Compositions and methods for preventing erythropoietin-associated hypertension   

This application is a divisional application of application Ser. No. 10/848,689, filed May 17, 2004 and claims priority from that application, which is also deemed incorporated by reference in its entirety in this application.

Not applicable

Erythropoietin is sold under the labels PROCRIT (epoetin), EPOGEN (epoetin), and ARANESP (darbepoetin). Erythropoietin is indicated for treatment of anemia, particularly anemia associated with chronic renal failure and cancer chemotherapy.

Erythropoietin (Epo), an angiogenic factor, increases hematocrit and hemoglobin concentrations via the stimulation of erythropoiesis, resulting in increased blood viscosity (1-5) and blood pressure (1-14). In clinical studies, approximately one-third of hemodialysis patients treated with recombinant human Epo have shown an increase in blood pressure. Epo has been postulated to increase peripheral vascular resistance and decrease cardiac output due to increased viscosity (6). Others have suggested additional mechanisms for Epo-caused hypertension, such as hypervolemia, increased plasma renin and angiotensin, along with adrenergic overactivity, increased plasma arginine vasopressin, decreased kinins and prostaglandins (15). An excessive, lasting elevation of circadian amplitude, blood pressure overswinging and an elevation of blood pressure may be based on vasomotor chronome (time structure) alteration. Hormones and other agents, in part on a genetic basis, may be contributing factors to the circadian blood pressure variation.

Hypertension is one of the most important risk factors in the development of cardiovascular complications. Hypertension is affected significantly by circadian rhythms. Hormonal concentration in the body fluctuates during the day and night with prominent spontaneous circadian (about-24-hour) changes that affect blood pressure and heart rate. There are also sufficiently important rhythms that can make the difference between the stimulation versus the inhibition of a malignancy.

Epo is a 34 kDa glycoprotein hormone that is produced by the interstitial cells in the peritubular capillary bed of the mammalian kidney and the perivenenous hepatocytes of the liver (3). Epo is secreted in response to hypoxia to coordinate erythropoiesis as a primary inducer and regulator of red cell differentiation by suppressing apoptosis and triggering cell division and terminal maturation of blood cell progenitors (16). These effects are mediated through the binding of Epo to specific cell surface receptors (17). The primary structure of human Epo has been known since the mid-1980s (18,19), but the structural features of the Epo molecule that confer its biological activity are largely unknown. Human Epo contains two disulfide bonds that link cysteine 29 with cysteine 33, and cysteine 7 with cysteine 161. Both bonds are essential for biological activity (18). Epoetin (recombinant human erythropoietin) was produced following isolation of the human gene and its expression in a Chinese hamster\'s ovarian cell line (4). The recombinant Epo is a 165-amino acid mature protein that differs from the mature native protein only in lacking the carboxyl terminus arginine of the native protein. Native human Epo is translated as a 193-amino acid peptide, from which a 27-amino-acid leader sequence is cleaved off (19,20). Recombinant Epo has an apparent molecular weight of about 30.4 kDa, appears to be immunologically equivalent to the endogenous hormone, and exhibits full biological activity (19).

Epo-treated humans and animals exhibit increased hematocrit % and increased hemoglobin via the stimulation of erythropoiesis (2-5). Some study results suggest that increased hematocrit levels are correlated with an increased blood pressure in humans (20). Other studies involving the treatment of anemia with Epo showed increased hematocrit concentrations and resulting elevated blood severe enough to require treatment with antihypertensive medication pressure in 20-30% of patients (5).

Hypertension is the most frequent and most important complication from treatment with erythropoietin. Furthermore, although the goal of Epo treatment is to increase hematocrit and hemoglobin, it has been found that the greater the increase of hematocrit, the greater the risk of mortality and cardiovascular events (PROCRIT warnings, www.rxlist.com/cgi/generic/epoetin-wcp.htm). This may be due to blood pressure rise, since the extent of blood pressure rise has been shown to correlate with the extent of hematocrit increase (20). The epoetin label warns that patients with uncontrolled hypertension should not be treated with epoetin.

New methods and compositions to prevent or treat blood pressure rise in patients treated with Epo are needed. New methods and compositions to treat anemia without causing hypertension are needed.

