.

Other pharmaceutical compositions of the invention include soluble Epo-binding proteins.

Typically, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier.

In one embodiment of the pharmaceutical composition comprising a recognition protein, the recognition protein is an antibody against SEQ ID NO: 2.

In one embodiment of the pharmaceutical composition comprising a soluble Epo-binding protein, the Epo-binding protein is SEQ ID NO: 2.

Another specific embodiment of the medicament, the soluble Epo-binding protein, a recognition protein that binds to the Epo receptor on the extracellular soluble portion of the Epo receptor, has been described for the methods of the present invention.

Antibody Provided

For antibody production, Epo receptors or Epo-bp can be administered directly to a mammal, or their protein or peptide fragments can be administered in combination with a carrier protein. Suitable carrier proteins include keyhole limbus hemocyanin, bovine serum albumin and ovalbumin. Methods of binding to the carrier protein include single step glutaraldehyde coupling and other methods described in Harlow Ed., Et al., Antibodies: a laboratory maunal, Cold Spring Harbor Laboratory (1988).

Immunogens are used to immunize vertebrates to induce vertebrate antibody production. Preferably, the immunogen is injected together with an adjuvant such as Freund's adjuvant to promote the immune response. Suitable vertebrates include rabbits, mice, mice, hamsters, goats, sheep and chickens.

Hybridomas for synthesizing monoclonal antibodies can be prepared by methods known in the art. See, eg, Wang H., et al. Antibody Expression and Engineering, Am. Chem, Soc., Washington, DC (1995). Polyclonal and monoclonal antibodies can be isolated by methods known in the art (e.g., by Harlow et al.).

The mutant antibodies are tetramers of two identical light (L) chains and two identical heavy (H) chains. The L and H chains have variable domains involved in antigen recognition and binding. Variability in the variable domain is concentrated in the complementary determining region (CDR).

Antibodies contemplated for use in the present invention may be any of a variety of forms, including antibody fragments such as whole immunoglobulins, Fv, Fab-like fragments, single chain antibodies of CDRs and the like, and are used herein. Includes all antibodies that fall within the broadest sense.

"Antibody fragment" means the antigen-binding portion of a full length antibody. Antibody fragments may be as small as about 4 amino acids, about 10 amino acids, about 30 amino acids, or more. Some types of antibody fragments are as follows:

(1) Fab is a fragment comprising a monovalent antigen-binding fragment of an antibody molecule. Fab fragments can be generated by treating the entire antibody with papain enzymes to create an intact light chain and one heavy chain site. Two Fab fragments are obtained per total antibody molecule.

(2) Fab ′ is an antibody fragment that can be obtained by treating a complete antibody with pepsin and then generating intact light and heavy chain portions by reduction. Two Fab 'fragments can be obtained from the complete antibody molecule. Fab 'fragments are distinguished from Fab fragments by the addition of several residues, such as one more cysteine at the carboxyl terminus of the heavy chain CH1 domain.

(3) F (ab ') ₂ are fragments obtained without reducing the complete antibody treated with pepsin. F (ab ') ₂ is held together linked by two disulfide bonds to the dimer of two Fab' fragments.

(4) Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. Fv consists of a dimer of one light chain and one heavy chain of tight non-covalent bonds (V _H -V _L dimer). In this arrangement it interacts with the three CDRs of each variable domain to delimit the antigen-binding site. In total, the six CDRs confer antigen-binding specificity to the antibody. However, even one variable domain (or half of the Fv consisting of 3 CDRs specific for the antigen) has the ability to bind the antigen (although it has a lower affinity than the full binding site).

(5) Single-chain antibodies (SCAs) are defined as genetically engineered molecules comprising the variable region of the heavy chain and the light region of the heavy chain, which are genetically linked by a suitable polypeptide linker fused into one chain molecule.

The preparation of polyclonal antibodies is well known to those skilled in the art. See, eg, Coligan et al., In Current Protocols in Immunology, section 2.4.1 (1992). The preparation of monoclonal antibodies is likewise customary. See, eg, Harlow et al., Page 726.

In vitro and in vivo manipulation methods of monoclonal antibodies are well known to those of skill in the art. For example, monoclonal antibodies used according to the invention can be made by the hybridoma method first described by Kohler and Milstein (Nature 256: 495 (1995)) or by recombinant methods. (Eg US Pat. No. 4,816,567). Monoclonal antibodies used according to the invention are also described in Nature 352: 624 (1991) by Clark et al. And J. Mol. Biol. The techniques described in 222: 581 (1991) can be used to extract from phage antibody libraries. Another method involves humanization of monoclonal antibodies by recombinant means to produce antibodies comprising human specific recognition sequences (see, eg, Holmes et al., J. Immunol. 158: 2192 (1997) and Vaswani). et al., Annals Allergy, Asthma & Immunol. 81: 105 (1998)).

Monoclonal antibodies specifically encompassed herein are “chimeric” antibodies in which the heavy and / or light chain moieties are identical or homologous to the corresponding sequences of the antibodies derived from the scoop, or in a particular antibody class or subclass. While the remaining chain (s) are identical or homologous to the corresponding sequence of an antibody derived from another species, antibodies belonging to different antibody classes or subclasses, and those exhibiting the desired biological activity. Fragments of antibodies (US Pat. No. 4,816,567; Morrison et al., Proc. Nat'l. Acad. Sci. 81: 6851 (1984)).

Methods of making antibody fragments are also known in the art (eg, Harlow and Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)). Antibody fragments of the present invention can be prepared by hydrolysis by proteolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by customary methods of treating whole antibodies with pepsin or papain. For example, antibody fragments can be produced by enzymatically cleaving an antibody with pepsin to produce a 5S fragment represented by F (ab ') ₂ . This fragment can be further cleaved using a thiol reducing agent and can optionally produce 3.5 S Fab 'monovalent fragments as a blocking group for the sulfhydryl group from cleavage of the disulfide bond. Alternatively, enzyme cleavage with pepsin directly produces two monovalent Fab fragments and one Fe fragment. These methods are described, for example, in US Pat. Nos. 4,036,945, 4,331,647 and the literature incorporated herein by reference.

Other methods of antibody cleavage, such as the separation of heavy chains, to form monovalent light chain-heavy chain fragments, in addition fragment cleavage, or other enzymatic, chemical or genetic techniques, may be used, in which fragments are recognized by an intact antibody. It can be used as long as it binds to the antigen. For example, Fv fragments consist of a combination of V _H and V _L chains. This bond may not be covalent, the variable chains may be linked by intermolecular disulfide bonds, or may be linked by cross linking with compounds such as glutaraldehyde. Preferably, the FV fragments comprise V _H and V _L chains linked by a peptide linker. These single chain antigen binding proteins (sFv) are prepared by loading structural genes comprising DNA sequences encoding V _H and V _L domains linked by oligonucleotides. The structural gene is then inserted into an expression vector that is introduced into a host cell, such as E. coli. Recombinant host cells synthesize a single peptide chain with a linker connecting two V domains. Methods for generating sFv are described, for example, by Whitlow et al., Methods: a Companion to Methods in Enzymology, 2:97 (1991); Bird et al., Science 242: 423 (1988); Ladner et al., US Pat. No. 4,946,778; And Pack et al., Bio / Technology 11: 1271 (1993).

