U.S. patent application number 14/593099 was filed with the patent office on 2015-05-07 for apelin peptides, antibodies thereto, and methods of use.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Ingalill Avis, Frank Cuttitta, David Salomon.
Application Number | 20150125459 14/593099 |
Document ID | / |
Family ID | 42667220 |
Filed Date | 2015-05-07 |
United States Patent
Application |
20150125459 |
Kind Code |
A1 |
Cuttitta; Frank ; et
al. |
May 7, 2015 |
APELIN PEPTIDES, ANTIBODIES THERETO, AND METHODS OF USE
Abstract
The present disclosure concerns the use of biologically active
apelin peptides and compositions that are processed from larger
precursor proteins and further post-translationally modified to
influence cell growth. Particular methods are useful for promoting
cell growth, while others are particularly useful for inhibiting
cell growth.
Inventors: |
Cuttitta; Frank; (Adamstown,
MD) ; Avis; Ingalill; (Gaithersburg, MD) ;
Salomon; David; (Frederick, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
42667220 |
Appl. No.: |
14/593099 |
Filed: |
January 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12715338 |
Mar 1, 2010 |
8946382 |
|
|
14593099 |
|
|
|
|
61156351 |
Feb 27, 2009 |
|
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Current U.S.
Class: |
424/139.1 ;
435/375; 530/387.3; 530/387.9 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/47 20130101; C07K 16/26 20130101; G01N 33/566 20130101;
A61P 9/12 20180101; A61P 11/00 20180101; A61P 3/10 20180101; C07K
2317/76 20130101; C07K 2317/24 20130101; A61P 27/02 20180101; C07K
2317/75 20130101; A61P 9/10 20180101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3; 435/375 |
International
Class: |
C07K 16/26 20060101
C07K016/26 |
Claims
1. An isolated antibody that binds to an epitope specific to
salcut-NH.sub.2 polypeptide (SEQ ID NO: 19), which isolated
antibody does not bind to apelin-36 (42-77), apelin-17 (61-77), or
apelin-13 (65-77).
2. A method of treating a tumor or a disease caused by abnormal
angiogenesis of a subject, comprising administering to the subject
a therapeutically effective amount of the isolated antibody of
claim 1, wherein the subject has a tumor or a disease caused by
abnormal angiogenesis and administration of the polypeptide
modifies tumor cell growth or endothelial cell growth in the
subject, thereby treating the tumor or disease caused by abnormal
angiogenesis of the subject.
3. The method of claim 2, wherein the disease caused by abnormal
angiogenesis comprises neoplasia, cardiovascular disease,
peripheral vascular disease, hypertension, preeclampsia syndrome,
abnormal angiogenesis, diabetes, ocular degeneration, idiopathic
pulmonary fibrosis, wound healing, altered mast cell migration,
chronic obstructive pulmonary disease, inflammatory diseases such
as arthritis (juvenile and rheumatoid) and inflammatory bowel
disease, cardiovascular disease, avascular or ischemic insult,
myocardial infarction, stroke, vasculititis/angiitis, systemic or
vascular sclerosis, gangrene, congelation (severe frostbite),
alopecia, eczema, ulcers, lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, or endometriosis.
4. The antibody of claim 1, wherein the antibody is a monoclonal
antibody.
5. The antibody of claim 4, wherein the monoclonal antibody is
humanized.
6. A method of modulating cell growth, comprising administering
isolated salcut-NH.sub.2 polypeptide (SEQ ID NO: 19) to a cell
sample, thereby modulating cell growth.
7. The method of claim 6, wherein cell growth is increased.
8. The method of claim 6, wherein the cell growth is decreased.
9. The method of claim 6, wherein the cells comprise endothelial
cells or tumor cells.
10. The method of claim 6, wherein the cell sample is in vivo.
11. An inhibitor of cell growth modulating activity of
salcut-NH.sub.2 polypeptide (SEQ ID NO: 19), comprising a peptide
or neutralizing antibody.
12. A method of treating a tumor or a disease caused by abnormal
angiogenesis of a subject, comprising administering to the subject
a therapeutically effective amount of the inhibitor of claim 11,
wherein the subject has a tumor or a disease caused by abnormal
angiogenesis and administration of the inhibitor modifies tumor
cell growth or endothelial cell growth in the subject, thereby
treating the tumor or disease caused by abnormal angiogenesis of
the subject.
14. An activator of cell growth modulating activity of
salcut-NH.sub.2 polypeptide (SEQ ID NO: 19), comprising a peptide
or activating antibody.
15. A method of treating a disease caused by abnormal angiogenesis
of a subject, comprising administering to the subject a
therapeutically effective amount of the activator of claim 14,
wherein the subject has a disease caused by abnormal angiogenesis
and administration of the activator modifies endothelial cell
growth in the subject, thereby treating the disease caused by
abnormal angiogenesis of the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of co-pending U.S. application Ser. No.
12/715,338, filed Mar. 1, 2010, which claims the benefit of the
earlier filing date of U.S. Provisional Application No. 61/156,351,
filed Feb. 27, 2009. Both prior applications are incorporated
herein by reference.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to the field of biologically active
peptides that are processed from larger precursor proteins and
further post-translationally modified. This disclosure also related
to methods of use of such post-translationally modified peptides,
for example for inhibiting cell growth or for enhancing cell
growth.
BACKGROUND
[0003] Biologically active peptides are generally first synthesized
as inactive, higher molecular weight precursors. Processing of a
proprotein precursor, by enzymatic cleavage and covalent
modifications, yields active peptide(s) from the larger proprotein.
It is not uncommon for a proprotein to be processed such that more
than one biologically active peptide is produced from the same
precursor molecule. For example, cholecystokinin,
proopiomelanocortin, calcitonin, proglucagon, and proadrenomedulin
each produce several different biologically active peptides.
[0004] Amidation is often a biologically important
post-translational modification, as the amidated form of a protein
generally is biologically active and more resistant to
carboxypeptidases. An amidation motif has been used to identify
potential cleavage/amidation sites in precursor proteins which may
result in the generation of biologically active amidated peptides
from a precursor protein (see, for example, Eberlein et al., J.
Biol. Chem., 267:1517-1521, 1992; Siegfried et al., Proc. Natl.
Acad. Sci. USA, 89:8107-8111, 1992; Quinn et al., Cancer Cells,
3:504-510, 1991; Cuttitta, The Anatomical Record, 236:87-93, 1993;
Fenger and Johnsen, Biochem. J., 250:781-788, 1988; Orskov et al.,
J. Biol. Chem., 264:12826-12829, 1989). The amidation motif
consists of an invariant glycine residue followed by a region of
basic amino acids on the carboxy-terminal side of the glycine
residue.
[0005] Although the free acid and amidated forms of a peptide are
difficult to distinguish structurally, the amide can be 100-1000
times more biologically active than the free acid form of the
peptide (Cuttitta, The Anatomical Record, 236:87-93, 1993).
Amidated peptides can exhibit the same type of biological activity
as other peptides processed from the same precursor protein,
although their activity may vary with peptide size (Tatemoto et
al., Biochem. Biophys. Res. Comm., 251:471-476, 1998).
SUMMARY OF THE DISCLOSURE
[0006] This disclosure provides apelin-36 (42-57) peptide (also
known as salcut) and variants thereof, and nucleic acid molecules
encoding these peptides, including cDNA sequences. In specific
embodiments, these amino acid sequences are post-translationally
modified, for example into an amide-derived form. The amide-derived
form of apelin-36 (42-57) (also known as salcut-NH.sub.2) has both
cell growth enhancing and inhibiting activity; depending for
instance on dosage. Thus, the molecules and compounds disclosed
herein are useful for modifying angiogenesis. In addition, the
molecules disclosed herein are useful for inhibiting or treating
tumor cell growth.
[0007] The molecules provided herein are further useful for
ameliorating, treating, detecting, prognosing, and diagnosing
diseases and conditions associated with abnormal apelin-36 (42-57)
levels, or more specifically abnormal amidated apelin-36 (42-57)
levels, such as neoplasia, hypertension, preeclampsia syndrome,
diabetes, ocular degeneration, idiopathic pulmonary fibrosis, wound
healing, abnormal angiogenesis, altered mast cell migration,
chronic obstructive pulmonary disease, inflammatory diseases such
as arthritis (juvenile and rheumatoid) and inflammatory bowel
disease, cardiovascular disease, avascular or ischemic insult,
myocardial infarction, stroke, vasculititis/angiitis, systemic or
vascular sclerosis, gangrene, congelation (severe frostbite),
alopecia, eczema, ulcers, lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, endometriosis, among others.
[0008] In some embodiments the disclosed molecules are used for
quantitating apelin-36 (42-57) levels, or more specifically
amidated apelin-36 (42-57) levels, in biological samples, such as
samples from cancer patients, in order to measure the severity of
the disease state. Antibodies specific for apelin-36 (42-57) can be
used to screen samples for the presence and quantity of the
disclosed peptides.
[0009] Also provided herein are inhibitors or antagonists of
amidated apelin-36 (42-57), for example apelin-36 (42-58) (also
referred to as salcut-Gly), free-acid form of apelin-36 (42-57),
small molecule inhibitors, and neutralizing monoclonal antibodies.
Activators of amidated apelin-36 (42-57) are also provided
herein.
[0010] The foregoing and other features and advantages of the
invention will become more apparent from the following detailed
description of a several embodiments which proceeds with reference
to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 illustrates amino acid sequences of apelin peptides;
these are aligned to assist in comparisons.
[0012] FIG. 2 is an alignment of the amino acid sequences of human
(SEQ ID NO: 8), dog (SEQ ID NO: 9), bovine (SEQ ID NO: 10, rat (SEQ
ID NO: 11), mouse (SEQ ID NO: 12), opossum (SEQ ID NO: 13), frog
(SEQ ID NO: 37), and zebra fish (SEQ ID NO: 14) apelin
preproprotein, demonstrating the evolutionary conservation of
apelin in mammals. Also shown are the regions of apelin which
correspond to the secretory signal peptide (residues 1-22),
apelin-36 (42-77), apelin-17 (61-77), apelin-13 (65-77), and
salcut-NH.sub.2/apelin-36 (42-57). Accession numbers for the gene,
cDNA, or protein sequences for each species are identified.
[0013] FIG. 3A-3O is a series of graphs demonstrating the
proliferative response of various endothelial cells and epithelial
cells to various concentrations of apelin-13 (65-77), apelin-36
(42-57) (salcut), the free acid form of apelin-36 (42-57)
(salcut-OH), or apelin-36 (42-58) (salcut-glycine). FIG. 3A shows
the proliferative response of the MCF-7 human breast cancer
(estrogen dependent) cell line in the presence of both apelin-13
(65-77) and apelin 36 (42-57). FIG. 3B shows the proliferative
response of the T47D human breast cancer (estrogen-independent)
cell line in the presence of both apelin-13 (65-77) and apelin-36
(42-57). FIG. 3C shows the proliferative response of the HTB 103
human gastric cancer cell line in the presence of both apelin-13
(65-77) and apelin 36 (42-57). FIG. 3D shows the proliferative
response of the A549 human lung cancer cell line in the presence of
both apelin-13 (65-77) and apelin 36 (42-57). FIG. 3E shows the
proliferative response of the HMEC-1 human blood vessel endothelial
cell line in the presence of both apelin-13 (65-77) and apelin 36
(42-57). FIG. 3F shows the proliferative response of the HMEC-1
human blood vessel endothelial cell line in the presence of both
the free acid form of apelin-36 (42-57) and apelin 36 (42-58). FIG.