SUMMARY

The inventors have discovered that a soluble Epo-binding protein, which is a soluble fragment of the membrane protein Epo receptor, when administered to mammals along with Epo, prevents the blood pressure increase ordinarily caused by Epo, while not affecting the rise in hemoglobin and hematocrit that is the goal of Epo treatment. An antibody against Epo-binding protein was also found to prevent the Epo-caused blood pressure increase while not affecting the Epo-induced hematocrit rise.

Accordingly, the invention provides a method of treating anemia in a mammal involving: administering erythropoietin (Epo) to the mammal; and administering to the mammal an agent selected from a soluble Epo-binding protein (Epo-bp), a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor, and a combination thereof.

Another embodiment of the invention provides a method of reducing hypertension in a mammal receiving Epo involving administering administering to the mammal an agent selected from a soluble Epo-binding protein (Epo-bp), a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor, and a combination thereof.

Another embodiment of the invention provides use of a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor in medical therapy.

Another embodiment of the invention provides use of a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor to prepare a medicament effective to reduce erythropoietin-induced hypertension.

Another embodiment of the invention provides use of a soluble erthyropoietin-binding protein in medical therapy.

Another embodiment of the invention provides use of a soluble erythropoietin-binding protein to prepare a medicament effective to reduce erythropoietin-induced hypertension.

Another embodiment of the invention provides a pharmaceutical composition including: erythropoietin; and an agent selected from a soluble Epo-binding protein (Epo-bp), a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor, and a combination thereof.

Another embodiment of the invention provides a pharmaceutical composition including: a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor.

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the average circadian blood pressure of each of the treatment groups of rats treated with Epo and other agents. Error bars represent standard error, SEM.

FIG. 2 shows the circadian blood pressure measurements for each of the treatment groups.

FIG. 3 is a representation of the circadian hematocrit variation in the treatment groups of rats.

FIG. 4 is the outline of photographs of spleens isolated from rats treated with Epo (panels A, B, and C), or saline (panel D).

FIG. 5 is a bar graph of optical density from immunodetection of Epo, ∀Epo, Epo-bp, and ∀Epo-bp in serum and plasma of human volunteers. Error bars represent standard error, SEM.

DETAILED DESCRIPTION

“Erythropoietin” as used herein includes erythropoietin isolated from natural sources and recombinant or engineered erythropoietin that has the biological activity of erythropoietin of stimulating red blood cell production. It includes epoetin and darbepoetin. Preferably, the erythropoietin has at least 70%, more preferably at least 90%, amino acid sequence identity with human erythropoietin, SEQ ID NO:3. Sequence identity is calculated using the default BLAST parameters for nucleotide sequence comparison at the PubMed website, www.ncbi.nlm.nih.gov/PubMed/.

As used herein, “a soluble Epo-binding protein” refers to a protein that is not an antibody, is water-soluble, binds erythropoietin with high affinity, and when administered with Epo to mammals is effective at reducing an Epo-induced blood pressure rise in the mammals. Preferably, the KD of the protein for binding with erythropoietin is less than 10:M, more preferably less than 1:M, more preferably still less than 100 nM, and most preferably less than 20 nM. KD can be determined by competition binding assays such as described in Example 6 of U.S. Pat. No. 5,843,726. Preferably, the soluble Epo-binding protein is or includes sequences from or sequences homologous to the soluble portion of an Epo receptor. The human Epo receptor sequence is SEQ ID NO:1 (Winkelmann, J. C., et al., 1990, Blood 76: 24-30). The soluble portion of the human Epo receptor is residues 25-250 of SEQ ID NO:1. In a particular embodiment, the residues of the Epo-binding protein responsible for binding Epo are at least 70% identical, more preferably at least 80% identical, more preferably at least 90% identical, most preferably identical, to the corresponding residues of SEQ ID NO:1.

As used herein, “an extracellular soluble portion of an Epo receptor” refers to the portion of the Epo receptor that is exposed on the extracellular surface of the cell in the aqueous environment. Specifically, it refers to SEQ ID NO:2, which is residues 25-250 of SEQ ID NO:1 (the human Epo receptor), or to the homologous soluble residues of another Epo receptor protein.