Another type of antibody fragment is a peptide with a single complementary-determining region (CDR). CDR peptides (“minimal recognition units”) are frequently involved in antigen recognition and binding. CDR peptides can be obtained by cloning or by constructing a gene encoding the CDR of the antibody of interest. Such genes are prepared by synthesizing variable regions from RNA of antibody-producing cells, for example using PCR. See, eg, Larrick et, al., Methods: a Companion to Methods in Enzymology 2: 106 (1991).

The present invention contemplates the humanized forms of human and non-human (eg murine) antibodies. Such humanized antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (Fv, Fab, Fab ', F (ab') ₂ or other antigen-binding sequences of the antibody) that are minimally derived from non-human immunoglobulins. Those containing sequences. Most humanized antibodies are human immunoglobulins in which the CDR residues of the recipient have been replaced by residues from the CDRs of non-human species (donating antibodies) such as mice, mice, goats, sheep and rabbits that have directed specificity, affinity and ability. (Receptive antibody).

In some instances, Fv framework residues of human immunoglobulin are replaced with corresponding nonhuman residues. Moreover, the humanized antibody may consist of residues that are not found in the recipient antibody or imported CDR or framework sequences. Their modifications make the role of antibody performance more refined and optimal. In general, a humanized antibody consists of substantially all or substantially all of the variable regions in which all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are human immune. Globulin consensus sequence. Humanized antibodies will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), which is typically of human immunoglobulin. For more details, see Jones et al., Nature 321: 522 (1986); Reichmann et al., Nature 332: 323 (1988); Presta, Curr. Opinion Struct. Biol. 2: 593 (1992); Holmes et al., J. Immunol. 158: 2192 (1997); And Vaswani et al., Annals Allergy, Asthma & Immunol. 81: 105 (1998).

Antibodies of the invention can also be mutated to optimize affinity, selectivity, binding strength or other directed properties. One method of antibody variation involves affinity variation using phage display. For affinity variation using phage display, see the procedure described by Lowmane et al. [Biochemistry 30: 10832 (1991)]; J. Mol. By Hawkins et al. Biol. See also 254: 889 (1992).

The invention will now be described in more detail by way of non-limiting examples.

Example 1

This example describes the preparation of Epo-bp, which is thought to have SEQ ID NO: 2, which is also described in US Pat. No. 5,843,726.

Construction of EpoR Production Vectors

Recombinant plasmid expression vector pJYL26 was constructed from the PCR product containing the extracellular lysis domain coding sequence of the human Epo receptor and the plasmid vector pGEX-2T purchased from Pharmacia.

PCR amplification was performed using the full-length human EpoR cDNA, SEQ ID NO: 4 as a template. 5'-TTGGATCCGCGCCCCCGCCTAAC-3 '(SEQ ID NO: 5) was used as the 5'-sense primer. This primer has a BamH1 linker sequence at the 5 'end, followed by a sequence encoding amino acids 25-29 of the full length human EpoR protein. 5'-TGAATTCGGGGTCCAGGTCGCT-3 '(SEQ ID NO: 6) was used as the 3'-antisense primer. This primer has an EcoR1 linker followed by a sequence complementary to the sequence encoding amino acids 250-246 of full length EpoR. PCR was performed as described in US Pat. No. 5,843,726.

The PCR product and pGEX-2T were digested with EcoR1 and BamH1, and the digested DNAs were purified by gel electrophoresis. The PCR product and pGEX-2T were ligated in a mixture containing 1 μg / μl each. The ligation product was found to be about 5.5 kb. The ligation plasmid mixture was used to transform E. coli JM 109. Colonies multiplied. DNA was extracted from each transgenic colony and analyzed. Plasmids were selected from one colony after confirming the expected size of the DNA treated with EcoR1 and EcoR1 Plus BamH1 on 1% agarose gel. The procedures were performed as described in US Pat. No. 5,843,726.

Purification of EpoRex-th Fusion Proteins

Transformed Escherichia coli including the recombinant vector pJYL26 was cultured and induced with IPTG. Cell extracts were applied to a GSH-agarose column. Bound EpoRex-th was eluted with reduced glutathione. This was done as described in US Pat. No. 5,843,726.

Purification of Epo-bp

EpoRex-th contains a thrombin-specific proteolytic cleavage recognition sequence that separates Epo-bp from glutathione-S-transferase. The amino acid sequence of the cleavage recognition sequence is Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 7). The fusion protein was cleaved with thrombin and Epo-bp was purified for binding affinity for the Epo-agarose column. This was done as described in US Pat. No. 5,843,726. The thrombin cleavage site was not determined experimentally because the amino acid terminus of Epo-bp was generated by this procedure. The thrombin cleavage is believed to produce Epo-bp of SEQ ID NO: 2. However, thrombin may also cleave another site, ie, between Arg and Gly of the recognition sequence, to produce a protein wherein the Gly-Ser peptide is attached to the amino terminus of SEQ ID NO: 2 (SEQ ID NO: 8).

Example 2

This example describes experiments that test the effect of fusion Epo-binding protein having the amino acid sequence of SEQ ID NO: 2 and Fab antibodies against Epo-bp by injecting mice with or without Epo.

Experimental procedure

<Material>

Glutathione (GSH) -agarose, pGEX-2T expression vector and Sephadex G-50 were purchased from Pharmacia (Mechanicsburg, PA). PCR reagents were purchased from Perkin Elmer Cetus (Norwalk, CT) and Affigel ^® 15 was purchased from BioRad: Richmond, CA. Bacteriophage T4 DNA ligase, restriction enzymes and isopropylthio-β-D-galactoside (IPTG) were purchased from BRL Gibco (Gaithersburg, MD). Geneclean II was purchased from Bin 101, La Jolla, CA. Nitrocellulose was purchased from Schleicher & Schuell Co. (Keene, NH). Chemiluminescence (ECL) reagents and ¹²⁵ I-Epo were purchased from Amersham (Arlington Heights, IL) and unlabeled Epo was donated from Chugai-Upjohn (Rosemont, IL). Thrombin, trypsin, phenylmethylsulfonylfluoride (PMSF), diisopropylfluorophosphate (DFP), triton X-100, 2,7-dichlorofluorescein, biotin-aminocaproyl hydrazoazide, alkaline phosphatase conjugates , Disodium p-nitrophenyl phosphate and o-phenylenediamine dihydrochloride (oPD) were purchased from Sigma Chemical Company (St. Louis, Mo.). Biotinylated rabbit anti-sheep antibodies and avidin-horses perradase and IgG purification kits were purchased from Pierce Co .: Rockford, IL. Streptavidin peroxidase was purchased from the Boehringer Mannheim Company (Indianapolis, Ind.) And microplates were purchased from Corning Costa (Cambridge, Mass.). All other reagents used reagent grade.

