U.S. patent application number 13/044818 was filed with the patent office on 2012-03-15 for compositions and methods for preventing erythropoietin-associated hypertension.
This patent application is currently assigned to Jong Y. Lee. Invention is credited to John S. Lee, Jong Y. Lee, Mary S. Lee.
Application Number | 20120064079 13/044818 |
Document ID | / |
Family ID | 35309674 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120064079 |
Kind Code |
A1 |
Lee; Jong Y. ; et
al. |
March 15, 2012 |
Compositions and Methods for 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) |
Assignee: |
Lee; Jong Y.
Minneapolis
MN
|
Family ID: |
35309674 |
Appl. No.: |
13/044818 |
Filed: |
March 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10848689 |
May 17, 2004 |
|
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|
13044818 |
|
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Current U.S.
Class: |
424/139.1 ;
514/15.7; 514/7.7; 530/387.9 |
Current CPC
Class: |
A61K 38/1816 20130101;
A61K 39/39533 20130101; C07K 16/2863 20130101; A61P 7/06 20180101;
A61K 2121/00 20130101; A61K 2039/505 20130101; A61K 2300/00
20130101; A61K 39/3955 20130101; A61K 38/179 20130101; A61P 9/12
20180101; C07K 2317/55 20130101 |
Class at
Publication: |
424/139.1 ;
514/7.7; 530/387.9; 514/15.7 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 7/06 20060101 A61P007/06; A61K 38/02 20060101
A61K038/02; A61P 9/12 20060101 A61P009/12; A61K 38/18 20060101
A61K038/18; C07K 16/28 20060101 C07K016/28 |
Claims
1. A method of treating anemia in a mammal comprising:
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.
2. The method of claim 1 wherein the agent is a soluble Epo-bp.
3. The method of claim 2 wherein the soluble Epo-bp comprises a
fragment of a soluble portion of a mammalian Epo receptor.
4. The method of claim 3 wherein the soluble Epo-bp comprises a
fragment of at least 30 residues of SEQ ID NO:2.
5. The method of claim 4 wherein the soluble Epo-bp is a fragment
of at least 30 residues of SEQ ID NO:2.
6. The method of claim 2 wherein the soluble Epo-bp comprises SEQ
ID NO:2.
7. The method of claim 6 wherein the soluble Epo-bp is SEQ ID
NO:2.
8. The method of claim 1 wherein the agent is a recognition protein
that binds Epo receptor on an extracellular soluble portion of the
Epo receptor.
9. The method of claim 8 wherein the recognition protein binds SEQ
ID NO:2.
10. The method of claim 8 wherein the recognition protein is an
antibody.
11. The method of claim 10 wherein the antibody is an antibody
fragment.
12. The method of claim 11 wherein the antibody fragment is
Fab.
13. The method of claim 1 wherein Epo and the agent are
administered together.
14. The method of claim 1 wherein Epo and the agent are
administered separately.
15. The method of claim 1 wherein the amount of agent administered
is at least equimolar with the amount of Epo administered.
16. The method of claim 1 wherein the amount of agent administered
is about equimolar with the amount of Epo administered.
17. The method of claim 1 wherein the agent reduces an
erythropoietin-induced blood pressure rise in the mammal.
18. A method of reducing hypertension in a mammal receiving
erythropoietin (Epo), the method comprising: administering to the
mammal an effective amount of an agent selected from a soluble
erythropoietin-binding protein (Epo-bp), a recognition protein that
binds Epo receptor on an extracellular soluble portion of the Epo
receptor, and a combination thereof.
19. Use of a recognition protein that binds erythropoietin (Epo)
receptor on an extracellular soluble portion of the Epo receptor in
medical therapy.
20. The use of claim 19 wherein the recognition protein is an
antibody against SEQ ID NO:2.
21. Use of a recognition protein that binds erythropoietin (Epo)
receptor on an extracellular soluble portion of the Epo receptor to
prepare a medicament effective to reduce erythropoietin-induced
hypertension.
22. The use of claim 21 wherein the recognition protein is an
antibody against SEQ ID NO:2.
23. Use of a soluble erthropoietin-binding protein (Epo-bp) in
medical therapy.
24. The use of claim 23 wherein the Epo-bp is SEQ ID NO:2.
25. Use of a soluble erythropoietin-binding protein (Epo-bp) to
prepare a medicament effective to reduce erythropoietin-induced
hypertension.
26. The use of claim 25 wherein the Epo-bp is SEQ ID NO:2.
27. A pharmaceutical composition comprising: erythropoietin (Epo);
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.
28. The pharmaceutical composition of claim 27 wherein the
recognition protein is an antibody against SEQ ID NO:2.
29. The pharmaceutical composition of claim 27 wherein the Epo-bp
is SEQ ID NO:2.
30. A pharmaceutical composition comprising: a recognition protein
that binds erythropoietin (Epo) receptor on an extracellular
soluble portion of the Epo receptor.
31. The pharmaceutical composition of claim 30 wherein the
recognition protein is an antibody against SEQ ID NO:2.
32. A pharmaceutical composition comprising: a soluble
erythropoietin-binding protein (Epo-bp).
33. The pharmaceutical composition of claim 32 wherein the Epo-bp
is SEQ ID NO:2.
34. The pharmaceutical composition of claim 32 wherein the 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.
Description
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] 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.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable
BACKGROUND
[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.
[0004] 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.
[0005] 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.
[0006] 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).
[0007] 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).
[0008] 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.
[0009] 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
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] Another embodiment of the invention provides use of a
soluble erthyropoietin-binding protein in medical therapy.
[0016] Another embodiment of the invention provides use of a
soluble erythropoietin-binding protein to prepare a medicament
effective to reduce erythropoietin-induced hypertension.
[0017] 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.
[0018] 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.
[0019] Another embodiment of the invention provides a
pharmaceutical composition including: a soluble Epo-binding
protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] 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.
[0021] FIG. 2 shows the circadian blood pressure measurements for
each of the treatment groups.
