U.S. patent application number 15/361453 was filed with the patent office on 2017-06-22 for compositions and methods of preventing erythropoietin associated hypertension.
The applicant listed for this patent is John S Lee, Jong Y Lee, Mary S Lee. Invention is credited to John S Lee, Jong Y Lee, Mary S Lee.
Application Number | 20170173150 15/361453 |
Document ID | / |
Family ID | 35309674 |
Filed Date | 2017-06-22 |
United States Patent
Application |
20170173150 |
Kind Code |
A1 |
Lee; Jong Y ; et
al. |
June 22, 2017 |
Compositions and Methods of Preventing Erythropoietin Associated
Hypertension
Abstract
The inventors have discovered that both soluble
erythropoietin-binding protein and antibodies against the
erythropoietin-binding protein, when they are administered to a
mammal along with erythropoietin (Epo), prevent or reduce the blood
pressure increase normally caused by erythropoietin, while not
affecting the hematocrit increase that is the purpose of Epo
treatment. 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. The invention also provides a method of
reducing hypertension in a mammal receiving Epo, and pharmaceutical
compositions containing a soluble Epo-bp and/or a recognition
protein that binds Epo receptor on an extracellular soluble portion
of the Epo receptor.
Inventors: |
Lee; Jong Y; (Minneapolis,
MN) ; Lee; Mary S; (Northbrook, IL) ; Lee;
John S; (Northbrook, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lee; Jong Y
Lee; Mary S
Lee; John S |
Minneapolis
Northbrook
Northbrook |
MN
IL
IL |
US
US
US |
|
|
Family ID: |
35309674 |
Appl. No.: |
15/361453 |
Filed: |
November 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14042702 |
Sep 30, 2013 |
9539308 |
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15361453 |
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13044818 |
Mar 10, 2011 |
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14042702 |
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10848689 |
May 17, 2004 |
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13044818 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 38/179 20130101; A61K 2039/505 20130101; A61K 38/1816
20130101; C07K 2317/55 20130101; C07K 16/2863 20130101; A61K
39/3955 20130101; A61P 9/12 20180101; A61K 2121/00 20130101; A61K
39/39533 20130101; A61P 7/06 20180101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/18 20060101 A61K038/18 |
Claims
1. A method of treating anemia in a patient, comprising:
administering to the patient both erythropoietin (Epo) and an
antibody that binds SEQ ID NO:2.
2. The method of claim 1 wherein the antibody is an antibody
fragment.
3. The method of claim 2 wherein the antibody fragment is Fab.
4. The method of claim 1 wherein the Epo and the antibody are
administered together.
5. The method of claim 1 wherein the Epo and the antibody are
administered separately.
6. The method of claim 1 wherein the amount of antibody
administered is at least equimolar with the amount of the Epo
administered.
Description
[0001] This nonprovisional patent application claims priority from
and is a copending continuation of U.S. application Ser. No.
14/042,702, filed Sep. 30, 2013, which was a copending continuation
of U.S. application Ser. No. 13/044,818 filed Mar. 10, 2011, which
was a copending continuation of U.S. application Ser. No.
10/848,689, filed May 17, 2004, which applications are incorporated
herein by reference thereto.
[0002] The research and development of the invention described
herein did not involve any federally sponsored funding.
BACKGROUND OF THE INVENTION
[0003] 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 over activity, increased plasma
arginine vasopressin, decreased kinins and prostaglandins (15). An
excessive, lasting elevation of circadian amplitude, blood pressure
over-swinging 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).
[0004] 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).
[0005] 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 and well
understood that the greater the increase of hematocrit, the greater
the risk of mortality and cardiovascular events. 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 OF THE INVENTION
[0006] 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 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
erythropoietin-binding protein in medical therapy.
[0007] 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
[0008] 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.
[0009] FIG. 2 shows the circadian blood pressure measurements for
each of the treatment groups.
[0010] FIG. 3 is a representation of the circadian hematocrit
variation in the treatment groups of rats.
[0011] FIG. 4 is the outline of photographs of spleens isolated
from rats treated with Epo (panels A, B, and C), or saline (panel
D).
[0012] FIG. 5 is a bar graph of optical density from
immunodetection of Epo, VEpo, Epo-bp, and VEpo-bp in serum and
plasma of human volunteers. Error bars represent standard error,
SEM.
DETAILED DESCRIPTION
Definitions
[0013] "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/.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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).
[0018] 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.
[0019] Description:
[0020] 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.
[0021] 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.
[0022] 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.
[0023] The Epo-bp of SEQ1O 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, vvwvv.ncbi.nlm.nih.gov/PubMed/.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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 SS, 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.
[0030] 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.
[0031] Epo and the agent may be administered separately or
together.
[0032] In particular embodiments, the amount of the agent
administered 1s 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.