Epo-bp, Fab antibody against Epo-bp (αEpo-bp) and Fab antibody against Epo (αEpo) were prepared in our laboratory. Epo-bp was prepared as described in US Pat. No. 5,843,726. Epo-bp had the amino acid sequence of SEQ ID NO: 2. The full length human receptor cDNA (SEQ ID NO: 4) was provided by Dr. Bernard G. Podget of Yale University. Oligonucleotides were synthesized in a microchemical research facility at the Institute of Human Genetics at the Minnesota School in Minneapolis, MN. All other reagents used reagent grade.

<Animal experiment>

Sprague-Dawley (SD) male rats were housed in animal facilities for university animal testing, providing free access to purina food and water. In the laboratory, which changed the day and night cycle from 4 am to 6 pm (04: 00-18: 00), the mice were protected, and the blood pressure, erythrocyte volume, weight (weight) and spleen weight of the mice were protected. Was influenced by the daily cycle of the Epo effect. To find an effective treatment time, each group of 5 rats, each group consisting of 6 subgroups, was placed in the control and treatment groups, with test times 00, 04, 08, 12, 16 and 20 o'clock. . Mice were randomly extracted and placed in each group of Epo treated (Rx) versus saline and other treatment groups, with no statistically significant differences in weight, blood pressure and erythrocyte volume ratios between groups prior to treatment.

Blood pressure, erythrocyte volume and body weight were measured immediately before the start of treatment and immediately after completion of 4 weeks of subcutaneous injection of Epo or saline twice a week. The amount of Epo, Epo-bp and αEpo-bp was determined according to the report of epoetin study (4). Epo amount was 50 units / kg body weight, and Epo-bp and αEpo-bp were administered in the same amount as Epo. Erythropoietin (epoetin) was purchased from Amgen company (Thousand Oaks, CA). Affinity-purified Epo-bp and αEpo-bp were made in our laboratory. Antibodies were treated with Fab fragments and Fab antibodies were purified.

For blood pressure measurement, the cannula was connected to the femoral artery under pentobarbitol (50 mg / kg) anesthesia. At the end of the experiment, the spleen was weighed and photographed. Weigh the brain, heart, aorta and both kidneys.

Detection of Ligand Binding Sites and Epo and EpoE in Progenitor Cells

We developed αEpo-bp by inoculating Epo-bp in sheep every 3-4 weeks for 3 months. After collecting the serum, the antibodies were purified and treated with papain to produce Fab antibodies. Fabs were purified. Fabs were labeled with fluorescein according to the manufacturer's instructions. These materials have been used to detect receptors in blood and / or tissue samples. Negative control cells had no antibodies added and positive control cells had Fab from IgG of pre-immune serum. To examine the antibody binding site (Epo receptor), bone marrow cells were washed with PBS and dispensed into round bottomed tubes at 1-3 × 10 ³ cells per well, followed by centrifugation at 500 × g for 2 minutes. The supernatant was removed and 100 μl of fluorescein conjugated Fab antibody was added. After mixing well, the mixture was incubated for 30 minutes in an ice bath. Cells were washed three times with 400 μl of PBS buffer containing 1% FCS and 0.01% NaN ₃ , and the supernatant was removed by centrifugation at 500 × g for 2 minutes for each wash. Washed cells were resuspended to a total amount of 50 μl of PBS and analyzed by inverted fluorescence microscopy.

Enzyme Immunoassay (EIA) is used to detect and measure levels of Epo, Epo-bp and antibodies against Epo and Epo-bp in untreated human subjects. EIA microplates were coated with 2 μl / well of anti-Epo-bp antibody (αEpo-bp) for Epo-bp detection and 2 μg / well anti-Epo for Epo detection. Wells were coated with 200 μl of 1:10 diluted serum or plasma PBS (pH 7.4) buffer to detect circulating anti-Epo and anti-αEpo-bp antibodies (αEpo-bp). Plates were incubated for 30 minutes at room temperature or overnight at 4 ° C. After coating the plate with antibody or serum, the wells were washed three times with 200 μl / well of PBST (0.05% TWEEN 20 in PBS). Nonspecific binding sites were blocked for 30 minutes at room temperature with 200 μl / well PBST containing 1% BSA. Wells were washed three times with 200 μl / well PBST. For bound antigen detection, the peroxidase-streptavidin label in our laboratory was used for Fab anti-Epo (for Epo detection), Epo (for anti-Epo antibody detection), Fab anti-Epo-bp ( To Epo-bp detection) and Epo-bp (for anti-Epo-bp antibody detection), respectively. 2 μg of the appropriate peroxidase-streptavidin labeled protein was added to each well with 200 μl PBST buffer. Wells were washed three times with 200 μl PBST buffer. A solution of O-phenylenediaminedihydrochloride (OPD) in citrate buffer (160 μl) was added to each well. (The solution contains 10 mg / ml in 24 mM citrate and 51 mM Na ₂ HPO ₄ , pH 6.0, and 0.4 ml of 3% H ₂ O ₂ is added to a 100 ml solution immediately before use). 40 µl of 5M NaOH was added to stop the reaction, and the absorbance was measured at 405 nm.

<statistics>

The data were analyzed by two-tailed Stndent's t test, cosineor method and linear least squares rhythmometry (21), which allowed the variables as a function of the data. Data are shown as mean ± SEM. It was considered significant when the p value was less than 0.05.

<result>

In Table 1, there was no statistically significant difference between groups of pre-treatment blood pressure, erythrocyte volume and body weight in all treatment groups. The overall body weight of the Epo-treated group was lower than that of the control group (295 vs, 313 g, p <0.01), and Epo treatment vs, control, Epo-bp and αEpo-bp (Epo-bp) in the baseline daily blood pressure comparison before treatment. Fab antibodies against) groups were not significant (87 ± 2.8 vs. 88.8 ± 3.4, 88.7 ± 2.5, 84.3 ± 2.3 mmHg). After treatment, the daily blood pressure increased significantly in the Epo-treated group. Epo treatment vs. Group comparisons between the control, Epo-bp and αEpo-bp treatment groups were as follows: 136.2 ± 2.3 vs. 116.2 ± 1.7, 118.4 ± 2.1 and 116.6 ± 2.1 mmHg, respectively. Each p <0.0001. However, when Epo-bp or αEpo-bp are administered with Epo. Blood pressure was maintained at a similar level to the saline control group: blood pressure values of 118.3 ± 1.7 in the Epo-bp + Ep group and 121.0 ± 2.0 mmHg in the αEpo-bp + Epo treatment group were significantly lower than those in the Epo treatment group (136.2 ± 2.3). It was found that each p <0.0001.

Table 1

Comprehensive effects on daily cycle weight, blood pressure, red blood cell volume and other organ systems in various treatments

In the table above,

: 평균 24-시간; αEpo-bp = 항-Epo-bp 항체; *p < 0.01; **p <0.001; ***p < 0.0001;

< 0.05; g = 그램; BW = 체중; BP = 혈압; Hct = 적혈구 용적율; SW = 비장 무게; W = 체중; R = 우측; L = 좌측.Rx: treatment; n = number of mice (30 per group);

: Average 24-hours; αEpo-bp = anti-Epo-bp antibody; * p <0.01; ** p <0.001; *** p <0.0001;

<0.05; g = grams; BW = body weight; BP = blood pressure; Hct = red blood cell volume ratio; SW = spleen weight; W = body weight; R = right; L = left.