[0022] FIG. 3 is a representation of the circadian hematocrit
variation in the treatment groups of rats.
[0023] FIG. 4 is the outline of photographs of spleens isolated
from rats treated with Epo (panels A, B, and C), or saline (panel
D).
[0024] FIG. 5 is a bar graph of optical density from
immunodetection of Epo, .A-inverted.Epo, Epo-bp, and
.A-inverted.Epo-bp in serum and plasma of human volunteers. Error
bars represent standard error, SEM.
DETAILED DESCRIPTION
Definitions.
[0025] "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/.
[0026] 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 K.sub.D 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. K.sub.D 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.
[0027] 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.
[0028] 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 10.sup.5 liters
per mole, more preferably 10.sup.6, more preferably at least
10.sup.7, most preferably at least 10.sup.8 liters per mole.
[0029] As used herein, the term "antibody" includes complete
antibodies and antigen-binding fragments of complete antibodies,
e.g., Fab or F(ab').sub.2 antibodies. The term "antibody" also
includes both monoclonal and polyclonal antibodies (e.g.,
antiserum).
[0030] 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.
Description:
[0031] 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.
[0032] In some embodiments of the invention, the agent is a soluble
Epo-bp.
[0033] In some embodiments, the soluble Epo-bp contains a fragment
of a soluble portion of a mammalian Epo receptor.
[0034] 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.
[0035] 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).
[0036] 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/.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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').sub.2, or Fv.
[0045] In particular embodiments, the antibody is an antibody
against SEQ ID NO:2.
[0046] 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.
[0047] 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.
[0048] Epo and the agent may be administered separately or
together.
[0049] In particular embodiments, the amount of the agent
administered is at least equimolar with the amount of Epo
administered.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] Another pharmaceutical composition of the invention includes
a soluble Epo-binding protein.
[0056] Typically, the pharmaceutical compositions include a
pharmaceutically acceptable diluent or carrier.
[0057] In one embodiment of the pharmaceutical compositions
containing the recognition protein, the recognition protein is an
antibody against SEQ ID NO:2.
[0058] In one embodiment of the pharmaceutical compositions
containing the soluble Epo-binding protein, the Epo-binding protein
is SEQ ID NO:2.
[0059] 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.
Raising Antibodies
[0060] 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).
[0061] 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.
[0062] 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.
[0063] 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).
[0064] 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.
[0065] 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:
[0066] (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.
[0067] (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.
[0068] (3) F(ab').sub.2 is the fragment that can be obtained by
digestion of whole antibody with pepsin, without reduction.
F(ab').sub.2 is a dimer of two Fab' fragments held together by two
disulfide bonds.
[0069] (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 (V.sub.H-V.sub.L 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.
[0070] (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.
[0071] 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.
[0072] 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).
[0073] 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)).
[0074] 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').sub.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.
[0075] 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 V.sub.H and
V.sub.L 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 V.sub.H and V.sub.L 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 V.sub.H and V.sub.L 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).
[0076] 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).
[0077] 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').sub.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.
[0078] 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).
[0079] 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).
[0080] The invention will now be illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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
.about.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.
[0085] 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.
[0086] 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).
Example 2
[0087] 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.
Experimental Procedures
Materials:
[0088] 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. 15 was from BioRad (Richmond, Calif.). Bacteriophage
T4 DNA ligase, restriction enzymes and
isopropylthio-.beta.-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 .sup.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.
[0089] Epo-bp, Fab antibody against Epo-bp (.alpha.Epo-bp), and Fab
antibody against Epo (.alpha.Epo) 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.
Animal Study:
[0090] 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.
[0091] 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 .alpha.Epo-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 .alpha.Epo-bp were
administered in an amount equimolar with Epo. The erythropoietin
(Epoetin) was from Amgen Company (Thousand Oaks, Calif.). Affinity
purified Epo-bp and .alpha.Epo-bp were prepared in our
laboratories. The antibodies were digested to Fab fragments, and
the Fab antibodies were purified.
[0092] 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.
Ligand Binding Site in Progenitor Cells and Detection of Epo and
EpoR:
[0093] We developed .alpha.Epo-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.times.10.sup.3 cells per well in round-bottomed tubes and
centrifuged into a pellet at 500 g for 2 minutes. Supernatant was
removed and 100 .mu.l 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 .mu.l of
buffer containing 1% FCS and 0.01% NaN.sub.3 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 .mu.l of PBS and analyzed
under an inverted fluorescence microscope.
[0094] 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 .mu.g/well of anti-Epo to detect Epo and 2 .mu.g/well of
anti-Epo-bp to detect Epo-bp. To detect circulating anti-Epo and
anti-Epo-bp antibodies, wells were coated with 200 .mu.l 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 .mu.l/well PBST (0.05% TWEEN 20 in PBS). Nonspecific
binding sites were blocked with 200 .mu.l/well 1% BSA in PBST for
30 min at room temperature. Wells were washed 3 times with 200
.mu.l/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 .mu.l PBST was added per well. The wells were washed
3 times with 200 .mu.l PBST. A solution (160 .mu.l) of
o-phenylenediaminedihydrochloride (OPD) in citrate buffer was added
to each well. (The solution contained 10 mg/ml in 24 mM citrate, 51
mM Na.sub.2HPO.sub.4, pH 6.0, with 0.4 ml of 3% H.sub.2O.sub.2
added to 100 ml of solution immediately before use.) The reaction
was stopped by adding 40 .mu.l of 5M NaOH, and the absorbance was
measured at 405 nm.