[0033] 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.
[0034] 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.
[0035] Raising Antibodies:
[0036] 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.
[0037] 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, DC (1995). Polyclonal and monoclonal antibodies can be
isolated by methods known in the art. See, for instance, id. and
Harlow et al.
[0038] 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.
[0039] 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:
[0040] 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.
[0041] 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 CHI domain
including one or more cysteines.
[0042] 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.
[0043] 4. Fv is the mmlmum 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 variable 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.
[0044] 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.
[0045] 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. Mot 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 Allerg y, Asthma & Immunol. 81:1050
(1998).
[0046] 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)).
[0047] 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
Pc 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).
[0048] Another form of an antibody fragment 1s 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).
[0049] 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.
[0050] 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 (Fe), 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., I Immunol. 158:2192 (1997); and
Vaswani et al., Annals Allergy, Asthma & Immunol 81:105
(1998).
[0051] 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., 1 Mol Biol.
254:889 (1992).
[0052] The invention will now be illustrated by the following
non-limiting examples.
EXAMPLE 1
[0053] 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
frill-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 BamHl 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 EcoR
1 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.
[0054] The PCR product and pGEX-2T were digested with EcoR 1 and
BamHl, the digested DNAs were purified with gel electrophoresis.
The ligation was done with a mixture containing 1:g/:1 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 EcoR 1 and EcoR 1 plus BamHl 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-QP.
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).
EXAMPLE 2
[0055] This Example describes experiments testing the effect of
administering Epobp, 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.
EXPERIMENTAL PROCEDURES
[0056] Materials: 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.RTM. was from BioRad (Richmond,
Calif.). Bacteriophage T4 DNA ligase, restriction enzymes and
isopropylthio-13-Dgalactoside (IPTG) were purchased from BRL Gibco
(Gaithersburg, Md.). GENECLEAN ||was from Bio 101, La Jolla,
Calif.. Nitrocellulose was from Schleicher & Schuell Co.
(Keene, N. H.). Chemiluminescence (ECL) reagents and 1251-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 Coming Costa (Cambridge, Mass.). All 5 other chemicals
were of reagent grade.
[0057] Epo-bp, Fab antibody against Epo-bp (aEpo-bp), and Fab
antibody against Epo (aEpo) 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, Minn. All
other chemicals were of reagent grade.
[0058] Animal Study:
[0059] 15 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 aEpo-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 aEpo-bp were
administered in an amount equimolar with Epo. The erythropoietin
(Epoetin) was from Amgen Company (Thousand Oaks, Calif.). Affinity
purified Epo-bp and aEpo-bp were prepared in our laboratories. The
antibodies were digested to Fab fragments, and the Fab antibodies
were purified.
[0060] 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.
[0061] Ligand Binding Site in Progenitor Cells and Detection of Epo
and EpoR:
[0062] We developed ocEpo-bp in sheep inoculated 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.times.103
cells per well in round-bottomed tubes and centrifuged into a
pellet at 500 g for 2 minutes. Supernatant was removed and 100 [1,1
of fluorescein-conjugated Fab antibodies were added. After mixing
well, the mixture was incubated on ice for 30 mM. The cells were
washed three times by adding 400 mill of buffer containing 1% PCS
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 1.tl 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 pg/well of anti-Epo to detect Epo and 21.1 g/well of
anti-Epo-bp to detect Epo-bp. To detect circulating anti-Epo and
anti-Epobp antibodies, wells were coated with 2001.11 of 1:10
diluted serum or plasma in PBS, pH 7.4. Plates were incubated at
room temperature for 30 minutes or at 4.degree. C. overnight. After
coating the plates with antibody or serum, wells were washed 3
times with 200 .tl/well PBST (0.05% TWEEN 20 in PBS). Nonspecific
binding sites were blocked with 200 p 1/well 1% BSA in PBST for 30
mM at room temperature. Wells were washed 3 times with 200 .tl/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
Epobp (for detecting anti-Epo-bp) in our laboratory. Two micrograms
of the appropriate
[0063] 5 peroxidase-streptavidin-labeled protein in 200 ill PBST
was added per well. The wells were washed 3 times with 200 piPBST.
A solution (160 LID 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%
1-1202 added to 100 ml of solution immediately before use.) The
reaction was stopped by adding 40 [Ll of 5M NaOH, and the
absorbance was measured at 405 nm.
[0064] Statistics:
[0065] 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.
RESULTS
[0066] 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 aEpo-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
ocEpo-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 aEpo-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 aEpo-bp +Epo treatment group, which were
significantly lower than that of the Epo treat group (136.2
.+-.2.3), each p <0.0001.
TABLE-US-00001 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. SO.I .+-. 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 aEpo-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 + a.Epo-bp 79.4 .+-. 1.5 88.9 .+-. 2.6 37.1 .+-.