3G shows the proliferative response of porcine aortic endothelial
(PAE) cells in the presence of both apelin-13 (65-77) and apelin 36
(42-57). FIG. 3H shows the proliferative response of human primary
microvascular dermal endothelial cells in the presence of both
apelin-13 (65-77) and apelin 36 (42-57). FIG. 3I shows the
proliferative response of the human lymphatic endothelial cell line
after a three day incubation in the presence of both apelin-13
(65-77) and apelin 36 (42-57). FIG. 3J shows the proliferative
response of the human lymphatic endothelial cell line after a five
day incubation in the presence of both apelin-13 (65-77) and apelin
36 (42-57). FIG. 3K shows the proliferative response of the CRL
1780 monkey endothelial cell line in the presence of both apelin-13
(65-77) and apelin 36 (42-57). FIG. 3L shows the proliferative
response of the HMC-1 human mast cell line in the presence of
apelin-13 (65-77). FIG. 3M shows the proliferative response of the
HMC-1 human mast cell line in the presence of apelin 36 (42-57).
FIG. 3N shows the proliferative response of the HTB 103 human
gastric cancer cell line in the presence of apelin 36 (42-57). FIG.
3O shows the proliferative response of the HTB 103 human gastric
cancer cell line in the presence of apelin 13 (65-77). RLU
(relative luminescent units) value is proportional to proliferation
or cell growth.
[0014] FIG. 4 is a pair of graphs demonstrating the proliferative
response of two cancer cell lines to apelin 36 (42-57)
(salcut-NH.sub.2) over a 1 nM to 1 .mu.M dose range. FIG. 4 (first
panel) shows a biphasic response curve of apelin 36 (42-57) on the
MCF-7 human breast cancer cell line. FIG. 4 (second panel) shows a
lack of a statistically significant proliferative response of the
A549 cells to various concentrations of apelin 36 (42-57), although
there appears to be a downward trend (inhibition) in proliferation
with increased concentration of apelin 36 (42-57). The resulting
biphasic response curve is indicative of either cell toxicity at a
higher dose range or a two receptor system (high affinity
receptor-mediating proliferation and low affinity receptor inducing
growth suppression; a homeostatic feedback mechanism).
[0015] FIG. 5 is a pair of graphs demonstrating the proliferative
response of two human cell lines to various concentrations of
apelin 36 (42-57) (salcut-NH.sub.2). FIG. 5 (first panel and second
panel) show biphasic response curves of the HMC-1 human mast cell
line and HTB 103 human gastric cancer cell line, respectively. The
resulting biphasic response curve is indicative of either cell
toxicity at a higher dose range or a two receptor system (high
affinity receptor-mediating proliferation and low affinity receptor
inducing growth suppression; a homeostatic feedback mechanism).
[0016] FIG. 6 is a pair of graphs demonstrating the proliferative
response of the HMEC-1 human blood vessel endothelial cell line to
various concentrations of apelin 36 (42-57) (salcut-NH.sub.2), the
free acid form of apelin 36 (42-57) (salcut OH), and apelin 37
(42-58) (salcut Gly). FIG. 6 (first panel) shows a biphasic
response curve of the HMEC-1 cells to apelin 36 (42-57). FIG. 6
(second panel) shows that salcut OH and salcut Gly have no effect
on HMEC-1 cell proliferation. The resulting biphasic response curve
is indicative of either cell toxicity at a higher dose range or a
two receptor system (high affinity receptor-mediating proliferation
and low affinity receptor inducing growth suppression; a
homeostatic feedback mechanism). Salcut-OH (free-acid) and the
salcut-Gly (glycine extended) derivative are non-responsive over
the dose range tested.
[0017] FIG. 7 is a series of immunofluorescence images
demonstrating the effect of various concentrations of apelin 36
(42-57) (salcut-NH.sub.2), the free acid form of apelin 36 (42-57),
or apelin 36 (42-58) on porcine aortic endothelial cell tube
formation.
[0018] FIG. 8 is a series of immunofluorescence images
demonstrating the effect of the APJ receptor antagonist
ala13-apelin 13 (also known as apelin-12 (F13A)) on endothelial
cell tube formation in the presence of apelin 36 (42-57)
(salcut-NH.sub.2) and apelin 13 (65-77).
[0019] FIG. 9 is a series of images demonstrating the dose response
of apelin 36 (42-57) (salcut-NH.sub.2) on aortic ring vessel
outcropping. The images show that the number of vessels formed
increase with increasing concentration of apelin 36 (42-57)
(salcut-NH.sub.2). Vessels begin to form with the ring at 10 nM
apelin 36 (42-57).
[0020] FIG. 10 is a graph demonstrating the sensitivity of the
salcut-NH.sub.2 quantitative ELISA when lowering the solid phase
concentration of c-salcut-NH.sub.2. As the solid phased
[c-salcut-NH.sub.2] is lowered, the resulting titration curve
becomes more linear and the sensitivity (detectable peptide)
increases.
[0021] FIG. 11 is a graph demonstrating an ELISA titration curve
using different absorption peptides and different solid phased
ligand targets. Antibodies with bovine serum albumin (BSA) or
Apelin-36 bind effectively to CSC-NH.sub.2
(cysteine-salcut-NH.sub.2, a modified cross-linker immunogen)
(.diamond., .box-solid.), but antibodies with CSC-NH.sub.2
dramatically block binding to CSC-NH.sub.2 (.tangle-solidup.).
Antibodies with BSA or CSC-NH.sub.2 bind to Apelin-36
(.largecircle., ), but antibodies with Apelin-36 block binding to
Apelin-36 (X).
[0022] FIG. 12 is a graph demonstrating effects of salcut-NH.sub.2
treatment on nude mouse xenograft growth of the human
bronchioloalveolar cancer cell line A549.
[0023] FIG. 13A-13B is a series of graphs demonstrating that the
apelin-13(F13A) antagonist blocks apelin-13 induced proliferation
but not salcut-NH.sub.2 mediated growth. FIG. 13A is a graph
showing the proliferative response of SV40 immortalized human
dermal microvascular endothelial cell line HMEC-1 to apelin-13 and
salcut-NH2 in the presence or absence of APJ receptor antagonist
apelin-13(F13A) (also known as ala13-apelin). FIG. 13B is a graph
showing the proliferative response of the spontaneously
immortalized rat aortic cell line PAE to apelin-13 and salcut-NH2
in the presence or absence of APJ receptor antagonist
apelin-13(F13A). Rx is apelin-13 or salcut-NH.sub.2; APL(F/A) is
apelin-13 (F13A); RLU is relative luminescent units.
[0024] FIG. 14A-C is a series of graphs showing the proliferative
response of the human breast cancer cell line MDA-MB43 to various
concentrations of salcut-NH2 and biotinylated salcut. FIG. 14A
shows the proliferative response of the MDA-MB43 human breast
cancer cell line in the presence of salcut-NH.sub.2 prepared by
Princeton Biomolecules (Langhorne, Pa.). FIG. 14B shows the
proliferative response of the MDA-MB43 human breast cancer cell
line in the presence of biotinylated amino terminal derivative of
salcut. FIG. 14C shows the proliferative response of the MDA-MB43
human breast cancer cell line in the presence of salcut prepared by
GenScript Corporation (Piscataway, N.Y.).
SEQUENCE LISTING
[0025] The nucleic and amino acid sequences listed herein and/or
herewith are shown using standard letter abbreviations for
nucleotide bases, and three letter code for amino acids, as defined
in 37 C.F.R. .sctn.1.822. Only one strand of each nucleic acid
sequence is shown, but the complementary strand is understood as
included by any reference to the displayed strand. The Sequence
Listing is submitted as an ASCII text file named Sequences.txt,
created on Dec. 29, 2014, 16 KB, which is incorporated by reference
herein.
[0026] SEQ ID NO: 1 shows the cDNA sequence of the human apelin
preproprotein, also presented as positions 308-541 of GenBank
Accession No. NM.sub.--017413.
[0027] SEQ ID NO: 2 shows the cDNA sequence of the dog apelin
preproprotein, also presented as Ensembl Accession No.
ENSCAFT00000029682.
[0028] SEQ ID NO: 3 shows the cDNA sequence of the bovine apelin
preproprotein, also presented as GenBank Accession No.
NM.sub.--174503 or Ensembl Accession No. ENSBTAT00000026630.
[0029] SEQ ID NO: 4 shows the cDNA sequence of the rat apelin
preproprotein, also presented as positions 327-560 of GenBank
Accession No. NM.sub.--031612.
[0030] SEQ ID NO: 5 shows the cDNA sequence of the mouse apelin
preproprotein, also presented as positions 348-581 of GenBank
Accession No. NM.sub.--013912.
[0031] SEQ ID NO: 6 shows the cDNA sequence of the opossum apelin
preproprotein, also presented as Ensembl Accession No.
ENSMODT00000016827.
[0032] SEQ ID NO: 7 shows the cDNA sequence of the zebra fish
apelin preproprotein, also presented as positions 73-306 of GenBank
Accession No. DQ062434.
[0033] SEQ ID NO: 8 shows the amino acid sequence of the human
apelin preproprotein, also presented as GenBank Accession No.
NP.sub.--059109 or Accession No. AAF25815.
[0034] SEQ ID NO: 9 shows the amino acid sequence of the dog apelin
preproprotein, also presented as Ensembl Accession No.
ENSCAFP00000027587.
[0035] SEQ ID NO: 10 shows the amino acid sequence of the bovine
apelin preproprotein, also presented as GenBank Accession No.
NP.sub.--776928 or Ensembl Accession No. ENSBTAP00000026630.
[0036] SEQ ID NO: 11 shows the amino acid sequence of the rat
apelin preproprotein, also presented as GenBank Accession No.
NP.sub.--113800.
[0037] SEQ ID NO: 12 shows the amino acid sequence of the mouse
apelin preproprotein, also presented as GenBank Accession No.
NP.sub.--038940.
[0038] SEQ ID NO: 13 shows the amino acid sequence of the opossum
apelin preproprotein, also presented as Ensembl Accession No.
ENSMODP00000016523.
[0039] SEQ ID NO: 14 shows the amino acid sequence of the zebra
fish apelin preproprotein, also presented as GenBank Accession No.
AAY46798.
[0040] SEQ ID NO: 15: shows the amino acid sequence of human
apelin-36.
[0041] SEQ ID NO: 16 shows the amino acid sequence of human
apelin-17.
[0042] SEQ ID NO: 17 shows the amino acid sequence of human
apelin-13.
[0043] SEQ ID NO: 18 shows the amino acid sequence of human
apelin-36 (42-57), also known as human salcut or salcut-OH (the
free-acid derivative of salcut).
[0044] SEQ ID NO: 19 shows the amino acid sequence of the amide
derivative of human apelin-36 (42-57), also known as human
salcut-NH.sub.2.
[0045] SEQ ID NO: 20 shows the amino acid sequence of a
glycine-extended form of human apelin-36 (42-57), also known as
human salcut-Gly or human apelin-36 (42-58).
[0046] SEQ ID NO: 21 shows the amino acid sequence of dog apelin-36
(42-57), also known as dog salcut or salcut-OH (the free-acid
derivative of salcut).
[0047] SEQ ID NO: 22 shows the amino acid sequence of the amide
derivative of dog apelin-36 (42-57), also known as dog
salcut-NH.sub.2.
[0048] SEQ ID NO: 23 shows the amino acid sequence of a
glycine-extended form of dog apelin-36 (42-57), also known as dog
salcut-Gly or dog apelin-36 (42-58).
[0049] SEQ ID NO: 24 shows the amino acid sequence of bovine
apelin-36 (42-57), also known as bovine salcut or salcut-OH (the
free-acid derivative of salcut).
[0050] SEQ ID NO: 25 shows the amino acid sequence of the amide
derivative of bovine apelin-36 (42-57), also known as bovine
salcut-NH.sub.2.