Statistics:
[0095] 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
[0096] 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 .alpha.Epo-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
.alpha.Epo-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 .alpha.Epo-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 .alpha.Epo-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. 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 .alpha.Epo-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 + .alpha.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 .alpha.Epo-bp 305.0 .+-. 4.9 116.6 .+-. 2.1 44.1 .+-. 0.7
0.85 .+-. 0.02 1.80 .+-. 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 +
.alpha.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
.alpha.Epo-bp 305.0 .+-. 4.9 116.6 .+-. 2.1*** 44.1 .+-. 0.7***
0.85 .+-. 0.02*** 1.80 .+-. 0.01 Epo + Epo-bp 303.4 .+-. 3.6 118.3
.+-. 1.7*** 58.0 .+-. 1.1.dagger-dbl. 1.62 .+-. 0.05 1.77 .+-. 0.02
Epo + .alpha.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
.alpha.E-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 + .alpha.Epo-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** .alpha.E-Epo-bp 1.04 .+-. 0.02** Epo +
Epo-bp 0.96 .+-. 0.02 Epo + .alpha.Epo-bp 0.99 .+-. 0.02* Rx:
treatment; n = number of rats (30 rats in each group); y: 24-h
average; .alpha.Epo-bp = anti-Epo-bp antibody; *p < 0.01; **p
< 0.001; ***p < 0.0001; .dagger-dbl.p < 0.05; g = gram; BW
= body weight; BP = blood pressure; Hct = hematocrit; SW = spleen
weight; W = weight; R = right; L = left
[0097] Epo treatment increased hematocrit markedly overall as
compared to the saline, Epo-bp or .alpha.Epo-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 .alpha.Epo-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+.alpha.Epo-bp Rx). But, significantly,
both Epo-bp and .alpha.Epo-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+.alpha.Epo-bp). Thus, both Epo-bp and .alpha.Epo-bp protected
the rats from the blood pressure rise caused by Epo treatment.
[0098] 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 .alpha.Epo-bp, each p<0.0001). Administering
Epo-bp or .alpha.Epo-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.
[0099] 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 .alpha.Epo-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 .alpha.Epo-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 .alpha.Epo-bp+Epo
groups' blood pressure levels were similar to the control
group.
[0100] Table 2 summarizes the circadian variations of body weight,
blood pressure, hematocrit and spleen weight in the 6 subgroups
after Epo, Epo-bp and .alpha.Epo-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 04 or 08 hours as
compared to control, Epo-bp and .alpha.Epo by Rx groups. Epo
treatment increased hematocrit markedly overall and at each of the
6 test times as compared to control, Epo-bp and .alpha.Epo-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 .alpha.Epo-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 .alpha.Epo-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 +
.alpha.Epo-bp 296 .+-. 13 286 .+-. 06 279 .+-. 04* 304 .+-. 12 305
.+-. 05 320 .+-. 13 BP (mmHg): 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.dagger-dbl. 140 .+-.
6.3* 137 .+-. 6.2.dagger-dbl. Epo-bp 118 .+-. 4.5 122 .+-. 5.5 118
.+-. 3.6 115 .+-. 4.0 118 .+-. 6.1 120 .+-. 7.7 .alpha.Epo-bp 113
.+-. 5.7 122 .+-. 5.1 117 .+-. 4.7 112 .+-. 3.4 113 .+-. 4.6 122
.+-. 6.8 Epo + Epo-bp 114 .+-. 2.2 121 .+-. 3.1 118 .+-. 6.9 121
.+-. 4.5.dagger-dbl. 119 .+-. 4.0 117 .+-. 3.8 Epo + .alpha.Epo-bp
116 .+-. 6.7 121 .+-. 4.1 120 .+-. 6.2 120 .+-. 5.0.dagger-dbl. 127
.+-. 5.6 122 .+-. 1.4 Epo vs. Epo-bp 118 .+-. 4.5 122 .+-. 5.5 118
.+-. 3.6* 115 .+-. 4.0.dagger-dbl. 118 .+-. 6.1.dagger-dbl. 120
.+-. 7.7 .alpha.Epo-bp 113 .+-. 5.7 122 .+-. 5.1 117 .+-. 4.7* 112
.+-. 3.4.dagger-dbl. 113 .+-. 4.6* 122 .+-. 6.8 Epo + Epo-bp 114
.+-. 2.2 121 .+-. 3.1 118 .+-. 6.9.dagger-dbl. 121 .+-. 4.5 119
.+-. 4.0.dagger-dbl. 117 .+-. 3.8.dagger-dbl. Epo + .alpha.Epo-bp
116 .+-. 6.7 121 .+-. 4.1 120 .+-. 6.2.dagger-dbl. 120 .+-. 5.0 127
.+-. 5.6 122 .+-. 1.4.dagger-dbl. 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 .alpha.Epo-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 .+-.
3.2.dagger-dbl. 53 .+-. 3.5 Epo + .alpha.Epo-bp 61 .+-. 2.0*** 64
.+-. 1.9*** 60 .+-. 2.7*** 57 .+-. 4.0.dagger-dbl. 57 .+-. 3.2* 55
.+-. 1.8* SW (gram): Saline vs. 0.88 .+-. 0.1 0.82 .+-. 0.1 0.88
.+-. 0.1 0.96 .+-. 0.1 0.73 .+-. 0.1 0.92 .+-. 0.1 Epo 1.65 .+-.
0.2* 1.70 .+-. 0.2** 1.69 .+-. 0.1*** 1.63 .+-. 0.1* 1.37 .+-. 0.1*
1.23 .+-. 0.1.dagger-dbl. Epo-bp 0.87 .+-. 0.0 0.87 .+-. 0.0 0.87
.+-. 0.0 0.94 .+-. 0.1 0.97 .+-. 0.1.dagger-dbl. 0.83 .+-. 0.1
.alpha.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 + .alpha.Epo-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**
Epo vs. Epo-bp 0.87 .+-. 0.0** 0.87 .+-. 0.0** 0.87 .+-. 0.0***
0.94 .+-. 0.1* 0.97 .+-. 0.1 0.83 .+-. 0.1.dagger-dbl.
.alpha.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 + .alpha.Epo-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 < 0.001; ***p <
0.0001; .dagger-dbl.p < 0.05; BP = Blood pressure; BW = Body
weight; Hct = Hematocrit; SW = Spleen weight.