Epo-treatment significantly increased the overall red blood cell volume (61.6 vs 42.7, 43.9 and 44.1%) compared to the saline, Epo-bp or αEpo-bp groups, with p <0.0001 in all six trials. Administration of Epo-bp or αEpo-bp with Epo had little effect on Epo-induced elevation of red blood cell volume (61.6% erythrocyte volume vs. 58.0% for Epo + Epo-bp and 59.1 for Epo + αEpo-bp treatment). %). Importantly, however, both Epo-bp and αEpo-bp virtually ruled out Epo-induced blood pressure elevation (136.2 mmHg in the Ep treatment group vs. 116.2 in the saline control group, 118.3 in Epo + Epo-bp and Epo + αEpo-bp). 121.0). Thus, Epo-bp and αEpo-bp protected mice from elevated blood pressure during Epo treatment.

Each rat in the Epo-treated group exhibited splenomegaly (total spleen weight 1.58 vs. saline 0.86, Epo-bp 0.89 and αEpo-bp 0.85 g, each p <0.0001). Administration of Epo-bp or αEpo-bp with Epo had no effect on splenomegaly. Although the weights of the aorta and kidneys were similar in each group, the brain and heart weights in the group treated with Epo were significantly lower than in all other groups.

Figure 1 shows the daily cycle blood pressure comparison in all groups as a bar graph ± standard error (SEM). The group treated with Epo significantly increased blood pressure compared to all five other groups. Each p <0.0001. 2 shows the daily cycle variation of MESOR (mean about 24 hours), amplitude and acrophase (time to peak) in each treatment group. Amplitudes in all groups were not significantly different, but the increase in daily cycle blood pressure (MESOR) due to Epo treatment was significant (all p <0.0001), after treatment, control, Epo-bp or αEpo- The peak time of blood pressure in Epo-treated rats was changed to daytime compared to the group treated with bp (19:40 vs. 04:08, 05:44, 05:16). Epo treatment resulted in a clear shift from nighttime peaks to daytime peaks in these night animals. When Epo-bp or αEpo-bp were administered with Epo, the blood pressure was similar to that of the control group, but the change in the peak time stayed at the same daytime as in the Epo-only group (14:48, 19:20). .

Table 2 summarizes the daily cycle changes in body weight, blood pressure, erythrocyte volume ratio and spleen weight in six subgroups after Epo, Epo-bp and αEpo-bp treatment. The weight difference was not statistically significant in the Epo-treated rats and other treatment groups during the six test times. Significantly elevated blood pressure in the Epo-treated group was detected at 12, 16, 20 and 00 hours except for 04 or 08 hours compared to the control, Epo-bp and αEpo-bp treated groups. Compared to the control Epo-bp and αEpo-bp treatment groups, Epo treatment significantly increased the overall red blood cell volume rate and the red blood cell volume rate at each of six test times. All, p <0.0001. The spleen weight was lower at each compared test time, but the spleen weight of the group mice treated with Epo was significantly higher at all test time points than those of the control, Epo-bp and αEpo-bp.

TABLE 2

Daily cycle changes in body weight, blood pressure, red blood cell volume, and spleen weight in various treatments

In the table above,

< 0.05; BP = 혈압; BW = 체중; Hct = 적혈구 용적율; SW = 비장 무게.Rx: treatment; n = 5 per each small group; * p <0.01; ** p <0.001; *** p <0.0001;

<0.05; BP = blood pressure; BW = body weight; Hct = red blood cell volume ratio; SW = spleen weight.

3 exemplifies by comparing the daily cyclic red blood cell volume ratio. There was a peak time shift change from night (20:15) to early morning (11:16) as well as an increase in erythrocyte volume fraction with Epo-treatment. In this graph, the a (control), c (Epo-bp) and d (αEpo-bp) groups were located in the night cycle plane but b (Epo), e (Epo + Epo-bp) and f (Epo + αEpo- bp) was located in the low period plane. To reiterate, these night animals produced a clear shift in daytime peak time from night in Epo treatment. Epo (61.6) vs control (42.7), Epo-bp (43.9) and αEpo-bp (44.1) treatments in the erythrocyte volume percentage (24-hour average) (MESOR) comparisons were statistically significant at each time point comparison ( Each p <0.0001). The peak-to-peak amplitude difference of the erythrocyte volume fraction was not significantly different from the difference between treatment groups. However, amplitude was greater in the Epo-treated group than in the non-Epo group (Epo 2.40, Epo + Epo-bp 4.41 and Epo + αEpo-bp 3.59 vs Control 1.65, Epo-bp 1.13 and αEpo-bp 1.73).

In FIG. 4, splenomegaly (a, b and c) was characterized in each rat treated with Epo as compared to saline treated rats (FIG. 4, panel d). Spleen weights were significantly higher in Epo-treated mice than those in the control, Epo-bp and αEpo-bp treated groups (Tables 1 and 2).

These results suggest that Epo treatment time, Epo-bp and / or αEpo-bp treatment may or may not be concurrently important may be important. Epo-bp and αEpo-bp protect against Epo-induced blood pressure rise without reducing the level of erythrocyte volume uplifted by Epo.

The amount of Epo used in the clinic to prevent systemic and local adverse effects such as high blood pressure and other organ damage should be reassessed.

The binding site of blood progenitor cells was identified using Epo-bp and antibodies against it. Fluorescein labeled αEpo-bp was used to visualize receptor sites in blood myeloid progenitor cells. No receptor was detected with fluorescein labeled pre-immune Fabs or negative control cells. However, labeled αEpo-bp detected binding sites on megakaryocytes, immature red blood cells, normal red blood cells and immature white blood cells (not shown).

Levels of Epo, Epo-bp and antibodies against Epo and Epo-bp were measured by enzyme-immunoassay (EIA) in untreated serum and plasma of humans and the results are shown in FIG. 5. Each absorbance (OD) measurement is expressed as mean ± SEM in duplicates of 8-14 specimens. The OD values shown in FIG. 5 were calculated by subtracting the OD value of the blank from the OD of each sample. Serum and plasma Epo and Epo-bp OD values were similar respectively: serum Epo 0.308 ± 0.026, serum Epo-bp 0.289 ± 0.022, plasma Epo 0.289 ± 0.028 and plasma Epo-bp 0.299 ± 0.015. Anti-Epo-bp antibody levels in plasma were significantly lower than the other three antibody categories: serum αEpo 0.058 ± 0.008, serum αEpo-bp 0.052 ± 0.006, plasma αEpo 0.054 ± 0.013 and plasma αEpo-bp 0.031 ± 0.004. Serum αEpo and αEpo-bp levels were similar, but the concentration of plasma αEpo-bp was significantly lower than that of serum αEpo, serum αEpo-bp or plasma αEpo (p <0.025). Epo and Epo-bp values were converted to known Epo concentrations using a control prepared in mU / ml on the same plate. Values in mU / ml were serum Epo 25.4 ± 2.17 mU, plasma Epo 24.2 ± 2.35 mU, serum Epo-bp 24.2 ± 1.84 mU, and plasma Epo-bp 25.0 ± 1.26 mU. This assay provides a simpler, more sensitive, and smaller SEM than Radioimmunoassay (Epo at 17.7 ± 6.3 mU / ml). In addition, the materials used in this preparation are more environmentally friendly than radioactive or other toxic chemicals used in conventional methods.