[0051] SEQ ID NO: 26 shows the amino acid sequence of a
glycine-extended form of bovine apelin-36 (42-57), also known as
bovine salcut-Gly or bovine apelin-36 (42-58).
[0052] SEQ ID NO: 27 shows the amino acid sequence of rat apelin-36
(42-57), also known as rat salcut or salcut-OH (the free-acid
derivative of salcut).
[0053] SEQ ID NO: 28 shows the amino acid sequence of the amide
derivative of rat apelin-36 (42-57), also known as rat
salcut-NH.sub.2.
[0054] SEQ ID NO: 29 shows the amino acid sequence of a
glycine-extended form of rat apelin-36 (42-57), also known as rat
salcut-Gly or rat apelin-36 (42-58).
[0055] SEQ ID NO: 30 shows the amino acid sequence of mouse
apelin-36 (42-57), also known as mouse salcut or salcut-OH (the
free-acid derivative of salcut).
[0056] SEQ ID NO: 31 shows the amino acid sequence of the amide
derivative of mouse apelin-36 (42-57), also known as mouse
salcut-NH.sub.2.
[0057] SEQ ID NO: 32 shows the amino acid sequence of a
glycine-extended form of mouse apelin-36 (42-57), also known as
mouse salcut-Gly or mouse apelin-36 (42-58).
[0058] SEQ ID NO: 33 shows the amino acid sequence of opossum
apelin-36 (42-57), also known as opossum salcut or salcut-OH (the
free-acid derivative of salcut).
[0059] SEQ ID NO: 34 shows the amino acid sequence of the amide
derivative of opossum apelin-36 (42-57) (based on the numbering of
the human apelin sequence), also known as opossum
salcut-NH.sub.2.
[0060] SEQ ID NO: 35 shows the amino acid sequence of a
glycine-extended form of opossum apelin-36 (42-57), also known as
opossum salcut-Gly or opossum apelin-36 (42-58) (based on the
numbering of the human apelin sequence).
[0061] SEQ ID NO: 36 shows the cDNA sequence of the frog apelin
preproprotein, also referred to as positions 97-327 of GenBank
Accession No. NM.sub.--001097924.
[0062] SEQ ID NO: 37 shows the amino acid sequence of the frog
apelin preproprotein, also referred to as GenBank Accession No.
NP.sub.--001091393.
[0063] SEQ ID NO: 38 shows cysteine linked salcut-NH.sub.2.
[0064] SEQ ID NO: 39 shows the cDNA sequence of the rhesus monkey
apelin preproprotein, also presented as Ensembl Accession No.
ENSMMUT00000003625.
[0065] SEQ ID NO: 40 shows the amino acid sequence of the rhesus
monkey apelin preproprotein, also presented as Ensembl Accession
No. ENSMMUP00000003428.
[0066] SEQ ID NO: 41 shows the amino acid sequence of rhesus monkey
apelin-36 (42-57), also known as rhesus monkey salcut or salcut-OH
(the free-acid derivative of salcut).
[0067] SEQ ID NO: 42 shows the amino acid sequence of the amide
derivative of rhesus monkey apelin-36 (42-57), also known as rhesus
monkey salcut-NH.sub.2.
[0068] SEQ ID NO: 43 shows the amino acid sequence of a
glycine-extended form of rhesus monkey apelin-36 (42-57), also
known as rhesus monkey salcut-Gly or rhesus monkey apelin-36
(42-58).
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
I. Abbreviations
[0069] CA Cancer
[0070] EC Endothelial cell
[0071] ELISA Enzyme-Linked ImmunoSorbent Assay
[0072] GFP Green Fluorescent Protein
[0073] HMC Human Mast Cell
[0074] HMEC Human microvascular endothelial cell
[0075] LEC Lymphatic endothelial cell
[0076] PAE Porcine aortic endothelial cell
[0077] RLU Relative Luminescent Units
II. Terms
[0078] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0079] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0080] Amidation or amide derivative: A post-translational
modification that leads to biological activity of an otherwise
inert peptide or that enhances the biological activity of the
peptide, wherein a peptide is post-translationally modified by
C-terminal amidation. The amino acid to be modified is always
followed by a glycine, which provides the amide group. The process
of post-translational amidation of a peptide derived from a
precursor proprotein is well characterized and involves three
enzymatic steps (Cuttitta, The Anatomical Record, 236:87-93, 1993).
Step one involves endoproteolytic cleavage at a pair of basic amino
acids near the carboxy terminus of the protein. Step two involves
carboxypeptidase-mediated removal of basic residues. Step three is
the amidation reaction, which involves oxidation of a terminal
glycine to form the amide of the neighboring carboxy terminal amino
acid. Glycine is the only known amino acid to function as an amide
donor for its neighboring amino acid. Although the free acid and
amidated forms of a peptide are difficult to distinguish
structurally, the amide can be 100-1000 times more biologically
active than the free acid form of the peptide (Cuttitta, The
Anatomical Record, 236:87-93, 1993). C-terminal amidation is
essential to the biological activity of many polypeptides,
including neuropeptides and hormones.
[0081] Animal: Living multicellular organisms, a category which
includes, for example, mammals, for example humans, and birds.
[0082] Antibody: Immunoglobulin (Ig) molecules and immunologically
active portions of Ig molecules, for instance, molecules that
contain an antigen binding site which specifically binds
(immunoreacts with) an antigen. In one embodiment the antigen is
CD34. Monoclonal, polyclonal, and humanized immunoglobulins are
encompassed by the disclosure. The disclosure also includes
synthetic and genetically engineered variants of these
immunoglobulins.
[0083] A naturally occurring antibody (for example, IgG) includes
four polypeptide chains, two heavy chains and two light chains
inter-connected by disulfide bonds. However, it has been shown that
the antigen-binding function of an antibody can be performed by
fragments of a naturally occurring antibody. Thus, these
antigen-binding fragments are also intended to be designated by the
term "antibody". Examples of binding fragments encompassed within
the term antibody include (i) an Fab fragment consisting of the
variable light (VL), variable heavy (VH), constant light (CL) and
constant heavy (CH)1 domains; (ii) an Fd fragment consisting of the
VH and CH1 domains; (iii) an Fv fragment consisting of the VL and
VH domains of a single arm of an antibody, (iv) a dAb fragment
(Ward et al., (1989) Nature 341:544-546) which consists of a VH
domain; and (v) an F(ab')2 fragment, a bivalent fragment comprising
two Fab fragments linked by a disulfide bridge at the hinge region.
Furthermore, although the two domains of the Fv fragment are coded
for by separate genes, a synthetic linker can be made that enables
them to be made as a single protein chain (known as single chain Fv
(scFv); Bird et al. (1988) Science 242:423-426; and Huston et al.
(1988) Proc. Natl. Acad. Sci. 85:5879-5883) by recombinant methods.
Such single chain antibodies, as well as dsFv, a disulfide
stabilized Fv (Bera et al. (1998) J. Mol. Biol. 281:475-483), and
dimeric Fvs (diabodies) that are generated by pairing different
polypeptide chains (Holliger et al. (1993) Proc. Natl. Acad. Sci.
90:6444-6448), are also included.
[0084] In one embodiment, antibody fragments for use in this
disclosure are those which are capable of cross-linking their
target antigen, for example, bivalent fragments such as F(ab')2
fragments. Alternatively, an antibody fragment which does not
itself cross-link its target antigen (for example, a Fab fragment)
can be used in conjunction with a secondary antibody which serves
to cross-link the antibody fragment, thereby cross-linking the
target antigen. Antibodies can be fragmented using conventional
techniques and the fragments screened for utility in the same
manner as described for whole antibodies. An antibody is further
intended to include humanized monoclonal molecules that
specifically bind the target antigen.
[0085] "Specifically binds" refers to the ability of individual
antibodies to specifically immunoreact with an antigen. This
binding is a non-random binding reaction between an antibody
molecule and the antigen. In one embodiment, the antigen is CD34.
Binding specificity is typically determined from the reference
point of the ability of the antibody to differentially bind the
antigen of interest and an unrelated antigen, and therefore
distinguish between two different antigens, particularly where the
two antigens have unique epitopes. An antibody that specifically
binds to a particular epitope is referred to as a "specific
antibody".
[0086] A variety of methods for attaching detectable labels to
antibodies are well known in the art. Detectable labels useful for
such purposes are also well known in the art, and include
radioactive isotopes such as .sup.32P, fluorophores,
chemiluminescent agents, and enzymes.
[0087] Antigen: Any molecule that can bind specifically with an
antibody. An antigen is also a substance that antagonizes or
stimulates the immune system to produce antibodies. Antigens are
often foreign substances such as allergens, bacteria or viruses
that invade the body.
[0088] Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA)
has two strands, a 5'->3' strand, referred to as the plus
strand, and a 3'->5' strand (the reverse complement), referred
to as the minus strand. Because RNA polymerase adds nucleic acids
in a 5'->3' direction, the minus strand of the DNA serves as the
template for the RNA during transcription. Thus, the RNA formed
will have a sequence complementary to the minus strand and
identical to the plus strand (except that U is substituted for
T).
[0089] Antisense molecules are molecules that are specifically
hybridizable or specifically complementary to either RNA or the
plus strand of DNA. Sense molecules are molecules that are
specifically hybridizable or specifically complementary to the
minus strand of DNA. Antigene molecules are either antisense or
sense molecules directed to a dsDNA target.
[0090] Apelin: The apelin gene has been identified in various
species, including human, dog, bovine, rat, mouse, rhesus monkey,
and zebra fish and codes for an apelin preproprotein of 77 amino
acids (also referred to herein as apelin (1-77)). In processing the
apelin preproprotein, a signal peptide corresponding to residues
1-22 of the apelin preproprotein is cleaved off, resulting in a 55
amino acid (residues 23-77) apelin proprotein (also referred to
herein as apelin (23-77)). Apelin-36 is a 36 amino acid long
peptide derived from the 55 amino acid long apelin (23-77)
proprotein (Tatemoto et al., Biochem. Biophys. Res. Comm.,
251:471-476, 1998) and corresponds to residues 42-77 of the
preproprotein (also referred to herein as apelin-36 (42-77).
Apelin-17 and apelin-13 are derived from the carboxy (C)-terminal
end of apelin. Apelin-17 corresponds to residues 61-77 of the
apelin proprotein and is also referred to as apelin-17 (61-77).
Apelin-13 corresponds to residues 65-77 of the apelin proprotein
and is also referred to as apelin-13 (65-77). Salcut (also referred
to as apelin-36 (42-57) or salcut-OH) is an apelin peptide derived
from residues 42-77 of the apelin proprotein or the 16 amino acids
from the amino terminal end of apelin-36 (42-77). Salcut can be
modified, for example by amidation or addition of a glycine residue
at the carboxy-terminal end of the peptide. In frogs and opossum,
apelin is 76 amino acids long (also referred to herein as apelin
(1-76)).
[0091] APJ receptor (apelin receptor): A member of the
seven-transmembrane-domain G-protein receptor family; this receptor
is structurally related to the angiotensin II receptor type I
(ATIR). Apelin-36 (42-77), apelin-17 (61-77), and apelin-13 (65-77)
all have a carboxy terminal phenylalanine residue and can bind the
APJ receptor. A peptide antagonist version of apelin-13 (65-77),
(Ala-13)-Apelin-13 (Lee et al., Endocrinology, 146:231-236, 2005),
has an alanine residue substituted for the carboxy terminal
phenylalanine. (Ala-13)-Apelin-13 binds to the APJ receptor but
does not stimulate APJ receptor activity.