[0101] FIG. 3 shows circadian hematocrit comparisons. 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 (.alpha.Epo-bp) are located in the dark cycle plane, while groups
of b (Epo), e (Epo+Epo-bp), and f (Epo.+-..alpha.Epo-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 % hematocrit in Epo (61.6)
vs. control (42.7), Epo-bp (43.9) and .alpha.Epo-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
.alpha.Epo-bp, versus 1.65 in control, 1.13 in Epo-bp and 1.73 in
.alpha.Epo-bp).
[0102] 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 .alpha.Epo-bp Rx
groups (Tables 1 and 2).
[0103] The results suggest that the time of the Epo treatment, with
or without Epo-bp and/or .alpha.Epo-bp treatment may be important.
Epo-bp and .alpha.Epo-bp protect against the Epo-caused blood
pressure rise, while not reducing the Epo-increased hematocrit
levels.
[0104] 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.
[0105] The binding sites of blood cell progenitors were identified
using Epo-bp and antibodies against it. Fluorescein-labeled
.alpha.Epo-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 .alpha.Epo-bp did detect binding sites on
megakaryocytes, erythroblasts, normoblasts, and myeloblasts (data
not shown).
[0106] 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 .alpha.Epo,
0.052.+-.0.006 serum .alpha.Epo-bp, 0.054.+-.0.013 plasma
.alpha.Epo, and 0.031.+-.0.004 plasma .alpha.Epo-bp. Serum
.alpha.Epo and .alpha.Epo-bp levels were similar but the
concentration of plasma .alpha.Epo-bp was significantly lower than
the concentration of serum .alpha.Epo, serum .alpha.Epo-bp, or
plasma .alpha.Epo (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
[0107] 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.
[0108] Remarkably, Epo-bp and .alpha.Epo-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 .alpha.Epo-bp might bind and eliminate the
toxic materials, since Epo-bp binds Epo or its degradation
products, and .A-inverted.Epo-bp might also bind certain products
induced by Epo treatment.
[0109] FIGS. 2 and 3 show that Epo, as well as combination
treatment with Epo+Epo-bp or Epo+.A-inverted.Epo-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+.A-inverted.Epo-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).
[0110] 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.
[0111] 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
[0112] Epo treatment increased hematocrit markedly overall as
compared to the saline control, Epo-bp, and anti-Epo-bp antibody
(.alpha.Epo-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 .alpha.Epo-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+.alpha.Epo-bp treatment groups (61.6 vs. 58.0 or 59.1%,
respectively). Thus, Epo-bp and .alpha.Epo-bp reduce or prevent the
Epo-induced rise in blood pressure.
[0113] 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 .alpha.Epo-bp, may also be
important.
[0114] 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: Epo
25.4.+-.2.17; 24.2.+-.2.35; and Epo-bp 24.2.+-.1.84; 25.0.+-.1.26
mU/ml, respectively. Serum .alpha.Epo and .alpha.Epo-bp levels were
similar, but the plasma .alpha.Epo-bp level was significantly lower
than that of serum or plasma .alpha.Epo or serum .alpha.Epo-bp.
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All cited patents, patent documents, and references are hereby
incorporated by reference.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 8 <210> SEQ ID NO 1 <211> LENGTH: 508 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <300>
PUBLICATION INFORMATION: <301> AUTHORS: Winkelmann, J.C. et
al. <302> TITLE: The gene for the human erthyropoietin
receptor <303> JOURNAL: Blood <304> VOLUME: 76
<305> ISSUE: 1 <306> PAGES: 24-30 <307> DATE:
1990-07-01 <400> SEQUENCE: 1 Met Asp His Leu Gly Ala Ser Leu
Trp Pro Gln Val Gly Ser Leu Cys 1 5 10 15 Leu Leu Leu Ala Gly Ala
Ala Trp Ala Pro Pro Pro Asn Leu Pro Asp 20 25 30 Pro Lys Phe Glu
Ser Lys Ala Ala Leu Leu Ala Ala Arg Gly Pro Glu 35 40 45 Glu Leu
Leu Cys Phe Thr Glu Arg Leu Glu Asp Leu Val Cys Phe Trp 50 55 60
Glu Glu Ala Ala Ser Ala Gly Val Gly Pro Gly Asn Tyr Ser Phe Ser 65
70 75 80 Tyr Gln Leu Glu Asp Glu Pro Trp Lys Leu Cys Arg Leu His