<Review>

As expected, erythrocyte volume ratios in mice treated with Epo were significantly elevated at all and at each six test points. In addition, the present inventors observed an increase in blood pressure in the Epo-treated group and splenomegaly in each mouse treated with Epo. Epo therapy not only raises blood pressure seriously, but also changes the peak time of blood pressure from night to day.

Remarkably, Epo-bp and αEpo-bp almost completely protected rats from Epo-induced blood pressure rise without reducing the percentage of red blood cell volume. The mechanism of this protective effect is unknown. However, the inventors speculate that repeated injections of epoetin (the recombinant Epo currently being used in clinical trials) may induce some toxic substances in living animals. Since Epo-bp binds to Epo or its degradation products, and αEpo-bp also binds to certain products induced by Epo treatment, Epo-bp and / or αEpo-bp can bind to and eliminate toxic substances. Seems to be.

2 and 3 result in peak time replacement of blood pressure within about 24 hours in combination treatment with Epo as well as Epo + Epo-bp or Epo + αEpo-bp. This suggests that treatment time for treatment with Epo, Epo + Epo-bp or Epo + αEpo-bp will significantly affect the treatment outcome. As shown in high blood pressure (22, 23) in Dahl rats due to salt sensitivity, individual genetic susceptibility must also be considered in endocrine therapy.

Cloning of human Epo-receptor recombinant vector JYL 26 and purification of pure human Epo-bp and its antibodies are important benchmarks for visualization of ligand binding sites and for identifying the cell types in which Epo receptors are located (Lee, US Pat. No. 5,483,726). ). To identify the ligand-binding sites we have developed a variety of sensitive and simple methods. These allow us to understand the structure of the Epo receptor, examine the factors involved in ligand binding, and identify other factors involved in controlling progenitor cells differentiation and proliferation. In this study we report the direct binding of Epo to purified human Epo-bp. We know that our Epo-bp and its antibodies are primarily purified pure human Epo receptor gene products that have the property of specifically binding to Epo and its antibodies at nM concentration.

The binding sites of blood progenitor cells were shown using Epo-bp and its antibodies. These data support the current proposal that all human blood progenitor cells have Epo receptors and bind to Epo. We do not yet know whether Epo's biophysical mechanisms or second messenger systems are involved in response to Epo-Epo receptor interactions. The methods listed in this report will help to identify defects related to Epo or Epo receptors and to reveal the role of Epo receptors (EpoR) in the progenitor cells' progression and ligand binding. These results will help to understand the structural and functional relationships of Epo-EpoR interactions in blood progenitor cells. Sensitive detection will help to understand the role of Epo-EpoR interactions and disease blood cell production in blood cell production, and will help to develop methods for the treatment of systemic cardiovascular diseases such as malignant blood disease and hypertension.

Conclusion

Compared to the control group, the group treated with Epo-bp and anti-Epo-bp antibody (αEpo-bp), Epo treatment significantly increased the erythrocyte volume fraction at each test time at both the overall and six test points (both p <0.0001 ). In mice treated with Epo, an increase in blood pressure was observed at 12, 16, 20, and 00 time points except for 04 or 08 time points. When Epo-bp or αEpo-bp were used with Epo treatment, blood pressure persisted at a level similar to that of the control group, but erythrocyte volume fraction was significant between groups treated with Epo vs Epo + Epo-bp or Epo + αEpo-bp. (61.6 vs. 58.0 or 59.1%, respectively). Thus, Epo-bp and αEpo-bp reduce or prevent Epo-induced blood pressure rise.

Body weight was lost by Epo treatment. Each rat treated with Epo was characterized by splenomegaly. Brain and heart weights in the group treated with Epo were significantly lower than those in the other groups. These data suggest that the amount of Epo must be reassessed to prevent further damage to the organ. Daily cycle results indicate that Epo treatment time may be important, either alone or in combination with Epo-bp and / or αEpo-bp.

Epo, Epo-bp and protein antibody levels in serum and plasma of untreated human volunteers were investigated. Serum and plasma Epo and Epo-bp levels were similar: Epo 25.4 ± 2.17 each; 24.2 ± 2.35; And Epo-bp each 24.2 ± 1.84; 25.0 ± 1.26 mU / ml. Serum αEpo and αEpo-bp levels were similar, but plasma αEpo-bp levels were significantly lower than those of serum or plasma αEpo or serum αEpo-bp.

Reference

All cited patents, patent documents and references are incorporated herein by reference.