[0092] Binding or stable binding: An oligonucleotide binds or
stably binds to a target nucleic acid if a sufficient amount of the
oligonucleotide forms base pairs or is hybridized to its target
nucleic acid, to permit detection of that binding. Binding can be
detected by either physical or functional properties of the
target:oligonucleotide complex. Binding between a target and an
oligonucleotide can be detected by any procedure known to one
skilled in the art, including both functional and physical binding
assays. Binding may be detected functionally by determining whether
binding has an observable effect upon a biosynthetic process such
as expression of a coding sequence, DNA replication, transcription,
amplification and the like.
[0093] Physical methods of detecting the binding of complementary
strands of DNA or RNA are well known in the art, and include such
methods as DNase I or chemical footprinting, gel shift and affinity
cleavage assays, Northern blotting, dot blotting and light
absorption detection procedures. For example, one method that is
widely used, because it is so simple and reliable, involves
observing a change in light absorption of a solution containing an
oligonucleotide (or an analog) and a target nucleic acid at 220 to
300 nm as the temperature is slowly increased. If the
oligonucleotide or analog has bound to its target, there is a
sudden increase in absorption at a characteristic temperature as
the oligonucleotide (or analog) and target disassociate from each
other, or melt.
[0094] The binding between an oligomer and its target nucleic acid
is frequently characterized by the temperature (T.sub.m) (under
defined ionic strength and pH) at which 50% of the target sequence
remains hybridized to a perfectly matched probe or complementary
strand. A higher (T.sub.m) means a stronger or more stable complex
relative to a complex with a lower (T.sub.m).
[0095] Other art-recognized forms of stable binding occur between
an antibody and antigen, a receptor and ligand, a binding protein
and ligand, an enzyme and substrate, and a lectin and a
carbohydrate (see, for example, Pio et al, J. Biol. Chem.,
276:12292-12300, 2001). Such interactions can be used as tools to
measure activators or inhibitors of activity (for example, salcut
cell growth modulator activity). In one embodiment, a
receptor-trap, wherein a soluble receptor or binding protein binds
ligand so that the ligand is no longer available to bind to its
cognate receptor, is used as a measure of binding or stability of
binding. In another embodiment, depending on type of glycosylation,
for example on a receptor (such as a salcut receptor), a lectin
would block or enhance ligand (such as salcut) binding.
[0096] Cell growth (proliferation): Relates to growth in cell
populations or cell number by means of cell reproduction or
division. Cell growth can be modulated by an agent, or a
combination of agents, in order to enhance, stimulate, or increase
cell growth (increase the number of cells in a population) or
inhibit, decrease cell growth (maintain or decrease the number of
cells in a population). An increase or decrease in cell growth or
proliferation can be quantified using any method known to those of
skill in the art. An increase or decrease in cell growth can be
expressed as a statistically significant change in the number or
percentage of cells in a cell population in the presence of an
agent or combination of agents, compared to the same cell
population in the absence of the agent or combination of
agents.
[0097] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and transcriptional regulatory
sequences. cDNA may also contain untranslated regions (UTRs) that
are responsible for translational control in the corresponding RNA
molecule. cDNA is usually synthesized in the laboratory by reverse
transcription from messenger RNA extracted from cells or other
samples.
[0098] DNA (deoxyribonucleic acid): DNA is a long chain polymer
which comprises the genetic material of most living organisms (some
viruses have genes comprising ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which comprises one of the four bases, adenine (A), guanine
(G), cytosine (C), and thymine (T) bound to a deoxyribose sugar to
which a phosphate group is attached. Triplets of nucleotides
(referred to as codons) code for each amino acid in a polypeptide,
or for a stop signal. The term codon is also used for the
corresponding (and complementary) sequences of three nucleotides in
the mRNA into which the DNA sequence is transcribed.
[0099] Unless otherwise specified, any reference to a DNA molecule
is intended to include the reverse complement of that DNA molecule.
Except where single-strandedness is required by the text herein,
DNA molecules, though written to depict only a single strand,
encompass both strands of a double-stranded DNA molecule. Thus, a
reference to the nucleic acid molecule that encodes a specific
protein, or a fragment thereof, encompasses both the sense strand
and its reverse complement. Thus, for instance, it is appropriate
to generate probes or primers from the reverse complement sequence
of the disclosed nucleic acid molecules.
[0100] Hybridization: Oligonucleotides and their analogs hybridize
by hydrogen bonding, which includes Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary bases.
Generally, nucleic acid consists of nitrogenous bases that are
either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or
purines (adenine (A) and guanine (G)). These nitrogenous bases form
hydrogen bonds between a pyrimidine and a purine, and the bonding
of the pyrimidine to the purine is referred to as "base pairing."
More specifically, A will hydrogen bond to T or U, and G will bond
to C. "Complementary" refers to the base pairing that occurs
between two distinct nucleic acid sequences or two distinct regions
of the same nucleic acid sequence.
[0101] "Specifically hybridizable" and "specifically complementary"
are terms that indicate a sufficient degree of complementarity such
that stable and specific binding occurs between the oligonucleotide
(or its analog) and the DNA or RNA target. The oligonucleotide or
oligonucleotide analog need not be 100% complementary to its target
sequence to be specifically hybridizable. An oligonucleotide or
analog is specifically hybridizable when binding of the
oligonucleotide or analog to the target DNA or RNA molecule
interferes with the normal function of the target DNA or RNA, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the oligonucleotide or analog to non-target
sequences under conditions where specific binding is desired, for
example under physiological conditions in the case of in vivo
assays or systems. Such binding is referred to as specific
hybridization.
[0102] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ concentration) of
the hybridization buffer will determine the stringency of
hybridization, though waste times also influence stringency.
Calculations regarding hybridization conditions required for
attaining particular degrees of stringency are discussed by
Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, chapters 9 and 11, herein incorporated by
reference.
[0103] For present purposes, "stringent conditions" encompass
conditions under which hybridization will only occur if there is
less than 25% mismatch between the hybridization molecule and the
target sequence. "Stringent conditions" may be broken down into
particular levels of stringency for more precise definition. Thus,
as used herein, "moderate stringency" conditions are those under
which molecules with more than 25% sequence mismatch will not
hybridize; conditions of "medium stringency" are those under which
molecules with more than 15% mismatch will not hybridize, and
conditions of "high stringency" are those under which sequences
with more than 10% mismatch will not hybridize. Conditions of "very
high stringency" are those under which sequences with more than 6%
mismatch will not hybridize.
[0104] Injectable composition: A pharmaceutically acceptable fluid
composition comprising at least one active ingredient, for example,
a peptide derived from apelin, such as the amide derivative of
apelin-36 (42-57). The active ingredient is usually dissolved or
suspended in a physiologically acceptable carrier, and the
composition can additionally comprise minor amounts of one or more
non-toxic auxiliary substances, such as emulsifying agents,
preservatives, and pH buffering agents and the like. Such
injectable compositions that are useful for use with the
compositions of this disclosure are conventional; appropriate
formulations are well known in the art.
[0105] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or portion of a tissue) that has
been substantially separated or purified away from other biological
components in the tissue or cell of the organism in which the
component naturally occurs. An "isolated" cell is a cell that has
been purified from the other cellular components of a tissue. Cells
can be isolated by, for instance mechanical and/or enzymatic
methods.
[0106] Nucleic acids and proteins that have been "isolated" include
nucleic acids and proteins purified by standard purification
methods. The term also embraces nucleic acids and proteins prepared
by recombinant expression in a host cell as well as chemically
synthesized nucleic acids.
[0107] Labeled: A biomolecule, such as a peptide or a specific
binding agent, attached covalently or noncovalently to a detectable
label or reporter molecule. Typical labels include radioactive
isotopes, enzyme substrates, co-factors, ligands, chemiluminescent
or fluorescent agents, haptens, and enzymes. Methods for labeling
and guidance in the choice of labels appropriate for various
purposes are discussed, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual, CSHL, New York, 1989 and Ausubel et
al., Current Protocols in Molecular Biology, Greene Publ. Assoc.
and Wiley-Intersciences, 1998.
[0108] Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects.
[0109] Nucleotide: "Nucleotide" includes, but is not limited to, a
monomer that includes a base linked to a sugar, such as a
pyrimidine, purine or synthetic analogs thereof, or a base linked
to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide
is one monomer in an oligonucleotide/polynucleotide. A nucleotide
sequence refers to the sequence of bases in an
oligonucleotide/polynucleotide.
[0110] The major nucleotides of DNA are deoxyadenosine
5'-triphosphate (dATP or A), deoxyguanosine 5'-triphosphate (dGTP
or G), deoxycytidine 5'-triphosphate (dCTP or C) and deoxythymidine
5'-triphosphate (dTTP or T). The major nucleotides of RNA are
adenosine 5'-triphosphate (ATP or A), guanosine 5'-triphosphate
(GTP or G), cytidine 5'-triphosphate (CTP or C) and uridine
5'-triphosphate (UTP or U). Inosine is also a base that can be
integrated into DNA or RNA in a nucleotide (dITP or ITP,
respectively).
[0111] Oligonucleotide: An oligonucleotide is a plurality of joined
nucleotides joined by native phosphodiester bonds, between about 6
and about 500 nucleotides in length. An oligonucleotide analog
refers to moieties that function similarly to oligonucleotides but
have non-naturally occurring portions. For example, oligonucleotide
analogs can contain non-naturally occurring portions, such as
altered sugar moieties or inter-sugar linkages, such as a
phosphorothioate oligodeoxynucleotide. Functional analogs of
naturally occurring polynucleotides can bind to RNA or DNA, and
include peptide nucleic acid (PNA) molecules.
[0112] Particular oligonucleotides and oligonucleotide analogs can
include linear sequences up to about 300 nucleotides in length, for
example a sequence (such as DNA or RNA) that is at least 6 bases,
for example at least 8, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50,
100 or even 200 or more bases long, or from about 6 to about 50
bases, for example about 8-25 bases, such as 10, 12, 15, 17, 20, or
25 bases.
[0113] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0114] Parenteral: Administered outside of the intestine, For
example, not via the alimentary tract. Generally, parenteral
formulations are those that will be administered through any
possible mode except ingestion. This term especially refers to
injections, whether administered intravenously, intrathecally,
intramuscularly, intraperitoneally, or subcutaneously, and various
surface applications including intranasal, intradermal, and topical
application, for instance.
[0115] Peptide: "Peptides," "polypeptides," and "oligopeptides" are
chains of amino acids (typically L-amino acids) whose alpha carbons
are linked through peptide bonds formed by a condensation reaction
between the carboxyl group of the alpha carbon of one amino acid
and the amino group of the alpha carbon of another amino acid. The
terminal amino acid at one end of the chain (for example, the amino
terminal) has a free amino group, while the terminal amino acid at
the other end of the chain (for example, the carboxy terminal) has
a free carboxyl group. As such, the term "amino terminus"
(abbreviated N-terminus) refers to the free alpha-amino group on
the amino acid at the amino terminal end of the peptide, or to the
alpha-amino group (imino group when participating in a peptide
bond) of an amino acid at any other location within the peptide.
The term "carboxy terminus" (abbreviated C-terminus) refers to the
free carboxyl group on the amino acid at the carboxy terminal end
of a peptide, or to the carboxyl group of an amino acid at any
other location within the peptide.
[0116] Typically, the amino acids making up a peptide are numbered
in order, starting at the amino terminus and increasing in the
direction toward the carboxy terminus of the peptide. Thus, when
one amino acid is said to "follow" another, that amino acid is
positioned closer to the carboxy terminal end of the peptide than
the preceding amino acid.
[0117] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the compounds herein disclosed.
[0118] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0119] A "pharmaceutical agent" or "drug" refers to a chemical
compound or other composition (including peptide based
pharmaceuticals) capable of inducing a desired therapeutic or
prophylactic effect when properly administered to a subject.