Gln Ala 85 90 95 Pro Thr Ala Arg Gly Ala Val Arg Phe Trp Cys Ser
Leu Pro Thr Ala 100 105 110 Asp Thr Ser Ser Phe Val Pro Leu Glu Leu
Arg Val Thr Ala Ala Ser 115 120 125 Gly Ala Pro Arg Tyr His Arg Val
Ile His Ile Asn Glu Val Val Leu 130 135 140 Leu Asp Ala Pro Val Gly
Leu Val Ala Arg Leu Ala Asp Glu Ser Gly 145 150 155 160 His Val Val
Leu Arg Trp Leu Pro Pro Pro Glu Thr Pro Met Thr Ser 165 170 175 His
Ile Arg Tyr Glu Val Asp Val Ser Ala Gly Asn Gly Ala Gly Ser 180 185
190 Val Gln Arg Val Glu Ile Leu Glu Gly Arg Thr Glu Cys Val Leu Ser
195 200 205 Asn Leu Arg Gly Arg Thr Arg Tyr Thr Phe Ala Val Arg Ala
Arg Met 210 215 220 Ala Glu Pro Ser Phe Gly Gly Phe Trp Ser Ala Trp
Ser Glu Pro Val 225 230 235 240 Ser Leu Leu Thr Pro Ser Asp Leu Asp
Pro Leu Ile Leu Thr Leu Ser 245 250 255 Leu Ile Leu Val Val Ile Leu
Val Leu Leu Thr Val Leu Ala Leu Leu 260 265 270 Ser His Arg Arg Ala
Leu Lys Gln Lys Ile Trp Pro Gly Ile Pro Ser 275 280 285 Pro Glu Ser
Glu Phe Glu Gly Leu Phe Thr Thr His Lys Gly Asn Phe 290 295 300 Gln
Leu Trp Leu Tyr Gln Asn Asp Gly Cys Leu Trp Trp Ser Pro Cys 305 310
315 320 Thr Pro Phe Thr Glu Asp Pro Pro Ala Ser Leu Glu Val Leu Ser
Glu 325 330 335 Arg Cys Trp Gly Thr Met Gln Ala Val Glu Pro Gly Thr
Asp Asp Glu 340 345 350 Gly Pro Leu Leu Glu Pro Val Gly Ser Glu His
Ala Gln Asp Thr Tyr 355 360 365 Leu Val Leu Asp Lys Trp Leu Leu Pro
Arg Asn Pro Pro Ser Glu Asp 370 375 380 Leu Pro Gly Pro Gly Gly Ser
Val Asp Ile Val Ala Met Asp Glu Gly 385 390 395 400 Ser Glu Ala Ser
Ser Cys Ser Ser Ala Leu Ala Ser Lys Pro Ser Pro 405 410 415 Glu Gly
Ala Ser Ala Ala Ser Phe Glu Tyr Thr Ile Leu Asp Pro Ser 420 425 430
Ser Gln Leu Leu Arg Pro Trp Thr Leu Cys Pro Glu Leu Pro Pro Thr 435
440 445 Pro Pro His Leu Lys Tyr Leu Tyr Leu Val Val Ser Asp Ser Gly
Ile 450 455 460 Ser Thr Asp Tyr Ser Ser Gly Asp Ser Gln Gly Ala Gln
Gly Gly Leu 465 470 475 480 Ser Asp Gly Pro Tyr Ser Asn Pro Tyr Glu
Asn Ser Leu Ile Pro Ala 485 490 495 Ala Glu Pro Leu Pro Pro Ser Tyr
Val Ala Cys Ser 500 505 <210> SEQ ID NO 2 <211> LENGTH:
226 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Ala Pro Pro Pro Asn Leu Pro Asp Pro Lys Phe
Glu Ser Lys Ala Ala 1 5 10 15 Leu Leu Ala Ala Arg Gly Pro Glu Glu
Leu Leu Cys Phe Thr Glu Arg 20 25 30 Leu Glu Asp Leu Val Cys Phe
Trp Glu Glu Ala Ala Ser Ala Gly Val 35 40 45 Gly Pro Gly Asn Tyr
Ser Phe Ser Tyr Gln Leu Glu Asp Glu Pro Trp 50 55 60 Lys Leu Cys
Arg Leu His Gln Ala Pro Thr Ala Arg Gly Ala Val Arg 65 70 75 80 Phe
Trp Cys Ser Leu Pro Thr Ala Asp Thr Ser Ser Phe Val Pro Leu 85 90
95 Glu Leu Arg Val Thr Ala Ala Ser Gly Ala Pro Arg Tyr His Arg Val
100 105 110 Ile His Ile Asn Glu Val Val Leu Leu Asp Ala Pro Val Gly
Leu Val 115 120 125 Ala Arg Leu Ala Asp Glu Ser Gly His Val Val Leu
Arg Trp Leu Pro 130 135 140 Pro Pro Glu Thr Pro Met Thr Ser His Ile
Arg Tyr Glu Val Asp Val 145 150 155 160 Ser Ala Gly Asn Gly Ala Gly
Ser Val Gln Arg Val Glu Ile Leu Glu 165 170 175 Gly Arg Thr Glu Cys
Val Leu Ser Asn Leu Arg Gly Arg Thr Arg Tyr 180 185 190 Thr Phe Ala
Val Arg Ala Arg Met Ala Glu Pro Ser Phe Gly Gly Phe 195 200 205 Trp
Ser Ala Trp Ser Glu Pro Val Ser Leu Leu Thr Pro Ser Asp Leu 210 215
220 Asp Pro 225 <210> SEQ ID NO 3 <211> LENGTH: 193
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION
NUMBER: NP_000790 <309> DATABASE ENTRY DATE: 2003-12-23
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(193) <400>
SEQUENCE: 3 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu
Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala
Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr
Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys
Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp
Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly
Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser
Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110
Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115
120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys
Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu
Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg
Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr
Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg <210> SEQ ID