0.4 y after Rx BW (g) BP (mm Hg) Hct (%) SW (g) Brain W (g) Control
(Saline) vs. 312.8 .+-. 4.9 116.2 .+-. 1.7 42.7 .+-. 0.8 0.86 .+-.
0.03 1.82 .+-. 0.01 Epo 294.9 .+-. 4.2* 136.2 .+-. 2.3*** 61.6 .+-.
1.3*** 1.58 .+-. 0.07*** 1.77 .+-. 0.02* Epo-bp 312.1 .+-. 3.9
118.4 .+-. 2.1 43.9 .+-. 0.6 0.89 .+-. 0.02 1.80 .+-. 0.02 aEpo-bp
305.0 .+-. 4.9 116.6 .+-. 2.1 44.1 .+-. 0.7 0.85 .+-. 0.02 I.SO
.+-. 0.01 Epo + Epo-bp 303.4 .+-. 3.6 118.3 .+-. 1.7 58.0 .+-.
1.1*** 1.62 .+-. 0.05*** 1.77 .+-. 0.02* Epo + a.Epo-bp 298.4 .+-.
4.4 121.0 .+-. 2.0 59.1 .+-. 1.1*** 1.79 .+-. 0.07*** 1.76 .+-.
0.01** Epo vs. 294.9 .+-. 4.2 136.2 .+-. 2.3 61.6 .+-. 1.3 1.58
.+-. 0.07 1.77 .+-. 0.02 Epo-bp 312.1 .+-. 3.9* 118.4 .+-. 2.1***
43.9 .+-. 0.6*** 0.89 .+-. 0.02*** 1.80 .+-. 0.02 aEpo-bp 305.0
.+-. 4.9 116.6 .+-. 2.1*** 44.1 .+-. 0.7*** 0.85 .+-. 0.02*** 1.80
.+-. 0.01 Epo + Epobp 303.4 .+-. 3.6 118.3 .+-. 1.7*** 58.0 .+-.
1.1, t 1.62 .+-. 0.05 1.77 .+-. 0.02 Epo + a.Epo-bp 298.4 .+-. 4.4
121.0 .+-. 2.0*** 59.1 .+-. 1.1 1.79 .+-. 0.07 1.76 .+-. 0.01 y
after Rx Heart W (g) Aorta W (g) R-Kidney W (g) L-Kidney W (g)
Control (Saline) vs. 1.03 .+-. 0.02 0.046 .+-. 0.002 1.107 .+-.
0.02 1.092 .+-. 0.02 Epo 0.93 .+-. 0.02** 0.046 .+-. 0.002 1.076
.+-. 0.02 1.084 .+-. 0.03 Epo-bp 1.03 .+-. 0.02 0.046 .+-. 0.002
1.106 .+-. 0.02 1.083 .+-. 0.03 aE-Epo-bp 1.04 .+-. 0.02 0.044 .+-.
0.002 1.112 .+-. 0.02 1.099 .+-. 0.02 Epo + Epo-bp 0.96 .+-. 0.02*
0.046 .+-. 0.002 1.098 .+-. 0.02 1.073 .+-. 0.02 Epo + aEpo-bp 0.99
.+-. 0.02 -0.046 .+-. 0.002 1.084 .+-. 0.02 1.044 .+-. 0.02 Epo vs.
0.93 .+-. 0.02 Epo-bp 1.03 .+-. 0.02** o:E-Epo-bp 1.04 .+-. 0.02**
Epo + Epo-bp 0.96 .+-. 0.02 Epo + aEpo-bp 0.99 .+-. 0.02* Rx:
treatment; n = number of rats (30 rats in each group); y: 24-h
average; a: Epo-bp = anti-Epo-bp antibody; *p < 0.01; **p <
0.001; ***p < 0.0001; tp < 0.05 g = gram; BW = body weight;
BP = blood pressure; HCT = hematocrit; SW = spleen weight; W =
weight; R = right; L = left
[0067] Epo treatment increased hematocrit markedly overall as
compared to the saline, Epobp or aEpo-bp groups (61.6 vs. 42.7,
43.9 and 44.1%, respectively) and at each of the 6 test times, all
p <0.0001. Administering Epo-bp or aEpo-bp together with Epo had
almost no effect on the Epo-induced hematocrit increase (61.6%
hematocrit in Epo vs. 58.0% in Epo +Epo-bp and 59.1% in Epo
+aEpo-bp Rx). But, significantly, both Epo-bp and aEpo-bp almost
eliminated the Epo-induced blood pressure rise (136.2 mm Hg in the
Epo-treated group, vs. 116.2 in saline control, 118.3 for
Epo+Epo-bp, and 121.0 in Epo+aEpo-bp). Thus, both Epo-bp and
aEpo-bp protected the rats from the blood pressure rise caused by
Epo treatment.