As used herein, “a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor” refers to a protein that binds the extracellular soluble portion of Epo receptor and that, when administered to mammals along with Epo, reduces an Epo-induced blood pressure rise in the mammals. The recognition protein can be a complete antibody raised against an Epo receptor or against an Epo-binding protein, where the antibody binds the soluble portion of Epo receptor, or a binding fragment of such a complete antibody. The recognition protein can also be a non-antibody protein or peptide (e.g., a protein or peptide selected by phage display binding) that binds to the extracellular soluble portion of the human Epo receptor or of another mammalian Epo receptor with a binding affinity of at least 105 liters per mole, more preferably 106, more preferably at least 107, most preferably at least 108 liters per mole.

As used herein, the term “antibody” includes complete antibodies and antigen-binding fragments of complete antibodies, e.g., Fab or F(ab′)2 antibodies. The term “antibody” also includes both monoclonal and polyclonal antibodies (e.g., antiserum).

The term “reducing hypertension” by administering an agent includes preventing or reducing an increase in blood pressure that otherwise occurs in a significant fraction of a population when the agent is not administered.

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

In some embodiments of the invention, the agent is a soluble Epo-bp.

In some embodiments, the soluble Epo-bp contains a fragment of a soluble portion of a mammalian Epo receptor.

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

In particular embodiments, the soluble Epo-bp includes or is SEQ ID NO:2. The soluble Epo-bp that is SEQ ID NO:2 can be expressed as described in U.S. Pat. No. 5,843,726. In general terms, SEQ ID NO:2 is expressed as a fusion protein with 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 with thrombin to release SEQ ID NO:2. The Epo-bp of SEQ ID NO:2 is found naturally in human serum and plasma, possibly produced as a cleavage product of Epo receptor (see Example 2 below).

In particular embodiments, the soluble Epo-bp has at least 70%, at least 80%, or at least 90% amino acid sequence identity to SEQ ID NO:2, as calculated using the default BLAST parameters for nucleotide sequence comparison at the PubMed website, www.ncbi.nlm.nih.gov/PubMed/.

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

In one embodiment of the invention, the soluble Epo-bp is a product of a process comprising: expressing a fusion protein and cleaving it with thrombin. The fusion protein consists essentially of a first polypeptide segment having a thrombin proteolytic cleavage site at its carboxyl terminus, and a second polypeptide segment consisting essentially of SEQ ID NO:2. The amino terminus of the second segment is covalently coupled 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 product of a process comprising: expressing a fusion protein comprising SEQ ID NO:2 linked at its amino terminus to a peptide sequence of Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:7), and cleaving the fusion protein with thrombin.

In one embodiment of the invention, the soluble Epo-bp is a product of a process comprising: expressing a fusion protein consisting of: (a) a first polypeptide segment having an amino terminus and a carboxyl terminus, said segment having SEQ ID NO:7 at its carboxyl terminus; and (b) a second polypeptide segment consisting of SEQ ID NO:2, the second polypeptide segment covalently coupled to the carboxyl terminus of the first polypeptide segment; and cleaving the fusion protein with thrombin.

In other particular embodiments of the methods of the invention, the agent is a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor.

The recognition protein may exert its effect of reducing the Epo-induced blood pressure increase by binding to the extracellular soluble portion of intact Epo receptor molecules in membranes, or it may exert its effect by binding to the soluble Epo-binding protein that exists naturally circulating in blood (which has the same amino acid sequence as the extracellular soluble portion of the Epo receptor, and may be a proteolytic product of the receptor), or by both of these mechanisms or other unknown mechanisms. Describing this embodiment of the agent as “a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor” is intended to describe a characteristic of the recognition protein, and not to necessarily describe the mechanism of action of the recognition protein.

In a particular embodiment, the recognition protein binds SEQ ID NO:2. That is, the recognition protein recognizes and binds to some sequence within SEQ ID NO:2. The recognition protein could, for instance, be an antibody raised against SEQ ID NO:2, an antibody raised against the Epo receptor where the antibody binds to SEQ ID NO:2, or an antibody raised against a peptide fragment of SEQ ID NO:2.