                         SEQUENCE LISTING <110> Lee, Jong Y.        Lee, Mary S.        Lee, John S.   <120> Compositions and Methods for Preventing Erytropoietin-Associated        Hypertension <130> 106.003WO1 <150> US 10 / 848,689 <151> 2004-05-17 <160> 10 <170> PatentIn version 3.3 <210> 1 <211> 508 <212> PRT <213> Homo sapiens <300> Winkelman, J. C., et al. <302> The gene for the human erythropoietin receptor: analysis of the        coding sequence and assignment to chromosome 19p. <303> Blood <304> 76 <305> 1 <306> 24-30 <307> 1990-07-01 <400> 1 Met Asp His Leu Gly Ala Ser Leu Trp Pro Gln Val Gly Ser Leu Cys 1 5 10 15 Leu Leu Leu Ala Gly Ala Ala Trp Ala Pro Pro Pro Asn Leu Pro Asp             20 25 30 Pro Lys Phe Glu Ser Lys Ala Ala Leu Leu Ala Ala Arg Gly Pro Glu         35 40 45 Glu Leu Leu Cys Phe Thr Glu Arg Leu Glu Asp Leu Val Cys Phe Trp     50 55 60 Glu Glu Ala Ala Ser Ala Gly Val Gly Pro Gly Asn Tyr Ser Phe Ser 65 70 75 80 Tyr Gln Leu Glu Asp Glu Pro Trp Lys Leu Cys Arg Leu His Gln Ala                 85 90 95 Pro Thr Ala Arg Gly Arg Val Arg Phe Trp Cys Ser Leu Pro Thr Ala             100 105 110 Asp Thr Ser Ser Phe Val Pro Leu Glu Leu Arg Val Thr Ala Ala Ser         115 120 125 Gly Ala Pro Arg Tyr His Arg Val Ile His Ile Asn Glu Val Val Leu     130 135 140 Leu Asp Ala Pro Val Gly Leu Val Ala Arg Leu Ala Asp Glu Ser Gly 145 150 155 160 His Val Val Leu Arg Trp Leu Pro Pro Pro Glu Thr Pro Met Thr Ser                 165 170 175 His Ile Arg Tyr Glu Val Asp Val Ser Ala Gly Asn Arg Pro Gly Ser             180 185 190 Val Gln Arg Val Glu Ile Leu Glu Gly Arg Thr Glu Cys Val Leu Ser         195 200 205 Asn Leu Arg Gly Arg Thr Arg Tyr Thr Phe Ala Val Arg Ala Arg Met     210 215 220 Ala Glu Pro Ser Phe Gly Gly Phe Trp Ser Ala Trp Ser Glu Pro Val 225 230 235 240 Ser Leu Leu Glu Pro Ser Asp Leu Asp Pro Leu Ile Leu Thr Leu Ser                 245 250 255 Leu Ile Leu Val Val Ile Leu Val Leu Leu Thr Val Leu Ala Leu Leu             260 265 270 Ser His Arg Arg Ala Leu Lys Gln Lys Ile Trp Pro Gly Ile Pro Ser         275 280 285 Pro Glu Ser Glu Phe Glu Gly Leu Phe Thr Thr His Lys Gly Asn Phe     290 295 300 Gln Leu Trp Leu Tyr Gln Asn Asp Gly Cys Leu Trp Trp Ser Pro Cys 305 310 315 320 Thr Pro Phe Thr Glu Asp Pro Pro Ala Ser Leu Glu Val Leu Ser Glu                 325 330 335 Arg Cys Trp Gly Thr Met Gln Ala Val Glu Pro Gly Thr Asp Asp Glu             340 345 350 Gly Pro Leu Leu Glu Pro Val Gly Ser Glu His Ala Gln Asp Thr Tyr         355 360 365 Leu Val Leu Asp Lys Trp Leu Leu Pro Arg Asn Pro Pro Ser Glu Asp     370 375 380 Leu Pro Gly Pro Gly Gly Ser Val Asp Ile Val Ala Met Asp Glu Gly 385 390 395 400 Ser Glu Ala Ser Ser Cys Ser Ser Ala Leu Ala Ser Lys Pro Ser Pro                 405 410 415 Glu Gly Ala Ser Ala Ala Ser Phe Glu Tyr Thr Ile Leu Asp Pro Ser             420 425 430 Ser Gln Leu Leu Arg Pro Trp Thr Leu Cys Pro Glu Leu Pro Pro Thr         435 440 445 Pro Pro His Leu Lys Tyr Leu Tyr Leu Val Val Ser Asp Ser Gly Ile     450 455 460 Ser Thr Asp Tyr Ser Ser Gly Asp Ser Gln Gly Ala Gln Gly Gly Leu 465 470 475 480 Ser Asp Gly Pro Tyr Ser Asn Pro Tyr Glu Asn Ser Leu Ile Pro Ala                 485 490 495 Ala Glu Pro Leu Pro Pro Ser Tyr Val Ala Cys Ser             500 505 <210> 2 <211> 226 <212> PRT <213> Homo sapiens <400> 2 Ala Pro Pro Pro Asn Leu Pro Asp Pro Lys Phe Glu Ser Lys Ala Ala 1 5 10 15 Leu Leu Ala Ala Arg Gly Pro Glu Glu Leu Leu Cys Phe Thr Glu Arg             20 25 30 Leu Glu Asp Leu Val Cys Phe Trp Glu Glu Ala Ala Ser Ala Gly Val         35 40 45 Gly Pro Gly Asn Tyr Ser Phe Ser Tyr Gln Leu Glu Asp Glu Pro Trp     50 55 60 Lys Leu Cys Arg Leu His Gln Ala Pro Thr Ala Arg Gly Arg Val Arg 65 70 75 80 Phe Trp Cys Ser Leu Pro Thr Ala Asp Thr Ser Ser Phe Val Pro Leu                 85 90 95 Glu Leu Arg Val Thr Ala Ala Ser Gly Ala Pro Arg Tyr His Arg Val             100 105 110 Ile His Ile Asn Glu Val Val Leu Leu Asp Ala Pro Val Gly Leu Val         115 120 125 Ala Arg Leu Ala Asp Glu Ser Gly His Val Val Leu Arg Trp Leu Pro     130 135 140 Pro Pro Glu Thr Pro Met Thr Ser His Ile Arg Tyr Glu Val Asp Val 145 150 155 160 Ser Ala Gly Asn Arg Pro Gly Ser Val Gln Arg Val Glu Ile Leu Glu                 165 170 175 Gly Arg Thr Glu Cys Val Leu Ser Asn Leu Arg Gly Arg Thr Arg Tyr             180 185 190 Thr Phe Ala Val Arg Ala Arg Met Ala Glu Pro Ser Phe Gly Gly Phe         195 200 205 Trp Ser Ala Trp Ser Glu Pro Val Ser Leu Leu Glu Pro Ser Asp Leu     210 215 220 Asp pro 225 <210> 3 <211> 193 <212> PRT <213> Homo sapiens <300> <308> GenBank NP 000790 <309> 2003-12-23 (313) (1) .. (193) <400> 3 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu             20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu         35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu     50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu                 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser             100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly         115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu     130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu                 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp             180 185 190 Arg      <210> 4 <211> 1527 <212> DNA <213> Homo sapiens <300> <301> Winkelman, J.C. <302> The gene for the human erythropoietin receptor: analysis of the        coding sequence and assignment to chromosome 19p. <303> Blood <304> 76 <305> 1 <306> 24-30 <307> 1990-07-01 <400> 4 atggaccacc tcggggcgtc cctctggccc caggtcggct ccctttgtct cctgctcgct 60 ggggccgcct gggcgccccc gcctaacctc ccggacccca agttcgagag caaagcggcc 120 ttgctggcgg cccgggggcc cgaagagctt ctgtgcttca ccgagcggtt ggaggacttg 180 gtgtgtttct gggaggaagc ggcgagcgct ggggtgggcc cgggcaacta cagcttctcc 240 taccagctcg aggatgagcc atggaagctg tgtcgcctgc accaggctcc cacggctcgt 300 ggtcgggtgc gcttctggtg ttcgctgcct acagccgaca cgtcgagctt cgtgccccta 360 gagttgcgcg tcacagcagc ctccggcgct ccgcgatatc accgtgtcat ccacatcaat 420 gaagtagtgc tcctagacgc ccccgtgggg ctggtggcgc ggttggctga cgagagcggc 480 cacgtagtgt tgcgctggct cccgccgcct gagacaccca tgacgtctca catccgctac 540 gaggtggacg tctcggccgg caaccggcca gggagcgtac agagggtgga gatcctggag 600 ggccgcaccg agtgtgtgct gagcaacctg cggggccgga cgcgctacac cttcgccgtc 660 cgcgcgcgta tggctgagcc gagcttcggc ggcttctgga gcgcctggtc ggagcctgtg 720 tcgctgctgg agcctagcga cctggacccc ctcatcctga cgctctccct catcctcgtg 780 gtcatcctgg tgctgctgac cgtgctcgcg ctgctctccc accgccgggc tctgaagcag 840 aagatctggc ctggcatccc gagcccagag agcgagtttg aaggcctctt caccacccac 900 aagggtaact tccagctgtg gctgtaccag aatgatggct gcctgtggtg gagcccctgc 960 acccccttca cggaggaccc acctgcttcc ctggaagtcc tctcagagcg ctgctggggg 1020 acgatgcagg cagtggagcc ggggacagat gatgagggcc ccctgctgga gccagtgggc 1080 agtgagcatg cccaggatac ctatctggtg ctggacaaat ggttgctgcc ccggaacccg 1140 cccagtgagg acctcccagg gcctggtggc agtgtggaca tagtggccat ggatgaaggc 1200 tcagaagcat cctcctgctc atctgctttg gcctcgaagc ccagcccaga gggagcctct 1260 gctgccagct