[0120] Polypeptide: A polymer in which the monomers are amino acid
residues that are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used, the L-isomers being preferred in
nature. The term polypeptide or protein as used herein encompasses
any amino acid sequence and includes, but may not be limited to,
modified sequences such as glycoproteins or amidated proteins. The
term polypeptide is specifically intended to cover naturally
occurring proteins, as well as those that are recombinantly or
synthetically produced.
[0121] Substantially purified polypeptide as used herein refers to
a polypeptide that is substantially free of other proteins, lipids,
carbohydrates or other materials with which it is naturally
associated. In one embodiment, the polypeptide is at least 50%, for
example at least 80% free of other proteins, lipids, carbohydrates
or other materials with which it is naturally associated. In
another embodiment, the polypeptide is at least 90% free of other
proteins, lipids, carbohydrates or other materials with which it is
naturally associated. In yet another embodiment, the polypeptide is
at least 95% free of other proteins, lipids, carbohydrates or other
materials with which it is naturally associated.
[0122] Conservative amino acid substitution tables providing
functionally similar amino acids are well known to one of ordinary
skill in the art. The following six groups are examples of amino
acids that are considered to be conservative substitutions for one
another:
[0123] 1) Alanine (A), Serine (S), Threonine (T);
[0124] 2) Aspartic acid (D), Glutamic acid (E);
[0125] 3) Asparagine (N), Glutamine (Q);
[0126] 4) Arginine (R), Lysine (K);
[0127] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0128] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0129] A non-conservative amino acid substitution can result from
changes in: (a) the structure of the amino acid backbone in the
area of the substitution; (b) the charge or hydrophobicity of the
amino acid; or (c) the bulk of an amino acid side chain.
Substitutions generally expected to produce the greatest changes in
protein properties are those in which: (a) a hydrophilic residue is
substituted for (or by) a hydrophobic residue; (b) a proline is
substituted for (or by) any other residue; (c) a residue having a
bulky side chain, e.g., phenylalanine, is substituted for (or by)
one not having a side chain, e.g., glycine; or (d) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histadyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl.
[0130] Variant amino acid sequences may, for example, be 80, 90 or
even 95 or 98% identical to the native amino acid sequence.
Programs and algorithms for determining percentage identity can be
found at the NCBI website.
[0131] Post-translational modification: The modification of a newly
formed protein; may involve deletion of amino acids, chemical
modification of certain amino acids (for example, amidation,
acetylation, phosphorylation, glycosylation, formation of
pyroglutamate, oxidation/reduction of sulfa group on a methionine,
or addition of similar small molecules) to certain amino acids.
[0132] Preproprotein: A biologically inert polypeptide that is
post-translationally modified to yield one or more biologically
active peptides. The maturation pathway of preproproteins involves
the proteolytic cleavage of an amino terminal signal peptide to
yield a proprotein, which can have biological activity. Enzymatic
processing of the resulting proprotein can yield one or more
biologically active peptides.
[0133] Probes and primers: Nucleic acid probes and primers can be
readily prepared based on the nucleic acid molecules provided as
indicators of disease or disease progression. It is also
appropriate to generate probes and primers based on fragments or
portions of these nucleic acid molecules. Also appropriate are
probes and primers specific for the reverse complement of these
sequences, as well as probes and primers to 5' or 3' regions.
[0134] A probe comprises an isolated nucleic acid attached to a
detectable label or other reporter molecule. Typical labels and
reporter molecules include radioactive isotopes, enzyme substrates,
co-factors, ligands, chemiluminescent or fluorescent agents,
haptens, and enzymes. Methods for labeling and guidance in the
choice of labels appropriate for various purposes are discussed,
e.g., in Sambrook et al. (In Molecular Cloning: A Laboratory
Manual, CSHL, New York, 1989) and Ausubel et al. (In Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
1998).
[0135] Primers are short nucleic acid molecules, for instance DNA
oligonucleotides 8 nucleotides or more in length. Longer DNA
oligonucleotides may be about 10, 12, 15, 17, 20, 25, 30 or 50
nucleotides or more in length. Primers can be annealed to a
complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand, and
then the primer extended along the target DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification of a
nucleic acid sequence, e.g., by the polymerase chain reaction (PCR)
or other in vitro nucleic-acid amplification methods known in the
art.
[0136] Methods for preparing and using nucleic acid probes and
primers are described, for example, in Sambrook et al. (In
Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989),
Ausubel et al. (ed.) (In Current Protocols in Molecular Biology,
John Wiley & Sons, New York, 1998), and Innis et al. (PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc., San Diego, Calif., 1990). Amplification primer pairs (for
instance, for use with polymerase chain reaction amplification) can
be derived from a known sequence such as the salcut sequences
described herein, for example, by using computer programs intended
for that purpose such as Primer (Version 0.5, .COPYRGT. 1991,
Whitehead Institute for Biomedical Research, Cambridge, Mass.).
[0137] One of ordinary skill in the art will appreciate that the
specificity of a particular probe or primer increases with its
length. Thus, for example, a primer comprising 15 consecutive
nucleotides of a salcut protein-encoding nucleotide will anneal to
a target sequence, such as another homolog of the designated salcut
protein, with a higher specificity than a corresponding primer of
only 8 or 10 nucleotides. Thus, in order to obtain greater
specificity, probes and primers can be selected that comprise at
least 12, 15, 17, 20, 23, 25, 27, 30 or more consecutive
nucleotides of a salcut protein-encoding nucleotide sequences.
[0138] Also provided are isolated nucleic acid molecules that
comprise specified lengths of the disclosed salcut nucleotide
sequences. Such molecules may comprise at least 8, 10, 12, 15, 20,
23, 25 or more consecutive nucleotides of these sequences. These
molecules may be obtained from any region of the disclosed
sequences.
[0139] Purified: A "purified" biological component (such as a
nucleic acid molecule, protein or portion of a tissue) that has
been substantially separated or purified away from other biological
components in the tissue or cell of the organism in which the
component naturally occurs. A "purified" cell is a cell that has
been purified from the other cellular components of a tissue. Cells
can be purified by, for instance mechanical and/or enzymatic
methods.
[0140] Nucleic acids and proteins that have been "purified" include
nucleic acids and proteins purified by standard purification
methods. The term also embraces nucleic acids and proteins prepared
by recombinant expression in a host cell as well as chemically
synthesized nucleic acids.
[0141] The term "purified" does not require absolute purity;
rather, it is intended as a relative term.
[0142] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination can be
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids, for
example, by genetic engineering techniques.
[0143] Salcut (Selective Apelin 36 Cutting): An apelin peptide
derived from the apelin preproprotein (1-77). Salcut generally
refers to any peptide derived from residues 42-57 of apelin (1-77).
Salcut is more specifically derived from the amino (N)-terminal
region (residues 42-57) of apelin-36 (42-77). Salcut can have a
modified C-terminal glycine that has an amino (--NH.sub.2) group
substituted for its hydroxyl (--OH) group. This modified peptide is
known as salcut-NH.sub.2 or apelin-36 (42-57)-NH.sub.2 (FIGS. 1 and
2). A salcut peptide can also be the free-acid derivative of
salcut-NH.sub.2 (salcut-OH; also referred to as apelin-36 (42-57))
or a glycine-extended apelin-36 (42-58) (salcut-glycine;
salcut-gly).
[0144] Sample: Includes biological samples such as those derived
from a human or other animal source (for example, blood, sweat,
tears, breast milk, bone marrow, stool, sera, urine, saliva, tears,
biopsy samples, bronchioalveolar lavage fluids, histology tissue
samples, cellular smears, moles, warts, body secretions etc.);
bacterial or viral preparations; cell cultures; forensic samples;
agricultural products; waste or drinking water; milk or other
processed foodstuff; air; and so forth. Samples containing a small
number of cells can be acquired by any one of a number of methods,
such as needle aspiration, biopsy, or tissue scrapes.
[0145] Sequence identity: The similarity between two nucleic acid
sequences, or two amino acid sequences, is expressed in terms of
the similarity between the sequences, otherwise referred to as
sequence identity. Sequence identity is frequently measured in
terms of percentage identity (or similarity or homology); the
higher the percentage, the more similar the two sequences are.
Homologs or orthologs of the disclosed apelin-36 (42-57) peptides,
and the corresponding cDNA sequences, will possess a relatively
high degree of sequence identity when aligned using standard
methods. This homology will be more significant when the
orthologous proteins or genes or cDNAs are derived from species
that are more closely related (e.g., human and chimpanzee
sequences), compared to species more distantly related (e.g., human
and C. elegans sequences).
[0146] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith & Waterman Adv. Appl. Math. 2: 482, 1981;
Needleman & Wunsch J. Mol. Biol. 48: 443, 1970; Pearson &
Lipman Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins &
Sharp Gene, 73: 237-244, 1988; Higgins & Sharp CABIOS 5:
151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988;
Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992;
and Pearson et al. Meth. Mol. Bio. 24, 307-31, 1994. Altschul et
al. (J. Mol. Biol. 215:403-410, 1990), presents a detailed
consideration of sequence alignment methods and homology
calculations.
[0147] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al. J. Mol. Biol. 215:403-410, 1990) is available from several
sources, including the National Center for Biotechnology
Information (NCBI, Bethesda, Md.) and on the Internet, for use in
connection with the sequence analysis programs blastp, blastn,
blastx, tblastn and tblastx. When aligning short peptides (fewer
than around 30 amino acids), the alignment is performed using the
Blast 2 sequences function, employing the PAM30 matrix set to
default parameters (open gap 9, extension gap 1 penalties).
[0148] An alternative indication that two nucleic acid molecules
are closely related is that the two molecules hybridize to each
other under stringent conditions. Stringent conditions are
sequence-dependent and are different under different environmental
parameters. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence remains
hybridized to a perfectly matched probe or complementary strand.
Conditions for nucleic acid hybridization and calculation of
stringencies can be found in Sambrook et al. (In Molecular Cloning:
A Laboratory Manual, CSHL, New York, 1989) and Tijssen (Laboratory
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York,
1993). Nucleic acid molecules that hybridize under stringent
conditions to a salcut protein-encoding sequence will typically
hybridize to a probe based on either an entire salcut
protein-encoding sequence or selected portions of the encoding
sequence under wash conditions of 2.times.SSC at 50.degree. C.
[0149] Nucleic acid sequences that do not show a high degree of
sequence identity may nevertheless encode similar amino acid
sequences, due to the degeneracy of the genetic code. It is
understood that changes in nucleic acid sequence can be made using
this degeneracy to produce multiple nucleic acid molecules that all
encode substantially the same protein.
[0150] Small molecule inhibitor: An inhibitor of at least one
function of a target molecule, with a molecular weight preferably
below about 1000 Daltons.
[0151] Specific binding agent: An agent that binds substantially
only to a defined target. Thus an apelin-36 (42-57) specific
binding agent is an agent that binds substantially to apelin-36
(42-57). In one embodiment, the specific binding agent is a
monoclonal antibody or a polyclonal antibody that specifically
apelin-36 (42-57). In particular a embodiment, the monoclonal
antibody is humanized.
[0152] Subject: Any vertebrate that has a vascular system and has
hematopoietic cells in the wild-type organism. The term subject
includes non-human mammals such as a monkey, mouse, rat, rabbit,
pig, goat, sheep or cow. It also includes humans. It is understood
that a cell or cell line in culture can be referred to as obtained
from a subject even though the cell has been in culture for a
length of time, even years.