In particular embodiments, the recognition protein is an antibody. In particular embodiments, the antibody is a complete antibody. In particular embodiments, the antibody is an antibody fragment. For instance, the antibody fragment may be an Fab, Fab′, or F(ab′)2, or Fv.

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

In other particular embodiments, the recognition protein is a non-antibody protein or peptide. For instance, it can be a recognition peptide or protein selected by phage display. Methods for selection of binding peptides using phage display are disclosed in Sidhu S S, Lowman H B, Cunningham B C, and Wells J A: Phage display for selection of novel binding peptides. Methods in Enzymology 2000; 328:333-363.

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

Epo and the agent may be administered separately or together.

In particular embodiments, the amount of the agent administered is at least equimolar with the amount of Epo administered.

In particular embodiments, the amount of the agent administered is about equimolar with the amount of Epo administered. For instance, the moles of the agent administered may be between 75% and 125% of the mole of Epo administered.

In particular embodiments of the method of treating anemia, the agent reduces an erythropoietin-induced blood pressure rise in the mammal. That is, the blood pressure of the mammal rises less when the mammal receives Epo and the agent, than when the mammal receives Epo alone.

Preferably, when Epo is administered to a mammal with an equimolar amount of the agent, blood pressure increases no more than 75% as much as it rises when Epo is administered alone to the mammal, more preferably no more than 50% as much as it increases when Epo is administered alone to the mammal.

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

Another pharmaceutical composition of the invention includes erythropoietin; and an agent selected from a soluble Epo-binding protein, a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor, and a combination thereof.

Another pharmaceutical composition of the invention includes a soluble Epo-binding protein.

Typically, the pharmaceutical compositions include a pharmaceutically acceptable diluent or carrier.

In one embodiment of the pharmaceutical compositions containing the recognition protein, the recognition protein is an antibody against SEQ ID NO:2.

In one embodiment of the pharmaceutical compositions containing the soluble Epo-binding protein, the Epo-binding protein is SEQ ID NO:2.

Other particular embodiments of the agent, the soluble Epo-binding protein, and the recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor are as described for the methods of the invention.

To generate antibodies, Epo receptor or Epo-bp can be administered directly to a mammal, or the proteins or peptide fragments thereof can be coupled to a carrier protein. Suitable carrier proteins include keyhole limpet hemocyanin, bovine serum albumin, and ovalbumin. Methods of coupling to the carrier protein include single step glutaraldehyde coupling and other methods disclosed in Harlow, Ed et al., Antibodies: a laboratory manual, Cold Spring Harbor Laboratory (1988).

The immunogen is used to immunize a vertebrate animal in order to induce the vertebrate to generate antibodies. Preferably the immunogen is injected along with an adjuvant such as Freund\'s adjuvant, to enhance the immune response. Suitable vertebrates include rabbits, mice, rats, hamsters, goats, sheep, and chickens.

Hybridomas to synthesize monoclonal antibodies can be prepared by methods known in the art. See, for instance, Wang, H., et al., Antibody Expression and Engineering, Am. Chem. Soc., Washington, D.C. (1995). Polyclonal and monoclonal antibodies can be isolated by methods known in the art. See, for instance, id. and Harlow et al.

Native antibodies are tetramers of two identical light (L) chains and two identical heavy (H) chains. The L and H chains each have variable domains that are responsible for antigen recognition and binding. The variability in the variable domains is concentrated in the complementarity determining regions (CDRs).

An antibody that is contemplated for use in the present invention can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody that includes the CDR, and like forms, all of which fall under the broad term “antibody” as used herein.

The term “antibody fragment” refers to an antigen-binding portion of a full-length antibody. Antibody fragments can be as small as about 4 amino acids, about 10 amino acids, or about 30 amino acids or more. Some types of antibody fragments are the following:

(1) Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. A Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain. Two Fab fragments are obtained per whole antibody molecule.

(2) Fab′ is the fragment of an antibody that can be obtained by treating whole antibody with pepsin, followed by reduction to yield an intact light chain and a portion of the heavy chain. Two Fab′ fragments are obtained per whole antibody molecule. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines.