ttgagtacac tatcctggac cccagctccc agctcttgcg tccatggaca 1320 ctgtgccctg agctgccccc taccccaccc cacctaaagt acctgtacct tgtggtatct 1380 gactctggca tctcaactga ctacagctca ggggactccc agggagccca agggggctta 1440 tccgatgggc cctactccaa cccttatgag aacagcctta tcccagccgc tgagcctctg 1500 ccccccagct atgtggcttg ctcttag 1527 <210> 5 <211> 23 <212> DNA <213> E. coli <400> 5 ttggatccgc gcccccgcct aac 23 <210> 6 <211> 22 <212> DNA <213> E. coli <400> 6 tgaattcggg gtccaggtcg ct 22 <210> 7 <211> 6 <212> PRT <213> E. coli <400> 7 Leu Val Pro Arg Gly Ser 1 5 <210> 8 <211> 228 <212> PRT <213> Artificial Sequence <220> Epo-bp SEQ ID NO: 2 with an additional Gly-Ser at the amino        terminus <400> 8 Gly Ser Ala Pro Pro Pro Asn Leu Pro Asp Pro Lys Phe Glu Ser Lys 1 5 10 15 Ala Ala Leu Leu Ala Ala Arg Gly Pro Glu Glu Leu Leu Cys Phe Thr             20 25 30 Glu Arg Leu Glu Asp Leu Val Cys Phe Trp Glu Glu Ala Ala Ser Ala         35 40 45 Gly Val Gly Pro Gly Asn Tyr Ser Phe Ser Tyr Gln Leu Glu Asp Glu     50 55 60 Pro Trp Lys Leu Cys Arg Leu His Gln Ala Pro Thr Ala Arg Gly Arg 65 70 75 80 Val Arg Phe Trp Cys Ser Leu Pro Thr Ala Asp Thr Ser Ser Phe Val                 85 90 95 Pro Leu Glu Leu Arg Val Thr Ala Ala Ser Gly Ala Pro Arg Tyr His             100 105 110 Arg Val Ile His Ile Asn Glu Val Val Leu Leu Asp Ala Pro Val Gly         115 120 125 Leu Val Ala Arg Leu Ala Asp Glu Ser Gly His Val Val Leu Arg Trp     130 135 140 Leu Pro Pro Pro Glu Thr Pro Met Thr Ser His Ile Arg Tyr Glu Val 145 150 155 160 Asp Val Ser Ala Gly Asn Arg Pro Gly Ser Val Gln Arg Val Glu Ile                 165 170 175 Leu Glu Gly Arg Thr Glu Cys Val Leu Ser Asn Leu Arg Gly Arg Thr             180 185 190 Arg Tyr Thr Phe Ala Val Arg Ala Arg Met Ala Glu Pro Ser Phe Gly         195 200 205 Gly Phe Trp Ser Ala Trp Ser Glu Pro Val Ser Leu Leu Glu Pro Ser     210 215 220 Asp Leu Asp Pro 225 <210> 9 <211> 508 <212> PRT <213> Homo sapiens <300> <308> GenBank / NM_000121 <309> 2004-10-26 (313) (1) .. (508) <400> 9 Met Asp His Leu Gly Ala Ser Leu Trp Pro Gln Val Gly Ser Leu Cys 1 5 10 15 Leu Leu Leu Ala Gly Ala Ala Trp Ala Pro Pro Pro Asn Leu Pro Asp             20 25 30 Pro Lys Phe Glu Ser Lys Ala Ala Leu Leu Ala Ala Arg Gly Pro Glu         35 40 45 Glu Leu Leu Cys Phe Thr Glu Arg Leu Glu Asp Leu Val Cys Phe Trp     50 55 60 Glu Glu Ala Ala Ser Ala Gly Val Gly Pro Gly Asn Tyr Ser Phe Ser 65 70 75 80 Tyr Gln Leu Glu Asp Glu Pro Trp Lys Leu Cys Arg Leu His Gln Ala                 85 90 95 Pro Thr Ala Arg Gly Ala Val Arg Phe Trp Cys Ser Leu Pro Thr Ala             100 105 110 Asp Thr Ser Ser Phe Val Pro Leu Glu Leu Arg Val Thr Ala Ala Ser         115 120 125 Gly Ala Pro Arg Tyr His Arg Val Ile His Ile Asn Glu Val Val Leu     130 135 140 Leu Asp Ala Pro Val Gly Leu Val Ala Arg Leu Ala Asp Glu Ser Gly 145 150 155 160 His Val Val Leu Arg Trp Leu Pro Pro Pro Glu Thr Pro Met Thr Ser                 165 170 175 His Ile Arg Tyr Glu Val Asp Val Ser Ala Gly Asn Gly Ala Gly Ser             180 185 190 Val Gln Arg Val Glu Ile Leu Glu Gly Arg Thr Glu Cys Val Leu Ser         195 200 205 Asn Leu Arg Gly Arg Thr Arg Tyr Thr Phe Ala Val Arg Ala Arg Met     210 215 220 Ala Glu Pro Ser Phe Gly Gly Phe Trp Ser Ala Trp Ser Glu Pro Val 225 230 235 240 Ser Leu Leu Thr Pro Ser Asp Leu Asp Pro Leu Ile Leu Thr Leu Ser                 245 250 255 Leu Ile Leu Val Val Ile Leu Val Leu Leu Thr Val Leu Ala Leu Leu             260 265 270 Ser His Arg Arg Ala Leu Lys Gln Lys Ile Trp Pro Gly Ile Pro Ser         275 280 285 Pro Glu Ser Glu Phe Glu Gly Leu Phe Thr Thr His Lys Gly Asn Phe     290 295 300 Gln Leu Trp Leu Tyr Gln Asn Asp Gly Cys Leu Trp Trp Ser Pro Cys 305 310 315 320 Thr Pro Phe Thr Glu Asp Pro Pro Ala Ser Leu Glu Val Leu Ser Glu                 325 330 335 Arg Cys Trp Gly Thr Met Gln Ala Val Glu Pro Gly Thr Asp Asp Glu             340 345 350 Gly Pro Leu Leu Glu Pro Val Gly Ser Glu His Ala Gln Asp Thr Tyr         355 360 365 Leu Val Leu Asp Lys Trp Leu Leu Pro Arg Asn Pro Pro Ser Glu Asp     370 375 380 Leu Pro Gly Pro Gly Gly Ser Val Asp Ile Val Ala Met Asp Glu Gly 385 390 395 400 Ser Glu Ala Ser Ser Cys Ser Ser Ala Leu Ala Ser Lys Pro Ser Pro                 405 410 415 Glu Gly Ala Ser Ala Ala Ser Phe Glu Tyr Thr Ile Leu Asp Pro Ser             420 425 430 Ser Gln Leu Leu Arg Pro Trp Thr Leu Cys Pro Glu Leu Pro Pro Thr         435 440 445 Pro Pro His Leu Lys Tyr Leu Tyr Leu Val Val Ser Asp Ser Gly Ile     450 455 460 Ser Thr Asp Tyr Ser Ser Gly Asp Ser Gln Gly Ala Gln Gly Gly Leu 465 470 475 480 Ser Asp Gly Pro Tyr Ser Asn Pro Tyr Glu Asn Ser Leu Ile Pro Ala                 485 490 495 Ala Glu Pro Leu Pro Pro Ser Tyr Val Ala Cys Ser             500 505 <210> 10 <211> 1849 <212> DNA <213> Homo sapiens <300> <308> GenBank / NM_000121 <309> 2004-10-26 (313) (1) .. (1849) <400> 10 acttagaggc gcctggtcgg gaagggcctg gtcagctgcg tccggcggag gcagctgctg 60 acccagctgt ggactgtgcc gggggcgggg gacggagggg caggagccct gggctccccg 120 tggcgggggc tgtatcatgg accacctcgg ggcgtccctc tggccccagg tcggctccct 180 ttgtctcctg ctcgctgggg ccgcctgggc gcccccgcct aacctcccgg accccaagtt 240 cgagagcaaa gcggccttgc tggcggcccg ggggcccgaa gagcttctgt gcttcaccga 300 gcggttggag gacttggtgt gtttctggga ggaagcggcg agcgctgggg tgggcccggg 360 caactacagc ttctcctacc agctcgagga tgagccatgg aagctgtgtc gcctgcacca 420 ggctcccacg gctcgtggtg cggtgcgctt ctggtgttcg ctgcctacag ccgacacgtc 480 gagcttcgtg cccctagagt tgcgcgtcac agcagcctcc ggcgctccgc gatatcaccg 540 tgtcatccac atcaatgaag tagtgctcct agacgccccc gtggggctgg tggcgcggtt 600 ggctgacgag agcggccacg tagtgttgcg ctggctcccg ccgcctgaga cacccatgac 660 gtctcacatc cgctacgagg tggacgtctc ggccggcaac ggcgcaggga gcgtacagag 720 ggtggagatc ctggagggcc gcaccgagtg tgtgctgagc aacctgcggg gccggacgcg 780 ctacaccttc gccgtccgcg cgcgtatggc tgagccgagc ttcggcggct tctggagcgc 840 ctggtcggag cctgtgtcgc tgctgacgcc tagcgacctg gaccccctca tcctgacgct 900 ctccctcatc ctcgtggtca tcctggtgct gctgaccgtg ctcgcgctgc tctcccaccg 960 ccgggctctg aagcagaaga tctggcctgg catcccgagc ccagagagcg agtttgaagg 1020 cctcttcacc acccacaagg gtaacttcca gctgtggctg taccagaatg atggctgcct 1080 gtggtggagc ccctgcaccc ccttcacgga ggacccacct gcttccctgg aagtcctctc 1140 agagcgctgc tgggggacga tgcaggcagt ggagccgggg acagatgatg agggccccct 1200 gctggagcca gtgggcagtg agcatgccca ggatacctat ctggtgctgg acaaatggtt 1260 gctgccccgg aacccgccca gtgaggacct cccagggcct ggtggcagtg tggacatagt 1320 ggccatggat gaaggctcag aagcatcctc ctgctcatct gctttggcct cgaagcccag 1380 cccagaggga gcctctgctg ccagctttga gtacactatc ctggacccca gctcccagct 1440 cttgcgtcca tggacactgt gccctgagct gccccctacc ccaccccacc taaagtacct 1500 gtaccttgtg gtatctgact ctggcatctc aactgactac agctcagggg actcccaggg 1560 agcccaaggg ggcttatccg atggccccta ctccaaccct tatgagaaca gccttatccc 1620 agccgctgag cctctgcccc ccagctatgt ggcttgctct taggacacca ggctgcagat 1680 gatcagggat ccaatatgac tcagagaacc agtgcagact caagacttat ggaacaggga 1740 tggcgaggcc tctctcagga gcaggggcat tgctgatttt gtctgcccaa tccatcctgc 1800 tcaggaaacc acaaccttgc agtattttta aatatgtata gtttttttg 1849  