[0068] Splenomegaly characterized each rat in the Epo-treated group
(spleen weight overall 1.58 vs. 0.86 for saline, 0.89 for Epo-bp,
and 0.85 grams for aEpo-bp, each p<0.0001). Administering Epo-bp
or aEpo-bp together with Epo did not affect the splenomegaly. Brain
and heart weights were significantly lower in the Epo Rx group as
compared to all other groups, although the aorta and kidney weights
were similar in each group.
[0069] FIG. 1 shows circadian blood pressures in all group
comparisons in bar graphs.+-.standard errors (SEM). The Epo-treated
group had a significantly increased blood pressure as compared to
all other 5 groups, each p<0.0001. FIG. 2 shows circadian
fluctuations of blood pressure in MESOR (about 24-h mean),
amplitude and acrophase (peak time) in each treatment group. Epo
treatment increased circadian blood pressure (MESOR) significantly
as compared to all other groups (all p <0.0001), although all
group amplitude comparisons were not significantly different. After
treatment, the peak time in the Epo-treated rats was shifted to the
daytime as compared to control, Epo-bp or ocEpo-bp treatment groups
(19:40 vs. 04:08, 05:44, 05: 16, respectively). It is an obvious
shift change from the night to the daytime peak with Epo treatment
in this nocturnal animal. When Epo-bp or ocEpo-bp was given
together with Epo, the shift change still remained in the same
daytime range as in the Epo-alone treatment group (14:48, 19:20,
respectively), although the Epo-bp+Epo and aEpo-bp+Epo groups'
blood pressure levels were similar to the control group.
[0070] Table 2 summarizes the circadian variations of body weight,
blood pressure, hematocrit and spleen weight in the 6 subgroups
after Epo, Epo-bp and aEpo-bp treatments. The body weight
difference between Epo-treated rats and any other treatment group
was not statistically significant among the 6 test times. A
significantly increased blood pressure in the Epo treated group was
detected at 12, 16, 20 and 00 hours, but not at 4.0 or 8.0 hours as
compared to control, Epo-bp and aEpo bp Rx groups. Epo treatment
increased hematocrit markedly overall and at each of the 6 test
times as compared to control, Epo-bp and aEpo-bp Rx groups, all
p<0.0001. The spleen weights were significantly higher in the
Epo-treated group rats than those of the control, Epo-bp and
aEpo-bp groups at all time points, although the body weight was
lower at each time comparison.
TABLE-US-00002 TABLE 2 Circadian Variations of Body Weight, Blood
Pressure, Hematocrit and Spleen Weight in Various Treatments Group
(Rx) 0400 0800 1200 1600 2000 0000 BW (gram): Saline vs. 313 .+-.
12 305 .+-. 09 324 .+-. 18 308 .+-. 13 310 .+-. 10 317 .+-. 13 Epo
305 .+-. 13 294 .+-. 07 294 .+-. 05 290 .+-. 05 295 .+-. 14 291
.+-. 14 Epo-bp 314 .+-. 11 310 .+-. 06 303 .+-. 04 312 .+-. 10 319
.+-. 11 319 .+-. 13 aEpo-bp 314 .+-. 10 297 .+-. 20 308 .+-. 13 299
.+-. 06 293 .+-. 05 320 .+-. 12 Epo + Epo-bp 297 .+-. 10 300 .+-.
04 301 .+-. 09 308 .+-. 11 301 .+-. 11 313 .+-. 09 Epo + a.Epo-bp
296 .+-. 13 286 .+-. 06 279 .+-. 04* 304 .+-. 12 305 .+-. 05 320
.+-. 13 BP (mm Hg): Saline vs. 116 .+-. 5.8 120 .+-. 4.6 117 .+-.
3.7 108 .+-. 1.0 119 .+-. 3.2 118 .+-. 4.1 Epo 131 .+-. 7.6 131
.+-. 4.8 139 .+-. 3.9''' 128 .+-. 8.1 t 140 .+-. 6.3* 137 .+-. 6.2t
Epo-bp 118 .+-. 4.5 122 .+-. 5.5 118 .+-. 3.6 115 .+-. 4.0 118 .+-.
6.1 120 .+-. 7.7 a.Epo-bp 113 .+-. 5.7 122 .+-. 5.1 117 .+-. 4.7
112 .+-. 3.4 113 .+-. 4.6 122 .+-. 6.8 Epo + Epo-bp I14 .+-. 2.2
121 .+-. 3.1 118 .+-. 6.9 121 .+-. 4.5t 119 .+-. 4.0 117 .+-. 3.8
.Epo + o:Epo-bp 116 .+-. 6.7 121 .+-. 4.1 120 .+-. 6.2 120 .+-.