(3) F(ab′)2 is the fragment that can be obtained by digestion of whole antibody with pepsin, without reduction. F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. Fv consists of a dimer of one H and one L chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single vaiable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to bind antigen, although at a lower affinity than the complete binding site.

(5) A single chain antibody (SCA) is defined as a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule.

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

Methods of in vitro and in vivo manipulation of monoclonal antibodies are well known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495 (1975), or may be made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clarkson et al., Nature 352:624 (1991), as well as in Marks et al., J. Mol. Biol. 222:581 (1991). Another method involves humanizing a monoclonal antibody by recombinant means to generate antibodies containing human specific and recognizable sequences. See, for review, Holmes et al., J. Immunol. 158:2192 (1997) and Vaswani et al., Annals Allergy, Asthma & Immunol. 81:105 (1998).

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. 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 (see, for example, Harlow and Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)). Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5 S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, in U.S. Pat. Nos. 4,036,945, and 4,331,647, and references contained therein.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of VH and VL chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker bridging the two V domains. Methods for producing sFvs 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., U.S. Pat. No. 4,946,778; and Pack et al., Bio/Technology 11:1271 (1993).

Another form of an antibody fragment is a peptide containing a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) are often involved in antigen recognition and binding. CDR peptides can be obtained by cloning or constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., Methods: a Companion to Methods in Enzymology, 2:106 (1991).

The invention contemplates human and humanized forms of non-human (e.g., murine) antibodies. Such humanized antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, goat, sheep, or rabbit having the desired specificity, affinity, and capacity.

In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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 their affinity, selectivity, binding strength or other desirable property. One method of mutating antibodies involves affinity maturation using phage display. Affinity maturation using phage display refers to a process described in Lowman et al., Biochemistry 30:10832 (1991); see also Hawkins et al., J. Mol. Biol. 254:889 (1992).

The invention will now be illustrated by the following non-limiting examples.

The Example describes the preparation of an Epo-bp thought to have SEQ ID NO:2, as is also described in U.S. Pat. No. 5,843,726.

Construction of EpoR recombinant vector. A recombinant plasmid expression vector, pJYL26, was constructed from a PCR product having the human Epo receptor extracellular soluble domain coding sequence and from the plasmid vector pGEX-2T, which was purchased from Pharmacia.

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

The PCR product and pGEX-2T were digested with EcoR1 and BamH1, the digested DNAs were purified with gel electrophoresis. The ligation was done with a mixture containing 1 :g/:l each of the PCR product and pGEX-2T. The ligated product was verified to be ˜5.5 kb. The ligated plasmid mixture was used to transform E. coli JM109. Colonies were grown. DNA was extracted from each transformed colony, and analyzed. Plasmid from one colony was selected after examining both EcoR1 and EcoR1 plus BamH1 digested DNA sizes in 1% agarose gels to confirm the predicted sizes. The procedures were carried out as described in U.S. Pat. No. 5,843,726.

Purification of EpoRex-th fusion protein. Transformed E. coli containing the recombinant vector pJYL26 was grown and induced with IPTG. Cell extract was passed through a GSH-agarose column. The bound EpoRex-th was eluted with reduced glutathione. This was performed as described in U.S. Pat. No. 5,843,726.

Purification of Epo-bp. EpoRex-th contains a thrombin-specific proteolytic cleavage recognition sequence separating 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 by affinity binding to an Epo-agarose column. This was performed as described in U.S. Pat. No. 5,843,726. The amino terminus of the Epo-bp produced by this procedure, and thus the thrombin cleavage site, was not experimentally determined. The thrombin cleavage is believed to produce Epo-bp of SEQ ID NO:2. But, thrombin may cleave at other sites, such as between the Arg and the Gly of the recognition sequence to produce a protein having the Gly-Ser peptide attached to the amino terminus of SEQ ID NO:2 (SEQ ID NO:8).

This Example describes experiments testing the effect of administering Epo-bp, which is a soluble Epo-binding protein having the amino acid sequence of SEQ ID NO:2, and an Fab antibody against Epo-bp, either with or without Epo, to rats.