[0153] Therapeutic: Therapeutic uses of apelin-36 (42-57) include
administration for the inhibition, reversal or prevention of
pathological conditions, such as neoplasia, hypertension,
preeclampsia syndrome, diabetes, ocular degeneration, idiopathic
pulmonary fibrosis, wound healing, abnormal angiogenesis, altered
mast cell migration, chronic obstructive pulmonary disease,
inflammatory diseases such as arthritis (juvenile and rheumatoid)
and inflammatory bowel disease, cardiovascular disease, avascular
or ischemic insult, myocardial infarction, stroke,
vasculititis/angiitis, systemic or vascular sclerosis, gangrene,
congelation (severe frostbite), alopecia, eczema, ulcers,
lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, or endometriosis.
[0154] Therapeutically effective amount: A quantity of compound,
such as the peptide salcut-NH.sub.2 (apelin-36 (42-57)) or a
specific inhibitor of salcut-NH.sub.2, sufficient to achieve a
desired effect in a subject being treated. For instance, this can
be the amount necessary to treat or ameliorate any one of a number
of diseases, such as neoplasia, hypertension, preeclampsia
syndrome, diabetes, ocular degeneration, idiopathic pulmonary
fibrosis, wound healing, abnormal angiogenesis, altered mast cell
migration, chronic obstructive pulmonary disease, inflammatory
diseases such as arthritis (juvenile and rheumatoid) and
inflammatory bowel disease, cardiovascular disease, avascular or
ischemic insult, myocardial infarction, stroke,
vasculititis/angiitis, systemic or vascular sclerosis, gangrene,
congelation (severe frostbite), alopecia, eczema, ulcers,
lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, or endometriosis, in a subject. In
some embodiments, it is the amount necessary to treat a subject by
a measurable amount over a period of time, or to measurably inhibit
progression of disease, in a subject. In other embodiments, a
therapeutically effective amount is the amount necessary to
prophylactically inhibit a disease.
[0155] An effective amount of salcut, for example salcut-NH.sub.2,
may be administered in a single dose, or in several doses, for
example daily, during a course of treatment. However, the effective
amount will be dependent on the compound applied, the subject being
treated, the severity and type of the affliction, and the manner of
administration of the compound.
[0156] Tumor: A neoplasm that may be either malignant or
non-malignant. Tumors originating in a particular organ (such as
breast, prostate, bladder or lung) are primary tumors. Tumors of
the same tissue type may be divided into tumor of different
sub-types (a classic example being bronchogenic carcinomas (lung
tumors) which can be an adenocarcinoma, small cell, squamous cell,
or large cell tumor).
[0157] Transformed: A transformed cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. As used herein, the term transformation encompasses all
techniques by which a nucleic acid molecule might be introduced
into such a cell, including transfection with viral vectors,
transformation with plasmid vectors, and introduction of naked DNA
by electroporation, lipofection, and particle gun acceleration.
[0158] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more selectable marker genes and other genetic
elements known in the art.
[0159] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents and other references mentioned herein are
incorporated by reference in their entirety. All sequence database
references are incorporated by reference as of Feb. 27, 2009,
unless specified otherwise. In case of conflict, the present
specification, including explanations of terms, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
III. Overview of Several Embodiments
[0160] Disclosed herein are isolated apelin-36 (42-57) and
apelin-36 (42-58) polypeptides having an amino acid sequence set
forth as SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:
30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. The
polypeptides have cell growth modulating activity and can have up
to four amino acid substitutions, which substitutions are not at
the last position of the amino acid sequence. Alternatively, the
polypeptides can have up to three, two, one, or no amino acid
substitutions.
[0161] Also provided herein are polypeptides comprising a
polypeptide comprising an amino acid sequence consisting of SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,
SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,
SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID
NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43, wherein the amino terminal
end of the amino acid sequence is attached to a heterologous amino
acid sequence, label, or reporter molecule.
[0162] Pharmaceutical compositions, comprising the disclosed
polypeptide in a pharmaceutically acceptable carrier are also
envisioned, as are inhibitors or activators of the cell growth
modulating activity of the polypeptides. Such inhibitors and
activators include peptides, small molecules, receptors, binding
proteins, salcut-Gly, free-acid form of apelin-36 (42-57), or
neutralizing antibodies. Also provided herein are methods of
treating a tumor or a disease caused by abnormal angiogenesis in a
subject. The method includes administering to a subject that has a
tumor or a disease caused by abnormal angiogenesis a
therapeutically effective amount of at least one of the disclosed
inhibitors. Administration of an inhibitor modifies tumor cell
growth or endothelial cell growth in the subject, thereby treating
the tumor or disease caused by abnormal angiogenesis of the
subject. The method also includes administering to a subject that
has a disease caused by abnormal angiogenesis a therapeutically
effective amount of the disclosed activators. Administration of an
activator modifies endothelial cell growth in the subject, thereby
treating the disease caused by abnormal angiogenesis of the
subject.
[0163] In addition, disclosed herein are isolated antibodies that
bind to an epitope that is specific to the disclosed polypeptides
and does not bind to apelin-36 (42-77), apelin-17 (61-77), or
apelin-13 (65-77). The antibodies can be monoclonal antibodies. In
one embodiment, the monoclonal antibody is humanized. Nucleic acid
sequences encoding the disclosed polypeptides are also provided
herein.
[0164] A method of modulating cell growth is provided herein. The
method comprises administering the disclosed polypeptides to a cell
sample, thereby modulating cell growth. A modulation in cell growth
can be an increase or a decrease in cell growth. The cells can be
endothelial cells or tumor cells. In one embodiment, the method
includes administering the disclosed polypeptides to a cell sample
in vivo.
[0165] It is further disclosed a method of treating a tumor or a
disease caused by abnormal angiogenesis of a subject, comprising
administering to the subject a therapeutically effective amount of
the disclosed polypeptides, wherein the subject has a tumor or a
disease caused by abnormal angiogenesis and administration of the
isolated polypeptide modifies tumor cell growth or endothelial cell
growth in the subject, thereby treating the tumor or disease caused
by abnormal angiogenesis of the subject.
[0166] A method of diagnosing severity of a disease is also
provided herein. The method comprises measuring the level of the
disclosed polypeptides in a biological sample, wherein a change in
the level of the isolated polypeptide correlates with severity of
disease, thereby diagnosing severity of a disease. In one
embodiment of the method, measuring the level of the isolated
polypeptide comprises contacting the biological sample with an
antibody that is specific to the disclosed polypeptide and that
does not bind to apelin-36 (42-77), apelin-17 (61-77), or apelin-13
(65-77).
[0167] Diseases treated or diagnosed, using the methods disclosed
herein, include neoplasia, cardiovascular disease, peripheral
vascular disease, hypertension, preeclampsia syndrome, abnormal
angiogenesis, diabetes, ocular degeneration, idiopathic pulmonary
fibrosis, wound healing, altered mast cell migration, chronic
obstructive pulmonary disease, inflammatory diseases such as
arthritis (juvenile and rheumatoid) and inflammatory bowel disease,
cardiovascular disease, avascular or ischemic insult, myocardial
infarction, stroke, vasculititis/angiitis, systemic or vascular
sclerosis, gangrene, congelation (severe frostbite), alopecia,
eczema, ulcers, lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, or endometriosis.
IV. Apelin and Related Peptides
[0168] Apelin cDNA has been identified in various species,
including human (NM.sub.--017413; SEQ ID NO: 1), dog
(ENSCAFT00000029682; SEQ ID NO: 2), bovine (NM.sub.--174503 or
ENSBTAT00000026630; SEQ ID NO: 3), rat (GenBank Accession No.
NM.sub.--031612; SEQ ID NO: 4), mouse (NM.sub.--013912; SEQ ID NO:
5), zebra fish (DQ062434; SEQ ID NO: 7), and rhesus monkeys
(ENSMMUT00000003625; SEQ ID NO: 39). Each of these cDNAs code for a
preproprotein of 77 amino acids (SEQ ID NOs: 8-14 and 40,
respectively; see also the following Accession Nos.:
human--NP.sub.--059109 or AAF25815; dog--ENSCAFP00000027587;
bovine--NP.sub.--776928 or ENSBTAP00000026630;
rat--NP.sub.--113800.1; mouse--NP.sub.--038940; zebra
fish--AAY46798; rhesus monkey--ENSMMUP00000003428). Frog cDNA
(NM.sub.--001097924; SEQ ID NO: 36) and opossum cDNA
(ENSMODT00000016827; SEQ ID NO: 6) encode for a preprotein of 76
amino acids (NP.sub.--001091393; SEQ ID NO: 37 and
ENSMODP00000016523, respectively). The content of the Accession
Numbers listed for human, dog, bovine, rat, mouse, opossum, frog,
and zebra fish is incorporated by reference as of Feb. 27, 2009.
The content of the Accession Numbers listed for rhesus monkey is
incorporated by reference as of Mar. 1, 2010. The amino acid
sequence of the apelin preproprotein (residues 1-77, or 1-76 for
opossum) is highly conserved across species, with identity in the
carboxy (C)-terminal region. Apelin-36 (see, for example, the human
sequence; SEQ ID NO: 15) is a 36 amino acid peptide derived from
the 77 amino acid apelin (1-77) preproprotein (Tatemoto et al.,
Biochem. Biophys. Res. Comm., 251:471-476, 1998) and corresponds to
residues 42-77 of the C-terminal region of the apelin (1-77)
preproprotein (FIG. 1). Apelin-17 (for example, SEQ ID NO: 16) and
apelin-13 (for example, SEQ ID NO: 17) are also derived from the
C-terminal end of apelin (residues 61-77 and 65-77, respectively;
FIG. 1).
[0169] Peptide-induced acidification rates of cells expressing the
apelin G protein receptor (known as APJ) have been shown to
increase in potency with decreased apelin peptide size, such that
apelin-36 is least potent, apelin-17 is moderately potent, and
apelin-13 is most potent (Tatemoto et al., Biochem. Biophys. Res.
Comm., 251:471-476, 1998). Apelin-13 has also been shown to have
mitogenic activity and to have a specific effect on
neoangiogenesis/endothelial cell growth (Kahn et al., Dev. Biol.,
305:599-614, 2007; Masri et al., FASEB Journal express article
10.1096/fj.04-1930fje. Published online Sep. 22, 2004, 26 pages;
Sorli et al., Oncogene, 26:7692-7699, 2007). Both apelin-36 and
apelin-13 bind the APJ apelin receptor and regulate the same set of
intracellular effectors; however, they display different
desensitization patterns on the APJ receptor, which may explain
their varying physiological responses (Masri et al., J. Biol. Chem.
281:18317-18326, 2006).
V. Apelin-36 (42-57) Peptides and Nucleic Acids
[0170] It has been surprisingly discovered that a new biologically
active peptide can be derived from apelin-36 (42-77). This sixteen
amino acid peptide of human apelin-36 (42-77) (referred to herein
as human salcut, human salcut-OH, or human apelin-36 (42-57); SEQ
ID NO: 18), derived from the amino (N)-terminal region (residues
42-57) of human apelin-36, optionally has a modified C-terminal
glycine that has an amino (--NH.sub.2) group substituted for its
hydroxyl (--OH) group. This C-terminal modified peptide is referred
to as an amidation or amide derivative of human apelin-36 (42-57),
and is also known as human salcut-NH.sub.2 or apelin-36
(42-57)-NH.sub.2 (SEQ ID NO: 19; FIGS. 1 and 2). The
glycine-extended human apelin-36 (42-58) (salcut-glycine;
salcut-gly; SEQ ID NO: 20) is also provided (FIG. 1).
[0171] An amidation or amide derivative of this peptide can also be
derived from the N-terminal region (residues 42-57) (based on the
numbering of the human apelin sequence) of apelin-36 from dog (SEQ
ID NO: 22), bovine (SEQ ID NO: 25), rat (SEQ ID NO: 28), mouse (SEQ
ID NO: 31), rhesus monkey (SEQ ID NO: 42), and opossum (SEQ ID NO:
34) apelin. In addition, a glycine-extended apelin-36 (42-58)
(based on the numbering of the human apelin sequence) sequences
from dog (SEQ ID NO: 23), bovine (SEQ ID NO: 26), rat (SEQ ID NO:
29), mouse (SEQ ID NO: 32), rhesus monkey (SEQ ID NO: 43), and
opossum (SEQ ID NO: 35) are provided.