5.0t 127 .+-. 5.6 122 .+-. 1.4 Epo vs. 118 .+-. 4.5 122 .+-. 5.5
118 .+-. 3.6''' 115 .+-. 4.0t 118 .+-. 6.1 t 120 .+-. 7.7 Epo-bp
aEpo-bp 113 .+-. 5.7 122 .+-. 5.1 117 .+-. 4.7* I12 .+-. 3.4t 113
.+-. 4.6* 122 .+-. 6.8 Epo + Epo-bp 114 .+-. 2.2 121 .+-. 3.1 118
.+-. 6.9t 121 .+-. 4.5 119 .+-. 4.0t I17 .+-. 3.8t Epo + a.Epo-bp
116 .+-. 6.7 121 .+-. 4.1 120 .+-. 6.2t 120 .+-. 5.0 127 .+-. 5.6
122 .+-. 1.41 Hct (%): Saline vs. 42 .+-. 2.6 41 .+-. 2.3 42 .+-.
1.6 44 .+-. 0.5 45 .+-. 1.4 43 .+-. 3.0 Epo 60 .+-. 4.5* 64 .+-.
2.2*** 66 .+-. 2.7*** 65 .+-. 1.5*** 61 .+-. 3.8* 64 .+-. 1.5**
Epo-bp 45 .+-. 1.4 45 .+-. 1.6 44 .+-. 1.1 41 .+-. 2.2 45 .+-. 0.6
43 .+-. 1.9 aEpo-bp 47 .+-. 1.0 45 .+-. 0.8 43 .+-. 0.8 43 .+-. 3.2
43 .+-. 2.2 44 .+-. 0.9 Epo + Epo-bp 58 .+-. 1.9** 62 .+-. 1.8***
60 .+-. 2.9** 62 .+-. 2.5*** 54 .+-. 3.2t 53 .+-. 3.5 Eoo +
o.Eoo-bo 61 .+-. 2.0*** 64 .+-. 1.9*** 60 .+-. 2.7*** 57 .+-. 4.0t
57 .+-. 3.2* 55 .+-. 1.8* SW (gram): Saline vs. 0.88 .+-. 0.1 0.82
.+-. 0.1 0.88 .+-. 0.1 0.96 .+-. 0.I 0.73 .+-. 0.1 0.92 .+-. 0.1
Epo 1.65 .+-. 0.2* 1.70 .+-. 0.2** 1.69 .+-.
0.1.sup..cndot..cndot..cndot. 1.63 .+-. 0.1* 1.37 .+-. 0.1 * 1.23
.+-. 0.1 t Epo-bp 0.87 .+-. 0.0 0.87 .+-. 0.0 0.87 .+-. 0.0 0.94
.+-. 0.1 0.97 .+-. 0.1 t 0.83 .+-. 0.1 a.Epo-bp 0.82 .+-. 0.0 0.92
.+-. 0.1 0.87 .+-. 0.0 0.80 .+-. 0.1 0.79 .+-. 0.1 0.89 .+-. 0.0
Epo + Epo-bp 1.50 .+-. 0.1*''' 1.58 .+-. 0.1 *** 1.67 .+-. 0.1***
1.62 .+-. 0.2* 1.48 .+-. 0.1*** 1.86 .+-. 0.1 *** Epo + aEpo-bp
1.69 .+-. 0.1 *** 1.54 .+-. 0.2** 1.53 .+-. 0.I*** 1.80 .+-.
0.1*''' 1.90 .+-. 0.2*** 2.27 .+-. 0.3** Epo vs. 0.87 .+-. 0.0**
0.87 .+-. 0.0** 0.87 .+-. 0.0*** 0.94 .+-. 0.1 * 0.97 .+-. 0.1 0.83
.+-. 0.1 t Epo-bp aEpo-bp 0.82 .+-. 0.0** 0.92 .+-. 0.1* 0.87 .+-.
0.0*** 0.80 .+-. 0.1** 0.79 .+-. 0.1* 0.89 .+-. 0.0* Epo + Epo-bp
1.50 .+-. 0.1 1.58 .+-. 0.1 1.67 .+-. 0.1 1.62 .+-. 0.2 1.48 .+-.
0.1 1.86 .+-. 0.1* Epo + aEpo-bp 1.69 .+-. 0.1 1.54 .+-. 0.2 1.53
.+-. 0.1 1.80 .+-. 0.1 1.90 .+-. 0.2 2.27 .+-. 0.3* Rx = Treatment;
n = 5 rats in each subgroup; *p < 0.01; **p < 0.00I; ***p
< 0.0001; tP < 0.05; BP = Blood pressure; BW = Body weight;
Hct = Hematocrit; SW = Spleen weight.