NO 4 <211> LENGTH: 1849 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <300> PUBLICATION INFORMATION:
<308> DATABASE ACCESSION NUMBER: NM_000121 <309>
DATABASE ENTRY DATE: 2003-12-20 <313> RELEVANT RESIDUES IN
SEQ ID NO: (1)..(1849) <400> SEQUENCE: 4 acttagaggc
gcctggtcgg gaagggcctg gtcagctgcg tccggcggag gcagctgctg 60
acccagctgt ggactgtgcc gggggcgggg gacggagggg caggagccct gggctccccg
120 tggcgggggc tgtatcatgg accacctcgg ggcgtccctc tggccccagg
tcggctccct 180 ttgtctcctg ctcgctgggg ccgcctgggc gcccccgcct
aacctcccgg accccaagtt 240 cgagagcaaa gcggccttgc tggcggcccg
ggggcccgaa gagcttctgt gcttcaccga 300 gcggttggag gacttggtgt
gtttctggga ggaagcggcg agcgctgggg tgggcccggg 360 caactacagc
ttctcctacc agctcgagga tgagccatgg aagctgtgtc gcctgcacca 420
ggctcccacg gctcgtggtg cggtgcgctt ctggtgttcg ctgcctacag ccgacacgtc
480 gagcttcgtg cccctagagt tgcgcgtcac agcagcctcc ggcgctccgc
gatatcaccg 540 tgtcatccac atcaatgaag tagtgctcct agacgccccc
gtggggctgg tggcgcggtt 600 ggctgacgag agcggccacg tagtgttgcg
ctggctcccg ccgcctgaga cacccatgac 660 gtctcacatc cgctacgagg
tggacgtctc ggccggcaac ggcgcaggga gcgtacagag 720 ggtggagatc
ctggagggcc gcaccgagtg tgtgctgagc aacctgcggg gccggacgcg 780
ctacaccttc gccgtccgcg cgcgtatggc tgagccgagc ttcggcggct tctggagcgc
840 ctggtcggag cctgtgtcgc tgctgacgcc tagcgacctg gaccccctca
tcctgacgct 900 ctccctcatc ctcgtggtca tcctggtgct gctgaccgtg
ctcgcgctgc tctcccaccg 960 ccgggctctg aagcagaaga tctggcctgg
catcccgagc ccagagagcg agtttgaagg 1020 cctcttcacc acccacaagg
gtaacttcca gctgtggctg taccagaatg atggctgcct 1080 gtggtggagc
ccctgcaccc ccttcacgga ggacccacct gcttccctgg aagtcctctc 1140
agagcgctgc tgggggacga tgcaggcagt ggagccgggg acagatgatg agggccccct
1200 gctggagcca gtgggcagtg agcatgccca ggatacctat ctggtgctgg
acaaatggtt 1260 gctgccccgg aacccgccca gtgaggacct cccagggcct
ggtggcagtg tggacatagt 1320 ggccatggat gaaggctcag aagcatcctc
ctgctcatct gctttggcct cgaagcccag 1380 cccagaggga gcctctgctg
ccagctttga gtacactatc ctggacccca gctcccagct 1440 cttgcgtcca
tggacactgt gccctgagct gccccctacc ccaccccacc taaagtacct 1500
gtaccttgtg gtatctgact ctggcatctc aactgactac agctcagggg actcccaggg
1560 agcccaaggg ggcttatccg atggccccta ctccaaccct tatgagaaca
gccttatccc 1620 agccgctgag cctctgcccc ccagctatgt ggcttgctct
taggacacca ggctgcagat 1680 gatcagggat ccaatatgac tcagagaacc
agtgcagact caagacttat ggaacaggga 1740 tggcgaggcc tctctcagga
gcaggggcat tgctgatttt gtctgcccaa tccatcctgc 1800 tcaggaaacc
acaaccttgc agtattttta aatatgtata gtttttttg 1849 <210> SEQ ID
NO 5 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 5 ttggatccgc gcccccgcct aac
23 <210> SEQ ID NO 6 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 6
tgaattcggg gtccaggtcg ct 22 <210> SEQ ID NO 7 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: E. coli
<400> SEQUENCE: 7 Leu Val Pro Arg Gly Ser 1 5 <210> SEQ
ID NO 8 <211> LENGTH: 228 <212> TYPE: PRT <213>
ORGANISM: E. coli <400> SEQUENCE: 8 Gly Ser Ala Pro Pro Pro
Asn Leu Pro Asp Pro Lys Phe Glu Ser Lys 1 5 10 15 Ala Ala Leu Leu
Ala Ala Arg Gly Pro Glu Glu Leu Leu Cys Phe Thr 20 25 30 Glu Arg
Leu Glu Asp Leu Val Cys Phe Trp Glu Glu Ala Ala Ser Ala 35 40 45
Gly Val Gly Pro Gly Asn Tyr Ser Phe Ser Tyr Gln Leu Glu Asp Glu 50
55 60 Pro Trp Lys Leu Cys Arg Leu His Gln Ala Pro Thr Ala Arg Gly
Ala 65 70 75 80 Val Arg Phe Trp Cys Ser Leu Pro Thr Ala Asp Thr Ser
Ser Phe Val 85 90 95 Pro Leu Glu Leu Arg Val Thr Ala Ala Ser Gly
Ala Pro Arg Tyr His 100 105 110 Arg Val Ile His Ile Asn Glu Val Val
Leu Leu Asp Ala Pro Val Gly 115 120 125 Leu Val Ala Arg Leu Ala Asp
Glu Ser Gly His Val Val Leu Arg Trp 130 135 140 Leu Pro Pro Pro Glu
Thr Pro Met Thr Ser His Ile Arg Tyr Glu Val 145 150 155 160 Asp Val
Ser Ala Gly Asn Gly Ala Gly Ser Val Gln Arg Val Glu Ile 165 170 175
Leu Glu Gly Arg Thr Glu Cys Val Leu Ser Asn Leu Arg Gly Arg Thr 180
185 190 Arg Tyr Thr Phe Ala Val Arg Ala Arg Met Ala Glu Pro Ser Phe
Gly 195 200 205 Gly Phe Trp Ser Ala Trp Ser Glu Pro Val Ser Leu Leu
Thr Pro Ser 210 215 220 Asp Leu Asp Pro 225
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 8 <210>
SEQ ID NO 1 <211> LENGTH: 508 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <300> PUBLICATION
INFORMATION: <301> AUTHORS: Winkelmann, J.C. et al.