[0172] The salcut consensus amidation motif (G-R-R or G-R-K) is
located, for example, at residues 58-60 of human, bovine, dog, rat,
mouse, and rhesus monkey apelin (1-77) and at residues 57-59 of
opossum apelin (1-76) (FIG. 2). In other embodiments, the salcut
amidation motif is G-K-K or G-K-R. The amidation motif includes a
glycine followed by no basic amino acid, or by one or more basic
amino acids. Salcut and its biologically active derivatives may be
isolated from nature, or synthesized in vitro using techniques well
known to those of skill in the art. Representative production
techniques are provided herein.
[0173] With the provision herein of the apelin-36 (42-57) amino
acid and cDNA sequences, in vitro nucleic acid amplification (such
as polymerase chain reaction (PCR)) may be utilized as a simple
method for producing apelin-36 (42-57) nucleic acid sequences, and
variants thereof. The selection of amplification primers will be
made according to the portion(s) of the apelin preproprotein cDNA
that is to be amplified, for example the portion of apelin (1-77)
including apelin-36 (42-57). In one embodiment, primers may be
chosen to amplify a segment of a cDNA that encodes the apelin-36
(42-57) polypeptide. Variations in amplification conditions may be
useful to optimize amplification conditions when using primers and
amplicons of differing lengths and composition; such considerations
are well known in the art and are discussed for instance in Innis
et al. (PCR Protocols, A Guide to Methods and Applications,
Academic Press, Inc., San Diego, Calif., 1990).
[0174] Re-sequencing of PCR products obtained by amplification
procedures optionally can be performed to facilitate confirmation
of the amplified sequence and provide information about natural
variation of this sequence in different populations or species.
Oligonucleotides derived from the known or provided apelin
sequences may be used in such sequencing methods.
[0175] In one embodiment, primers or oligonucleotides may comprise
a sequence of at least 8 consecutive nucleotides of the apelin
(1-77) or apelin-36 (42-57) nucleic acid sequence. If these primers
or oligonucleotides are used with an in vitro amplification
procedure (such as PCR), lengthening the primers or
oligonucleotides may enhance amplification specificity. Thus, in
other embodiments, oligonucleotides or primers comprising at least
10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, or more consecutive
nucleotides of these sequences may be used. These oligonucleotides
or primers, for instance, may be obtained from any region of the
disclosed sequences.
VI. Apelin-36 (42-57) Sequence Variants
[0176] With the provision of apelin-36 (42-57) protein (amidated or
free-acid forms) and corresponding nucleic acid sequences herein,
the creation of variants of these sequences is now enabled.
[0177] In one embodiment, variant apelin-36 (42-57) proteins
include proteins that differ in amino acid sequence from the
apelin-36 (42-57) sequences disclosed but that share at least 72%
amino acid sequence identity with the provided apelin-36 (42-57)
protein. In other embodiments, other variants will share at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, or at least 99% amino acid sequence identity.
Manipulation of the nucleotide sequence of apelin-36 (42-57) using
standard procedures, including in one specific, non-limiting,
embodiment, site-directed mutagenesis or in another specific,
non-limiting, embodiment, PCR, can be used to produce such
variants. The simplest modifications involve the substitution of
one or more amino acids for amino acids having similar biochemical
properties. These so-called conservative substitutions are likely
to have minimal impact on the activity of the resultant protein.
One would avoid substituting the residues of the salcut consensus
amidation motif (for example, G-R-R or G-R-K), as well as any
residues that have not diverged among mammalian species (FIG.
2).
[0178] Orthologs of apelin-36 (42-57) (amidated or free-acid forms;
based on the numbering of the human sequence) can be isolated. In
one embodiment, orthologs will generally share at least 65%
sequence identity with the disclosed apelin-36 (42-57) cDNA. Where
the orthologous species is more closely related to the subject
species, the sequence identity will in general be greater. In other
embodiments, closely related orthologous apelin-36 (42-57)
molecules may share at least 70%, at least 75%, at least 80% at
least 85%, at least 90%, at least 91%, at least 93%, at least 95%,
at least 98%, or at least 99% sequence identity with the disclosed
apelin-36 (42-57) nucleotide or amino acid sequences.
[0179] Additional aspects of the disclosure include analogs,
derivatives, and mimetics based on the amino acid sequence of the
apelin-36 (42-57) peptides (amidated or free-acid forms) disclosed
herein. Typically, mimetic compounds are synthetic compounds having
a three-dimensional structure (of at least part of the mimetic
compound) that mimics, for example, the primary, secondary, and/or
tertiary structural, and/or electrochemical characteristics of a
selected peptide, structural domain, active site, or binding region
(e.g., a homotypic or heterotypic binding site, a catalytic active
site or domain, a receptor or ligand binding interface or domain,
or a structural motif) thereof. The mimetic compound will often
share a desired biological activity with a native peptide, as
discussed herein (e.g., cell growth modulating activity).
Typically, at least one subject biological activity of the mimetic
compound is not substantially reduced in comparison to, and is
often the same as or greater than, the activity of the native
peptide on which the mimetic was modeled.
[0180] A variety of techniques well known to one of skill in the
art are available for constructing peptide mimetics with the same,
similar, increased, or reduced biological activity as the
corresponding native peptide. Often these analogs, variants,
derivatives and mimetics will exhibit one or more desired
activities that are distinct or improved from the corresponding
native peptide, for example, improved characteristics related to
the modulation of cell growth.
[0181] In another embodiment, more substantial changes in apelin-36
(42-57) activity may be obtained by selecting amino acid
substitutions that are less conservative than conservative
substitutions. In one specific, non-limiting, embodiment, such
changes include changing residues that differ more significantly in
their effect on maintaining polypeptide backbone structure (e.g.,
sheet or helical conformation) near the substitution, charge or
hydrophobicity of the molecule at the target site, or bulk of a
specific side chain. The following specific, non-limiting, examples
are generally expected to produce the greatest changes in protein
properties: (a) a hydrophilic residue (e.g., seryl or threonyl) is
substituted for (or by) a hydrophobic residue (e.g., leucyl,
isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine or
proline is substituted for (or by) any other residue; (c) a residue
having an electropositive side chain (e.g., lysyl, arginyl, or
histadyl) is substituted for (or by) an electronegative residue
(e.g., glutamyl or aspartyl); or (d) a residue having a bulky side
chain (e.g., phenylalanine) is substituted for (or by) one lacking
a side chain (e.g., glycine).
[0182] In other embodiments, changes in apelin-36 (42-57) activity
or other protein features may be obtained by mutating, substituting
or deleting regions of apelin-36 (42-57) that have a known
function, regions where the function is yet to be determined, or
regions that are known to be highly conserved or not conserved.
[0183] In another embodiment, a detectable moiety can be linked to
the apelin-36 (42-57) peptides disclosed herein, creating a
peptide-detectable moiety conjugate or fusion protein. Detectable
moieties suitable for such use include any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. The detectable moieties
contemplated for the present disclosure can include, but are not
limited to, a fluorescent moiety (e.g., fluorescein, rhodamine,
Texas red, and the like), a radioactive moiety (e.g., .sup.3H,
.sup.32P, .sup.125I, .sup.35S), an enzyme moiety (e.g., horseradish
peroxidase, alkaline phosphatase), a colorimetric moiety (e.g.,
colloidal gold, biotin, colored glass or plastic, and the like).
The detectable moiety can be linked to the apelin-36 (42-57)
peptide at either the N- and/or C-terminus. Optionally, a linker
can be included between the apelin-36 (42-57) peptide and the
detectable moiety.
[0184] Means of detecting such moieties are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
illumination. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
[0185] Variant apelin-36 (42-57)-encoding sequences may be produced
by standard DNA mutagenesis techniques. In one specific,
non-limiting, embodiment, M13 primer mutagenesis is performed.
Details of these techniques are provided in Sambrook et al. (In
Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ch.
15. By the use of such techniques, variants may be created that
differ in minor ways from the apelin-36 (42-57) sequences
disclosed. In one embodiment, DNA molecules and nucleotide
sequences that are derivatives of those specifically disclosed
herein, and which differ from those disclosed by the deletion,
addition, or substitution of nucleotides while still encoding a
protein that has at least 65% sequence identity with the apelin-36
(42-57) sequences disclosed, are comprehended by this disclosure.
In other embodiments, more closely related nucleic acid molecules
that share at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 98%, or at least 99%
nucleotide sequence identity with the disclosed apelin-36 (42-57)
sequences are comprehended by this disclosure. Alternatively,
specific examples of related nucleic acid molecules will have no
more than 2, 3, 5, 7, 10, 12, 15, 20, 25, or 30 nucleic acid
changes compared to the sequences disclosed herein. In one
embodiment, such variants differ from the disclosed sequences by
alteration of the coding region to fit the codon usage bias of the
particular organism into which the molecule is to be
introduced.
[0186] In other embodiments, the coding region may be altered by
taking advantage of the degeneracy of the genetic code to alter the
coding sequence such that, while the nucleotide sequence is
substantially altered, it nevertheless encodes a protein having an
amino acid sequence substantially similar to the disclosed
apelin-36 (42-57) protein sequences. For example, because of the
degeneracy of the genetic code, four nucleotide codon triplets
(CCU, CCC, CCA, and CCG) code for proline. The coding sequence of
any specific proline residue within the apelin-36 (42-57) protein,
therefore, could be changed to any of these alternative codons
without affecting the amino acid composition or characteristics of
the encoded protein. Based upon the degeneracy of the genetic code,
variant DNA molecules may be derived from the cDNA and gene
sequences disclosed herein using standard DNA mutagenesis
techniques as described above, or by synthesis of DNA sequences.
Thus, this disclosure also encompasses nucleic acid sequences that
encode an apelin-36 (42-57) protein, but which vary from the
disclosed nucleic acid sequences by virtue of the degeneracy of the
genetic code.
[0187] Nucleic acid molecules that are derived from the apelin-36
(42-57) cDNA nucleic acid sequences include molecules that
hybridize under low stringency, high stringency, or very high
stringency conditions to the disclosed apelin-36 (42-57) nucleic
acid molecules, and fragments thereof.
[0188] Apelin-36 (42-57) nucleic acid encoding molecules, and
orthologs and homologs of these sequences, may be incorporated into
transformation or expression vectors.
VII. Synthesis, Purification, and Post-Translational Modification
of Peptides
[0189] With the provision of apelin (1-77) (such as the cDNAs shown
in SEQ ID NOs: 1-7 and 36) and apelin-36 nucleic acid sequences
(FIG. 1), the synthesis and purification of apelin-36 (42-57)
(salcut) peptides by standard laboratory techniques is now enabled.
The apelin-36 (42-57) (salcut and salcut-NH.sub.2) and apelin-36
(42-58) (salcut-Gly) peptides of the disclosure can be prepared
using virtually any technique known to one of ordinary skill in the
art for the preparation of peptides. For example, the peptides can
be prepared using step-wise solution or solid phase peptide
syntheses, or recombinant DNA techniques, or the equivalents
thereof. Purified apelin-36 (42-57) protein, as well as its
amidated and free-acid derivatives, may be used for functional
analyses, antibody production, diagnostics, and patient therapy.
Purified apelin-36 (42-58) may be used as an antagonist of the
amidated form of apelin-36 (42-57). In another embodiment, the
free-acid form of apelin-36 (42-57) can be used as an antagonist of
the amidated form of apelin-36 (42-57).