[0071] FIG. 3 shows circadian hematocrit compansons. There was not
only an increased hematocrit but also the peak time of hematocrit
shifted from night (20: 15) to a late morning hour (11:16) with
Epo-treatment. In the graph, groups of a (control), c (Epo-bp) and
d (aEpo-bp) are located in the dark cycle plane, while groups of b
(Epo), e (Epo+Epo-bp), and f (Epo.+-.aEpo-bp) are located in the
light cycle plane. Again, it is an obvious shift change from the
night to the daytime peak in Epo Rx in this nocturnal animal. MESOR
comparisons in cYo hematocrit in Epo (61.6) vs. control (42.7),
Epo-bp (43.9) and aEpo-bp (44.1) treatment were all statistically
significant in each time point comparison (each p<0.0001). The
amplitudes of the circadian peak-to-peak differences in hematocrit
were not significantly different between the treatment groups. But
the amplitudes were larger in the Epo-treated groups than in the
groups that did not receive Epo (2.40 in Epo, 4.41 in Epo+Epo-bp,
and 3.59 in Epo.+-.aEpo-bp, versus 1.65 in control, 1.13 in Epo-bp
and 1.73 in aEpo-bp).
[0072] In FIG. 4, splenomegaly (a, b and c) characterized each Epo
treated rat when compared with the saline treated rats (FIG. 4,
panel d). The spleen weight was significantly higher in Epo treated
rats, as compared to those of control, Epo-bp and aEpo-bp Rx groups
(Tables 1 and 2). The results suggest that the time of the Epo
treatment, with or without Epo-bp and/or aEpo-bp treatment may be
important. Epo-bp and aEpo-bp protect against the Epo-caused blood
pressure rise, while not reducing the Epo-increased hematocrit
levels. Epo dose in clinical use should be reevaluated to prevent
further systemic and local adverse effects, such as high blood
pressure and other organ damages. The binding sites of blood cell
progenitors were identified using Epo-bp and antibodies against it.
Fluorescein-labeled aEpo-bp was used to visualize receptor sites of
bone marrow progenitor cells. No receptors were detected with
fluorescein-labeled preimmune Fab, or in negative control cells.
But labeled aEpo-bp did detect binding sites on megakaryocytes,
erythroblasts, normoblasts, and myeloblasts (data not shown).
[0082] The levels of Epo, Epo-bp, and antibodies against Epo and
Epo-bp were measured in serum and plasma in healthy untreated
humans by enzyme immunoassay (EIA The EIA results are presented in
FIG. 5. Optical density (OD) of each measurement is presented as
the mean.+-.SEM of 8-14 individual samples in duplicates. The OD
values presented in FIG. 5 were calculated by subtracting the OD
value of the blanks from the OD of each sample. Serum and plasma
Epo and Epo-bp OD values were similar to each other: 0.308.+-.0.026
serum Epo, 0289.+-.0.022 serum Epo-bp, 0.289.+-.0.028 plasma Epo,
and 0.299.+-.0.015 plasma Epo-bp. The plasma level of anti-Epo-bp
antibody was significantly lower than those of the other three
antibody categories: 0.058.+-.0.008 serum aEpo, 0.052.+-.0.006
serum aEpo-bp, 0.054.+-.0.013 plasma aEpo, and 0.031.+-.0.004
plasma aEpo-bp. Serum aEpo and aEpo-bp levels were similar but the
concentration of plasma aEpo-bp was significantly lower than the
concentration of serum aEpo, serum aEpo-bp, or plasma aEpo
(p<0.025). The Epo and Epo-bp values were converted with known
Epo concentrations prepared as controls in the same plate to mU/ml.
The converted values in mU/ml were 25.4.+-.2.17 mU serum Epo,
24.2.+-.2.35 mU plasma Epo; 24.2.+-.1.84 mU serum Epo-bp,
25.0.+-.1.26 mU plasma Epo-bp. This assay method is simple and more
sensitive than the radioimmunoassay (17.7.+-.6.3 mU/ml of Epo) and
gives a much smaller SEM. Furthermore, the materials used in the
preparation are more environmentally friendly than radioactive or
other toxic chemicals used in conventional methods.
DISCUSSION
[0073] As expected, we observed an increase in blood pressure in
the Epo-treated group. In addition, the hematocrit was markedly
increased overall and at each of the 6 test times in the
Epo-treated rats, and splenomegaly characterized each rat with the
Epo treatment. Epo treatment not only significantly increased blood
pressure but also shifted the peak time of blood pressure from the
night to the daytime. Remarkably, Epo-bp and aEpo-bp protected the
rats almost completely from the Epo-induced rise in blood pressure,
while not reducing hematocrit percent. The mechanism of this
protective effect is not known. We could speculate, however, that
Epoetin (recombinant Epo currently in clinical use) may induce some
toxic materials in the living animal body when repetitively
injected. Epo-bp and/or ocEpo-bp might bind and eliminate the toxic
materials, since Epo-bp binds Epo or its degradation products, and
VEpo-bp might also bind certain products induced by Epo
treatment.