Claims (31)

(a) erythropoietin (Epo); And (b) a pharmaceutical composition for treating anemia, comprising a medicament selected from soluble Epo-binding protein (Epo-bp) of SEQ ID NO: 2, an antibody to soluble Epo-binding protein, and a complex thereof. The pharmaceutical composition of claim 1, wherein the medicament is a soluble Epo-binding protein of SEQ ID NO: 2 (Epo-bp). delete delete delete delete delete The pharmaceutical composition of claim 1, wherein the medicament is an antibody against soluble Epo-binding protein. The pharmaceutical composition of claim 8, wherein the antibody against soluble Epo-binding protein binds SEQ ID NO: 2. 10. delete delete The pharmaceutical composition of claim 1, wherein the antibody against soluble Epo-binding protein is a Fab antibody against soluble Epo-binding protein. The pharmaceutical composition of claim 1 comprising Epo and a medicament in a mixed form. The pharmaceutical composition of claim 1 comprising the Epo and the medicament in separate forms. The pharmaceutical composition of claim 1, wherein the dosage of the medicament is 75-125% molar amount of the Epo dosage. The pharmaceutical composition of claim 1, wherein the dosage of the medicament is equimolar to the dosage of Epo. 2. The pharmaceutical composition of claim 1, wherein the medicament reduces blood pressure elevation induced by erythropoietin in a mammal. Drugs for reducing hypertension in mammals receiving erythropoietin (Epo), comprising an effective amount of a drug selected from soluble Epo-binding protein (Epo-bp) of SEQ ID NO: 2, an antibody against soluble Epo-binding protein, and a complex thereof Pharmaceutical composition. (a) erythropoietin (Epo); And (b) hypertension as compared to a composition containing no component (b), comprising an agent selected from soluble Epo-binding protein (Epo-bp) of SEQ ID NO: 2, an antibody against soluble Epo-binding protein, and a complex thereof; A pharmaceutical composition for treating less anemia. The pharmaceutical composition of claim 19, wherein the antibody against soluble Epo-binding protein is an antibody against SEQ ID NO: 2. delete delete delete delete delete The pharmaceutical composition of claim 1, wherein the soluble Epo-binding protein (Epo-bp) is the product of a process comprising the following steps: (a) a first polypeptide segment containing an amino terminus and a carboxyl terminus and having a SEQ ID NO: 7 at the carboxyl terminus; And (b) expressing a fusion protein consisting of SEQ ID NO: 2 and consisting of a second polypeptide segment covalently linked to the carboxyl terminus of the first polypeptide segment; And Cleaving the fusion protein with thrombin. delete delete delete delete delete

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