Glutathione (GSH)-agarose, pGEX-2T expression vector and SEPHADEX G-50 were purchased from Pharmacia (Mechanicsburg, Pa.). PCR reagents were from Perkin-Elmer Cetus (Norwalk, Conn.) and AFFIGEL® 15 was from BioRad (Richmond, Calif.). Bacteriophage T4 DNA ligase, restriction enzymes and isopropylthio-β-D-galactoside (IPTG) were purchased from BRL Gibco (Gaithersburg, Md.). GENECLEAN II was from Bio 101, La Jolla, Calif. Nitrocellulose was from Schleicher & Schuell Co. (Keene, N.H.). Chemiluminescence (ECL) reagents and 125I-Epo were from Amersham (Arlington Heights, Ill.) and unlabeled Epo was a gift of Chugai-Upjohn (Rosemont, Ill.). Thrombin, trypsin, phenylmethylsulfonylfluoride (PMSF), diisopropylfluorophosphate (DFP), TRITON X-100, 2,7-Dichlorofluoresein, biotin-amidocaproyl hydroazide, alkaline phosphatase conjugate, disodium p-nitrophenyl phosphate, and o-phenylenediaminedihydrochloride (oPD) were from Sigma (St. Louis, Mo.). Biotinylated rabbit anti-sheep antibodies, Avidin-horse radish peroxidase, and IgG purification Kit were from Pierce Co. (Rockford, Ill.). Streptavidin peroxidase was purchased from Boehringer Manheim Corp (Indianapolis, Ind.), and microplates were from Corning Costa (Cambridge, Mass.). All other chemicals were of 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 U.S. Pat. No. 5,843,726. Epo-bp had the amino acid sequence of SEQ ID NO:2. A full-length human EpoR cDNA (SEQ ID NO:4) was from Dr. Bernard G. Forget, Yale University. Oligonucleotides were synthesized by the microchemical facility of the Institute of Human Genetics, University of Minnesota, Minneapolis. All other chemicals were of reagent grade.

Male Sprague-Dawley (SD) rats were housed at the University Animal Care facility with Purina Chow and drinking water freely accessible. We examined any circadian stage-dependence of Epo effects on the blood pressure, hematocrit, body weight and spleen weight of the rats kept in an alternating light-darkness cycle from 04:00 to 18:00 for light. To seek an effective treatment time, 5-week-old rats were. assigned to control or treatment groups, each group consisting of 6 subgroups, each of 5 rats, in 6 test times at 00, 04, 08, 12, 16 and 20 hours. The rats were randomly distributed into groups such that the baseline inter-group differences in body weight, blood pressure and hematocrit of Epo Rx versus saline and other Rx groups were not statistically significant.

Blood pressure, hematocrit, and body weight were measured just before and immediately after the completion of a 4-week course of twice-weekly Epo (50 U/kg BW) or physiological saline subcutaneous injections. Epo, Epo-bp, and αEpo-bp dosage was determined based on Epoetin study reports (4). The Epo dose was 50 units per kg body weight, and an Epo-bp and αEpo-bp were administered in an amount equimolar with Epo. The erythropoietin (Epoetin) was from Amgen Company (Thousand Oaks, Calif.). Affinity purified Epo-bp and αEpo-bp were prepared in our laboratories. The antibodies were digested to Fab fragments, and the Fab antibodies were purified.

For blood pressure measurement, the femoral artery was cannulated under pentobarbital (50 mg/kg) anesthesia. At the end of the study, spleens were weighed and photographed. The weights of the brain, heart, aorta, and L- and R-kidneys were also obtained.

We developed αEpo-bp in sheep innoculated with Epo-bp every 3-4 week for 3 months. After collecting serum, the antibodies were purified and digested to generate Fab antibodies. The Fab were purified. Fab were fluorescein labeled according to the manufacturer\'s description. These materials were used to detect Epo receptor in blood and/or tissue samples. Negative control cells had no antibodies added and positive control cells had Fab from IgG of preimmune serum. To test for antibody binding sites (Epo receptor) bone marrow cells were washed in PBS and dispensed at 1-3×103 cells per well in round-bottomed tubes and centrifuged into a pellet at 500 g for 2 minutes. Supernatant was removed and 100 μl of fluorescein-conjugated Fab antibodies were added. After mixing well, the mixture was incubated on ice for 30 min. The cells were washed three times by adding 400 μl of buffer containing 1% FCS and 0.01% NaN3 in PBS and centrifuged at 500 g for 2 minutes to remove supernatant. The cells were resuspended in a total volume of up to 50 μl of PBS and analyzed under an inverted fluorescence microscope.