<302> TITLE: The gene for the human erthyropoietin receptor
<303> JOURNAL: Blood <304> VOLUME: 76 <305>
ISSUE: 1 <306> PAGES: 24-30 <307> DATE: 1990-07-01
<400> SEQUENCE: 1 Met Asp His Leu Gly Ala Ser Leu Trp Pro Gln
Val Gly Ser Leu Cys 1 5 10 15 Leu Leu Leu Ala Gly Ala Ala Trp Ala
Pro Pro Pro Asn Leu Pro Asp 20 25 30 Pro Lys Phe Glu Ser Lys Ala
Ala Leu Leu Ala Ala Arg Gly Pro Glu 35 40 45 Glu Leu Leu Cys Phe
Thr Glu Arg Leu Glu Asp Leu Val Cys Phe Trp 50 55 60 Glu Glu Ala
Ala Ser Ala Gly Val Gly Pro Gly Asn Tyr Ser Phe Ser 65 70 75 80 Tyr
Gln Leu Glu Asp Glu Pro Trp Lys Leu Cys Arg Leu His Gln Ala 85 90
95 Pro Thr Ala Arg Gly Ala Val Arg Phe Trp Cys Ser Leu Pro Thr Ala
100 105 110 Asp Thr Ser Ser Phe Val Pro Leu Glu Leu Arg Val Thr Ala
Ala Ser 115 120 125 Gly Ala Pro Arg Tyr His Arg Val Ile His Ile Asn
Glu Val Val Leu 130 135 140 Leu Asp Ala Pro Val Gly Leu Val Ala Arg
Leu Ala Asp Glu Ser Gly 145 150 155 160 His Val Val Leu Arg Trp Leu
Pro Pro Pro Glu Thr Pro Met Thr Ser 165 170 175 His Ile Arg Tyr Glu
Val Asp Val Ser Ala Gly Asn Gly Ala Gly Ser 180 185 190 Val Gln Arg
Val Glu Ile Leu Glu Gly Arg Thr Glu Cys Val Leu Ser 195 200 205 Asn
Leu Arg Gly Arg Thr Arg Tyr Thr Phe Ala Val Arg Ala Arg Met 210 215
220 Ala Glu Pro Ser Phe Gly Gly Phe Trp Ser Ala Trp Ser Glu Pro Val
225 230 235 240 Ser Leu Leu Thr Pro Ser Asp Leu Asp Pro Leu Ile Leu
Thr Leu Ser 245 250 255 Leu Ile Leu Val Val Ile Leu Val Leu Leu Thr
Val Leu Ala Leu Leu 260 265 270 Ser His Arg Arg Ala Leu Lys Gln Lys
Ile Trp Pro Gly Ile Pro Ser 275 280 285 Pro Glu Ser Glu Phe Glu Gly
Leu Phe Thr Thr His Lys Gly Asn Phe 290 295 300 Gln Leu Trp Leu Tyr
Gln Asn Asp Gly Cys Leu Trp Trp Ser Pro Cys 305 310 315 320 Thr Pro
Phe Thr Glu Asp Pro Pro Ala Ser Leu Glu Val Leu Ser Glu 325 330 335
Arg Cys Trp Gly Thr Met Gln Ala Val Glu Pro Gly Thr Asp Asp Glu 340
345 350 Gly Pro Leu Leu Glu Pro Val Gly Ser Glu His Ala Gln Asp Thr
Tyr 355 360 365 Leu Val Leu Asp Lys Trp Leu Leu Pro Arg Asn Pro Pro
Ser Glu Asp 370 375 380 Leu Pro Gly Pro Gly Gly Ser Val Asp Ile Val
Ala Met Asp Glu Gly 385 390 395 400 Ser Glu Ala Ser Ser Cys Ser Ser
Ala Leu Ala Ser Lys Pro Ser Pro 405 410 415 Glu Gly Ala Ser Ala Ala
Ser Phe Glu Tyr Thr Ile Leu Asp Pro Ser 420 425 430 Ser Gln Leu Leu
Arg Pro Trp Thr Leu Cys Pro Glu Leu Pro Pro Thr 435 440 445 Pro Pro
His Leu Lys Tyr Leu Tyr Leu Val Val Ser Asp Ser Gly Ile 450 455 460
Ser Thr Asp Tyr Ser Ser Gly Asp Ser Gln Gly Ala Gln Gly Gly Leu 465
470 475 480 Ser Asp Gly Pro Tyr Ser Asn Pro Tyr Glu Asn Ser Leu Ile
Pro Ala 485 490 495 Ala Glu Pro Leu Pro Pro Ser Tyr Val Ala Cys Ser
500 505 <210> SEQ ID NO 2 <211> LENGTH: 226 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
2 Ala Pro Pro Pro Asn Leu Pro Asp Pro Lys Phe Glu Ser Lys Ala Ala 1
5 10 15 Leu Leu Ala Ala Arg Gly Pro Glu Glu Leu Leu Cys Phe Thr Glu
Arg 20 25 30 Leu Glu Asp Leu Val Cys Phe Trp Glu Glu Ala Ala Ser
Ala Gly Val 35 40 45 Gly Pro Gly Asn Tyr Ser Phe Ser Tyr Gln Leu
Glu Asp Glu Pro Trp 50 55 60 Lys Leu Cys Arg Leu His Gln Ala Pro
Thr Ala Arg Gly Ala Val Arg 65 70 75 80 Phe Trp Cys Ser Leu Pro Thr
Ala Asp Thr Ser Ser Phe Val Pro Leu 85 90 95 Glu Leu Arg Val Thr
Ala Ala Ser Gly Ala Pro Arg Tyr His Arg Val 100 105 110 Ile His Ile
Asn Glu Val Val Leu Leu Asp Ala Pro Val Gly Leu Val 115 120 125 Ala
Arg Leu Ala Asp Glu Ser Gly His Val Val Leu Arg Trp Leu Pro 130 135
140 Pro Pro Glu Thr Pro Met Thr Ser His Ile Arg Tyr Glu Val Asp Val
145 150 155 160 Ser Ala Gly Asn Gly Ala Gly Ser Val Gln Arg Val Glu
Ile Leu Glu 165 170 175 Gly Arg Thr Glu Cys Val Leu Ser Asn Leu Arg
Gly Arg Thr Arg Tyr 180 185 190 Thr Phe Ala Val Arg Ala Arg Met Ala
Glu Pro Ser Phe Gly Gly Phe 195 200 205 Trp Ser Ala Trp Ser Glu Pro
Val Ser Leu Leu Thr Pro Ser Asp Leu 210 215 220 Asp Pro 225
<210> SEQ ID NO 3 <211> LENGTH: 193 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <300> PUBLICATION
INFORMATION: <308> DATABASE ACCESSION NUMBER: NP_000790
<309> DATABASE ENTRY DATE: 2003-12-23 <313> RELEVANT
RESIDUES IN SEQ ID NO: (1)..(193) <400> SEQUENCE: 3 Met Gly
Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15
Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20
25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys
Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser
Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe
Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu
Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg
Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro
Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser
Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu 130 135 140 Ala
Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150
155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe
Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg
Thr Gly Asp 180 185 190 Arg <210> SEQ ID NO 4 <211>
LENGTH: 1849 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <300> PUBLICATION INFORMATION: <308> DATABASE
ACCESSION NUMBER: NM_000121 <309> DATABASE ENTRY DATE:
2003-12-20 <313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(1849)
<400> SEQUENCE: 4 acttagaggc gcctggtcgg gaagggcctg gtcagctgcg
tccggcggag gcagctgctg 60 acccagctgt ggactgtgcc gggggcgggg
gacggagggg caggagccct gggctccccg 120 tggcgggggc tgtatcatgg
accacctcgg ggcgtccctc tggccccagg tcggctccct 180 ttgtctcctg
ctcgctgggg ccgcctgggc gcccccgcct aacctcccgg accccaagtt 240
cgagagcaaa gcggccttgc tggcggcccg ggggcccgaa gagcttctgt gcttcaccga
300 gcggttggag gacttggtgt gtttctggga ggaagcggcg agcgctgggg
tgggcccggg 360 caactacagc ttctcctacc agctcgagga tgagccatgg
aagctgtgtc gcctgcacca 420 ggctcccacg gctcgtggtg cggtgcgctt
ctggtgttcg ctgcctacag ccgacacgtc 480 gagcttcgtg cccctagagt
tgcgcgtcac agcagcctcc ggcgctccgc gatatcaccg 540 tgtcatccac
atcaatgaag tagtgctcct agacgccccc gtggggctgg tggcgcggtt 600
ggctgacgag agcggccacg tagtgttgcg ctggctcccg ccgcctgaga cacccatgac
660 gtctcacatc cgctacgagg tggacgtctc ggccggcaac ggcgcaggga
gcgtacagag 720 ggtggagatc ctggagggcc gcaccgagtg tgtgctgagc
aacctgcggg gccggacgcg 780 ctacaccttc gccgtccgcg cgcgtatggc
tgagccgagc ttcggcggct tctggagcgc 840 ctggtcggag cctgtgtcgc
tgctgacgcc tagcgacctg gaccccctca tcctgacgct 900 ctccctcatc
ctcgtggtca tcctggtgct gctgaccgtg ctcgcgctgc tctcccaccg 960
ccgggctctg aagcagaaga tctggcctgg catcccgagc ccagagagcg agtttgaagg
1020 cctcttcacc acccacaagg gtaacttcca gctgtggctg taccagaatg
atggctgcct 1080 gtggtggagc ccctgcaccc ccttcacgga ggacccacct
gcttccctgg aagtcctctc 1140 agagcgctgc tgggggacga tgcaggcagt
ggagccgggg acagatgatg agggccccct 1200 gctggagcca gtgggcagtg
agcatgccca ggatacctat ctggtgctgg acaaatggtt 1260 gctgccccgg
aacccgccca gtgaggacct cccagggcct ggtggcagtg tggacatagt 1320
ggccatggat gaaggctcag aagcatcctc ctgctcatct gctttggcct cgaagcccag
1380 cccagaggga gcctctgctg ccagctttga gtacactatc ctggacccca
gctcccagct 1440 cttgcgtcca tggacactgt gccctgagct gccccctacc
ccaccccacc taaagtacct 1500 gtaccttgtg gtatctgact ctggcatctc
aactgactac agctcagggg actcccaggg 1560 agcccaaggg ggcttatccg
atggccccta ctccaaccct tatgagaaca gccttatccc 1620 agccgctgag
cctctgcccc ccagctatgt ggcttgctct taggacacca ggctgcagat 1680
gatcagggat ccaatatgac tcagagaacc agtgcagact caagacttat ggaacaggga
1740 tggcgaggcc tctctcagga gcaggggcat tgctgatttt gtctgcccaa
tccatcctgc 1800 tcaggaaacc acaaccttgc agtattttta aatatgtata
gtttttttg 1849 <210> SEQ ID NO 5 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: E. coli <400>
SEQUENCE: 5 ttggatccgc gcccccgcct aac 23 <210> SEQ ID NO 6
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
E. coli <400> SEQUENCE: 6 tgaattcggg gtccaggtcg ct 22
<210> SEQ ID NO 7 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: E. coli <400> SEQUENCE: 7 Leu Val Pro
Arg Gly Ser 1 5 <210> SEQ ID NO 8 <211> LENGTH: 228
<212> TYPE: PRT <213> ORGANISM: E. coli <400>
SEQUENCE: 8 Gly Ser Ala Pro Pro Pro Asn Leu Pro Asp Pro Lys Phe Glu
Ser Lys 1 5 10 15 Ala Ala Leu Leu Ala Ala Arg Gly Pro Glu Glu Leu
Leu Cys Phe Thr 20 25 30 Glu Arg Leu Glu Asp Leu Val Cys Phe Trp
Glu Glu Ala Ala Ser Ala 35 40 45 Gly Val Gly Pro Gly Asn Tyr Ser
Phe Ser Tyr Gln Leu Glu Asp Glu 50 55 60 Pro Trp Lys Leu Cys Arg
Leu His Gln Ala Pro Thr Ala Arg Gly Ala 65 70 75 80 Val Arg Phe Trp
Cys Ser Leu Pro Thr Ala Asp Thr Ser Ser Phe Val 85 90 95 Pro Leu
Glu Leu Arg Val Thr Ala Ala Ser Gly Ala Pro Arg Tyr His 100 105 110
Arg Val Ile His Ile Asn Glu Val Val Leu Leu Asp Ala Pro Val Gly 115
120 125 Leu Val Ala Arg Leu Ala Asp Glu Ser Gly His Val Val Leu Arg
Trp 130 135 140 Leu Pro Pro Pro Glu Thr Pro Met Thr Ser His Ile Arg
Tyr Glu Val 145 150 155 160 Asp Val Ser Ala Gly Asn Gly Ala Gly Ser
Val Gln Arg Val Glu Ile 165 170 175 Leu Glu Gly Arg Thr Glu Cys Val
Leu Ser Asn Leu Arg Gly Arg Thr 180 185 190 Arg Tyr Thr Phe Ala Val
Arg Ala Arg Met Ala Glu Pro Ser Phe Gly 195 200 205 Gly Phe Trp Ser
Ala Trp Ser Glu Pro Val Ser Leu Leu Thr Pro Ser 210 215 220 Asp Leu
Asp Pro 225
* * * * *
References