[0074] FIGS. 2 and 3 show that Epo, as well as combination
treatment with Epo+Epo-bp or Epo+VEpo-bp caused a shift in the
circadian time of peak blood pressure. This suggests that treatment
time for treatment with Epo, Epo +Epo-bp, or Epo +VEpo-bp may
markedly affect the outcome. An individual's genetic susceptibility
to endocrine treatment, as shown by salt susceptibility to
hypertension in Dahl rats, also must be considered (22,23).
[0075] The cloning of the human Epo-receptor recombinant vector
JYL26 and purification of the pure human Epo-bp and its antibodies
were important benchmarks to allow us to visualize the ligand
binding sites and to identify the cell type where the Epo receptor
is located (Lee, U.S. Pat. No. 5,843,726). To identify the ligand
binding site, we developed several sensitive and simple methods.
These may allow us to understand the structure of the Epo receptor,
and examine the factors involved in ligand binding, as well as to
identify other factors involved in regulating differentiation and
proliferation of the progenitor cells. In this study, we report the
direct binding of Epo to our purified human Epo-bp. Our Epo-bp and
its antibodies are to our knowledge the first purified pure human
Epo receptor gene products, which are characterized in specific
binding of Epo and its antibodies in nM concentrations. The binding
sites of blood progenitor cells were elaborated using Epo-bp and
its antibodies. These data support the current proposal that all
human progenitor blood cells contain Epo receptors and bind Epo. We
do not know what the biophysiological mechanisms of Epo or the
second messenger system involved in response to the Epo-Epo
receptor interaction are. The methods presented in this report will
help identify defects related to Epo or Epo receptor, and elucidate
the role of Epo receptor (EpoR) in progenitor processes and ligand
binding. The results may help in understanding the structural and
functional relationship of Epo-EpoR interactions in blood cell
progenitors. The sensitive detection may help us to understand the
role of the Epo-EpoR interaction in blood cell production and
diseases of blood cell production and help to develop treatment
methods for hematological malignancies and some systemic
cardiovascular diseases, such as high blood pressure.
CONCLUSIONS
[0076] Epo treatment increased hematocrit markedly overall as
compared to the saline control, Epo-bp, and anti-Epo-bp antibody
(aEpo-bp) treated groups, and did so at each of the 6 test times,
all p<0.0001. Increased blood pressure was detected at 12, 16,
20 and 00 hours, but not at 04 or 08 hours in rats treated with
Epo. When Epo-bp or aEpo-bp was given in conjunction with Epo
treatment, blood pressure was maintained at similar levels to the
control group. However, hematocrit levels were not significantly
changed in Epo treatment vs. Epo+Epo-bp or Epo+aEpo-bp treatment
groups (61.6 vs. 58.0 or 59.1%, respectively). Thus, Epo-bp and
aEpo-bp reduce or prevent the Epo-induced rise in blood
pressure.
[0077] Body weight was lowered by Epo treatment. Splenomegaly
characterized each rat in Epo treatment. Brain and heart weights
were significantly lower in the Epo treated group as compared to
all other groups. These data suggest that Epo dose should be
reevaluated to prevent further organ damage. The circadian results
indicate that the time of the Epo treatment, alone or in
combination of Epo-bp and/or aEpobp, may also be important.
[0078] Serum and plasma levels of Epo, Epo-bp, and antibodies
against the proteins in untreated human volunteers were determined.
Serum and plasma Epo and Epo-bp levels were similar:
Epo25.4.+-.2.17; 24.2.+-.2.35; and Epo-bp 24.2.+-.1.84;
25.0.+-.1.26 mU/ml, respectively. Serum aEpo and aEpo-bp levels
were similar, but the plasma aEpo-bp level was significantly lower
than that of serum or plasma aEpo or serum aEpo-bp.
REFERENCES CITED
[0079] 1. lfudu 0, Dawood M, Homel P: Erythropoietin-induced
elevation in blood pressure is immediate and dose dependent.
Nephron 1998; 79(4):486-487.
[0080] 2. Schiffl H, Lang S M: Hypertension induced by recombinant
human erythropoietin (rHU-Epo) can be prevented by indomethacin.
Pathogenetic role of cytosolic calcium. European J Med Res 1997;
2(3):97-100.
[0081] 3. Gobel B O, Schulte-Gebel A, Weisser B, Glanzer K, Vetter
H, Dusing R: Arterial blood pressure: Correlation with erythrocyte
count, hematocrit, and hemoglobin concentration. Am. J. Hypertens
1991; 4(1):14-19.