Enzyme immunoassay (EIA) was used to detect and measure the levels of Epo, Epo-bp, and antibodies against Epo, and Epo-bp in healthy untreated human subjects. EIA microplates were coated with 2 μg/well of anti-Epo to detect Epo and 2 μg/well of anti-Epo-bp to detect Epo-bp. To detect circulating anti-Epo and anti-Epo-bp antibodies, wells were coated with 200 μl of 1:10 diluted serum or plasma in PBS, pH 7.4. Plates were incubated at room temperature for 30 minutes or at 4° C. overnight. After coating the plates with antibody or serum, wells were washed 3 times with 200 μl/well PBST (0.05% TWEEN 20 in PBS). Nonspecific binding sites were blocked with 200 μl/well 1% BSA in PBST for 30 min at room temperature. Wells were washed 3 times with 200 μl/well PBST. To detect bound antigen, peroxidase-streptavidin label was attached to Fab anti-Epo (for detecting Epo), Epo (for detecting anti-Epo antibodies), Fab anti-Epo-bp (for detecting Epo-bp), and Epo-bp (for detecting anti-Epo-bp) in our laboratory. Two micrograms of the appropriate peroxidase-streptavidin-labeled protein in 200 μl PBST was added per well. The wells were washed 3 times with 200 μl PBST. A solution (160 μl) of o-phenylenediaminedihydrochloride (OPD) in citrate buffer was added to each well. (The solution contained 10 mg/ml in 24 mM citrate, 51 mM Na2HPO4, pH 6.0, with 0.4 ml of 3% H2O2 added to 100 ml of solution immediately before use.) The reaction was stopped by adding 40 μl of 5M NaOH, and the absorbance was measured at 405 nm.

Data were analyzed by two-tailed Student\'s t test, the cosinor method and the linear least square rhythmometry (21), allowing variation as a function of the data. Data are expressed as mean±SEM. A p value of less than 0.05 was considered significant.

In Table 1, before treatment, the inter-group differences for blood pressure, hematocrit, and body weight in all treatment groups were not statistically significant. Overall, body weight was lowered by Epo compared to control (295 vs. 313 grams, p<0.01). The reference circadian blood pressure differences in Epo treatment versus control, Epo-bp, and αEpo-bp (Fab antibody against Epo-bp) treatment groups before treatment were not statistically significant (87±2.8 vs. 88.8±3.4, 88.7±2.5, 84.3±2.3 mm Hg). After treatment, the circadian blood pressure was significantly increased in the Epo treated group. The group comparisons between Epo treatment versus 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 mm Hg, respectively, each p<0.0001. When Epo-bp or αEpo-bp was given along with Epo, however, blood pressure was maintained at similar levels to the saline control group: 118.3±1.7 in the Epo-bp+Epo group and 121.0±2.0 mm Hg in the αEpo-bp+Epo treatment group, which were significantly lower than that of the Epo treat group (136.2±2.3), each p<0.0001.

TABLE 1 Overall Effects upon Circadian Body Weight, Blood Pressure, Hematocrit and Other Organ Systems in Various Treatments. Group (Rx) y before Rx (all group n = 30) BW (g) BP (mm Hg) Hct (%) Control (Saline) vs. 80.1 ± 1.7 88.8 ± 3.4 36.2 ± 0.7 Epo 80.2 ± 1.4 87.1 ± 2.8 37.0 ± 0.6 Epo-bp 81.6 ± 1.5 88.7 ± 2.5 36.5 ± 0.7 αEpo-bp 81.2 ± 1.3 84.3 ± 2.3 36.1 ± 0.4 Epo + Epo-bp 81.0 ± 1.0 84.3 ± 3.4 36.3 ± 0.6 Epo + αEpo-bp 79.4 ± 1.5 88.9 ± 2.6 37.1 ± 0.4 y after Rx

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