[0082] 4. Faulds D, Sorkin E M: Epoetin (Recombinant Human
Erythropoietin). A review of its pharmacodynamic and
pharmacokinetic properties and therapeutic potential in anemia and
the stimulation of erythropoiesis. Drugs 1989; 38(6):863-899.
[0083] 5. Raine A E G: Hypertension, blood viscosity, and
cardiovascular morbidity in renal failure: implications of
erythropoietin therapy. Lancet 1988; 1:97-100.
[0084] 6. Buckner F S, Eschbach J W, Haley N R, Davidson R C,
Adamson J W: Hypertension following erythropoietin therapy in
anemic hemodialysis patients. Am J Hypertens 1990; 3:947-955.
[0085] 7. Kong D H, Yoon Kl, Han D S: Acute effects of recombinant
human erythropoietin on plasma levels of proendothelin-1 and
endothelin-1 in haemodialysis patients. Nephrol, Dialysis,
Transplantation 1998; 13(11):2877-2883.
[0086] 8. Lebel M, Lacasse M S, Lariviere R, Kingma I, Grose J H:
Plasma and blood vessel endothelin-1 concentration in hypertensive
uremic rats treated with erythropoietin. Clin & Exp Hypertens
1998; 20(8):939-951.
[0087] 9. Vogal V, Kramer H J, Backer A, Meyer-Lehnert H, Jelkmann
W, Frandrey J: Effects of erythropoietin on endothelin-1 synthesis
and the cellular calcium messenger system in vascular endothelial
cells. Am J Hypertens 1997; 10(3):289-296.
[0088] 10. Carlini R G, Dusso A S, Obialo C L, Alvarez U M,
Rothstein M: Recombinant human erythropoietin increases
endothelin-1 release by endothelial cells. Kidney Int 1993; 43:
1010-1014.
[0089] 11. Schmieder R E, Langenfeld M R, Hilgers K F: Endogenous
erythropoietin correlates with blood pressure in essential
hypertension. Am J Kidney Dis 1997; 29(3):376-382.
[0090] 12. Canadian erythropoietin study group: Effect of
recombinant human erythropoietin therapy on blood pressure in
hemodialysis patients. Am J Nephrol 1991; 11:23-26.
[0091] 13. Nowicki M: Erythropoietin and hypertension. J Hum
Hypertens 1995; 9:81-88.
[0092] 14. Abraham P A, Macres M G: Blood pressure in hemodialysis
patients during amelioration of anemia with erythropoietin. J Am
Soc Nephrol 1991; 2:927-936.
[0093] 15. Barrett J D, Zhang Z, Zhu J H, Lee DBN, Ward H J,
Jamgotchian N, Hu M S, Fredal A, Giordani M, Eggena P:
Erythropoietin upregulates angiotensin receptors in cultured rat
vascular smooth muscle cells. J Hypertens 1998; 16:1749-1757.
[0094] 16. Fisher J W: Erythropoietin: Physiologic and
Pharmacologic aspects. Proc Soc Exp biol Med 1997; 216:358-369.
[0095] 17. Mayeux P, Billat C, Jacquot R: The erythropoietin
receptor of rat erythroid progenitor cells. Characterization and
affinity cross-linkage. J Biol Chem 1987; 262:13985-13990.
[0096] 18. Wang F F, Kung CK-H, Goldwater E: Some chemical
properties of human erythropoietin. Endocrinology 1985; 116;
2286-2292.
[0097] 19. Egrie J C, Strickland T W, Lane J, Aoki K, Cohen A M,
Smalling R. Trail G. Lin F K. Browne J K. Hines D K:
Characterization and biological effects of recombinant human
erythropoietin. Immunology 1986; 172:213-224.
[0098] 20. Lin F K, Lin C H, Lai P H, Browne J K, Egrie J C,
Smalling R, Fox G M, Chen K K, Castro M, Suggs S: Monkey
erythropoietin gene: cloning, expression and comparison with the
human erythropoietin gene. Gene 1986; 44(2-3):201-209.
[0099] 21. Mojon A, Fernandes J R, Hermida R C: Chronolab: An
Interactive software package for chronobiologic time series
analysis written for the Macintosh computer. Chronobiol Intern
1992; 9(6):403-412.
[0100] 22. Dahl L, Heine M, Tassimiani M: Role of genetic factors
in susceptibility to essential hypertension due to chronic excess
salt ingestion. Nature 1962; 194:480- 482.
[0101] 23 .Lee J Y, Tobian L, Hanlon S, Hamer R, Johnson M A, Iwai
J: How is the NaCl signal transmitted in NaCl-induced hypertension?
Hypertension 1989;13:668-675.
[0102] All of the patents, patent documents, and references cited
herein are hereby incorporated by reference thereto.
* * * * *
References