[0190] A. Chemical Synthesis
[0191] Apelin-36 (42-57) and apelin-36 (42-58) peptides, and
modified versions of these peptides (for example, amidated forms of
the peptides) can be readily synthesized by automated solid phase
procedures well known in the art. Techniques and procedures for
solid phase synthesis are described in Solid Phase Peptide
Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard,
published by IRL, Oxford University Press, 1989. Alternatively,
apelin-36 (42-57) peptides may be prepared by way of segment
condensation, as described, for example, in Liu et al., Tetrahedron
Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc.
117:1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res.
45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992;
Liu and Tam, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tam,
Proc. Natl. Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and
Li, Int. J. Peptide Protein Res. 31:322-334, 1988). Other methods
useful for synthesizing the apelin-36 (42-57) peptides of the
disclosure are described in Nakagawa et al., J. Am. Chem. Soc.
107:7087-7092, 1985.
[0192] Additional exemplary techniques known to those of ordinary
skill in the art of peptide synthesis are taught by Bodanszky, M.
and Bodanszky, A., The Practice of Peptide Synthesis, Springer
Verlag, New York, 1994; and by Jones, J., Amino Acid and Peptide
Synthesis, 2nd ed., Oxford University Press, 2002. The Bodanszky
and Jones references detail the parameters and techniques for
activating and coupling amino acids and amino acid derivatives.
Moreover, the references teach how to select, use and remove
various useful functional and protecting groups.
[0193] Peptides of the disclosure can also be readily purchased
from commercial suppliers of synthetic peptides. Such suppliers
include, for example, Advanced ChemTech (Louisville, Ky.), Applied
Biosystems (Foster City, Calif.), Anaspec (San Jose, Calif.), and
Cell Essentials (Boston, Mass.).
[0194] B. Recombinant Synthesis
[0195] The disclosed apelin-36 (42-57) (amidated or free-acid
forms) and apelin-36 (42-58) peptides can also be synthesized using
conventional recombinant genetic engineering techniques. For
recombinant production, a polynucleotide sequence encoding the
apelin-36 (42-57) or apelin-36 (42-58) peptide is inserted into an
appropriate expression vehicle, that is, a vector which contains
the necessary elements for the transcription and translation of the
inserted coding sequence, or in the case of an RNA viral vector,
the necessary elements for replication and translation. The
expression vehicle is then transfected into a suitable target cell
which will express the apelin-36 (42-57) or apelin-36 (42-58)
peptide. Depending on the expression system used, the expressed
peptide is then isolated by procedures well-established in the art.
Methods for recombinant protein and peptide production are well
known in the art (see, e.g., Sambrook et al. (ed.), Molecular
Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, Ch. 17 and
Ausubel et al. Short Protocols in Molecular Biology, 4.sup.th ed.,
John Wiley & Sons, Inc., 1999).
[0196] DNA sequences can be manipulated with standard procedures
such as restriction enzyme digestion, fill-in with DNA polymerase,
deletion by exonuclease, extension by terminal deoxynucleotide
transferase, ligation of synthetic or cloned
[0197] DNA sequences, site-directed sequence-alteration via
single-stranded bacteriophage intermediate or with the use of
specific oligonucleotides in combination with nucleic acid
amplification. These techniques are known to those of ordinary
skill.
[0198] Methods for expressing large amounts of protein from a
cloned gene or cDNA sequence introduced into Escherichia coli (E.
coli) may be utilized for the purification of proteins. By way of
example, fusion proteins consisting of amino terminal peptides
encoded by a portion of the E. coli lacZ or trpE gene linked to
apelin-36 (42-57) proteins may be used to prepare polyclonal and
monoclonal antibodies (including humanized monoclonal antibodies)
against these proteins. Thereafter, these antibodies may be used in
other embodiments to purify proteins by immunoaffinity
chromatography, in diagnostic assays to quantitate the levels of
protein and to localize proteins in tissues and individual cells by
immunofluorescence. Such antibodies may be specific for epitope
tags, which can be added to the expression construct for instance
for identification and/or purification purposes.
[0199] To increase efficiency of production, the polynucleotide can
be designed to encode multiple units of the apelin-36 (42-57) or
apelin-36 (42-58) peptide separated by enzymatic cleavage sites.
The resulting polypeptide can be cleaved (e.g., by treatment with
the appropriate enzyme) in order to recover the peptide units. This
can increase the yield of peptides driven by a single promoter. In
one embodiment, a polycistronic polynucleotide can be designed so
that a single mRNA is transcribed which encodes multiple peptides,
each coding region operatively linked to a cap-independent
translation control sequence, for example, an internal ribosome
entry site (IRES). When used in appropriate viral expression
systems, the translation of each peptide encoded by the mRNA is
directed internally in the transcript, for example, by the IRES.
Thus, the polycistronic construct directs the transcription of a
single, large polycistronic mRNA which, in turn, directs the
translation of multiple, individual peptides. This approach
eliminates the production and enzymatic processing of polyproteins
and can significantly increase yield of peptide driven by a single
promoter.
[0200] A variety of host-expression vector systems may be utilized
to express the peptides described herein. These include, but are
not limited to, microorganisms such as bacteria transformed with
recombinant bacteriophage DNA or plasmid DNA expression vectors
containing an appropriate coding sequence; yeast or filamentous
fungi transformed with recombinant yeast or fungi expression
vectors containing an appropriate coding sequence; insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus) containing an appropriate coding sequence; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing an appropriate coding sequence; or animal cell
systems.
[0201] The transfer of DNA into eukaryotic, in particular human or
other mammalian cells, is now a conventional technique. Recombinant
expression vectors can be introduced into the recipient cells as
pure DNA (transfection) by, for example, precipitation with calcium
phosphate (Graham and vander Eb, Virology 52:466, 1973) or
strontium phosphate (Brash et al., Mol. Cell Biol. 7:2013, 1987),
electroporation (Neumann et al., EMBO J 1:841, 1982), lipofection
(Feigner et al., Proc. Natl. Acad. Sci USA 84:7413, 1987), DEAE
dextran (McCuthan et al., J. Natl. Cancer Inst. 41:351, 1968),
microinjection (Mueller et al., Cell 15:579, 1978), protoplast
fusion (Schafner, Proc. Natl. Acad. Sci. USA 77:2163-2167, 1980),
or pellet guns (Klein et al., Nature 327:70, 1987). In another
embodiment, the cDNA, or fragments thereof, can be introduced by
infection with virus vectors. Systems are developed that use, for
example, retroviruses (Bernstein et al., Gen. Engr'g 7:235, 1985),
adenoviruses (Ahmad et al., J. Virol. 57:267, 1986), or Herpes
virus (Spaete et al., Cell 30:295, 1982). Techniques of use in
packaging long transcripts can be found in Kochanek et al. (Proc.
Natl. Acad. Sci. USA 93:5731-5739, 1996), Parks et al. (Proc. Natl.
Acad. Sci. USA 93:13565-13570, 1996) and Parks and Graham (J.
Virol. 71:3293-3298, 1997). In yet another embodiment, apelin-36
(42-57) encoding sequences can be delivered to target cells in
vitro via non-infectious systems, for instance liposomes.
[0202] Methods and plasmid vectors for producing fusion proteins or
native proteins in bacteria are described in Sambrook et al.
(Sambrook et al., In Molecular Cloning: A Laboratory Manual, Ch.
17, CSHL, New York, 1989). Such proteins may be made in large
amounts, are easy to purify, and can be used to elicit antibody
response. In one embodiment, apelin-36 (42-57) proteins can be
produced in bacteria by placing a strong, regulated promoter and an
efficient ribosome binding site upstream of the cloned gene. If low
levels of protein are produced, additional steps may be taken to
increase protein production; if high levels of protein are
produced, purification is relatively easy. Suitable methods are
presented in Sambrook et al. (In Molecular Cloning: A Laboratory
Manual, CSHL, New York, 1989) and are well known in the art. In one
embodiment, proteins expressed at high levels are found in
insoluble inclusion bodies. Methods for extracting proteins from
these aggregates are described by Sambrook et al. (In Molecular
Cloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989).
[0203] It is appreciated that, for mutant or variant apelin-36
(42-57) and apelin-36 (42-58) sequences, similar systems are
employed to express and produce the mutant/variant product. It is
also appreciated that the recombinant apelin-36 (42-57) sequence
can be further modified (for example, by amidation).
[0204] C. Purification
[0205] The apelin-36 (42-57) (amidated or free-acid forms) and
apelin-36 (42-58) peptides can be purified by many techniques well
known in the art, such as reverse phase chromatography, high
performance liquid chromatography, ion exchange chromatography,
size exclusion chromatography, affinity chromatography, gel
electrophoresis, and the like. The actual conditions used to purify
a particular apelin-36 (42-57) or apelin-36 (42-58) peptide, or
their modified forms, will depend, in part, on synthesis strategy
and on factors such as net charge, hydrophobicity, hydrophilicity,
and the like, and will be apparent to those of ordinary skill in
the art.
[0206] D. Post-Translational Modifications
[0207] Proteins can be altered by a chemical modification after
translation by any means known in the art. Examples of
post-translational modifications include addition of functional
groups (such as amide, acetate, phosphate, lipids, or
carbohydrates), removal of a portion of a protein (such as a signal
sequence or the initial methionine residue) or formation of
alternatively spliced variants, and formation of a disulfide bond.
The modifications can be disease specific and differences in the
extent of the modification can be diagnostic.
[0208] Proteins or protein fragments can be isolated and/or
enriched based on a post-translational modification, for example
using antibodies specific for the post-translational modification.
In particular embodiments, apelin-36 (42-57) peptides (amidated or
free-acid forms) can be isolated based on the presence or absence
of the amidated glycine in salcut-NH.sub.2 by using an antibody
that specifically binds the amidated glycine. Such antibodies can
be used to distinguish salcut-NH.sub.2 (the amidated apelin-36
(42-57) protein) from the unmodified apelin-36 (42-57), the
glycine-extended apelin-36 (42-58) form, or the larger apelin-36
(42-77) proteins. In other embodiments, cells that express a
receptor that binds an apelin-36 (42-57) peptide can be isolated
and/or enriched when an antibody specific for a post-translational
modification (such as the amidated glycine of salcut-NH.sub.2, the
free-acid group of salcut, or the extended glycine of salcut-gly)
binds the apelin-36 (42-57) peptide bound to its cell-surface
receptor.
VIII. Apelin-36 (42-57) (Salcut) and Derivatives Thereof as
Modulators of Cell Growth
[0209] It has been surprisingly demonstrated that apelin-36 (42-57)
(salcut) has biological activity and can modulate the growth (or
proliferation) of cells. In particular embodiments,
post-translationally modified forms of salcut (for example,
salcut-NH.sub.2) have a modified proliferative activity, compared
to an unmodified salcut. Modulation of cell growth (or a modified
proliferative activity) includes enhancing, stimulating,
increasing, augmenting cell growth, or inhibiting, decreasing,
reducing cell growth.
[0210] The cell growth enhancing or inhibiting activity of salcut
occurs in a dose-dependent fashion. In some embodiments, the dose
response is a biphasic (bell-shaped rise and fall) proliferative
response. Without being bound by theory, a biphasic response is
indicative of two different receptors involved in modulating the
proliferative response: (i) a high affinity receptor involved in
cell growth stimulation/proliferation at lower concentrations of
salcut and (ii) a low affinity receptor involved in cell growth
inhibition or suppression at higher concentrations of salcut. In
other embodiments, the dose response increases (a proliferative
response) or decreases (a cell growth suppressive response) with
increasing concentrations of salcut, rather than exhibiting a
biphasic effect. Salcut-NH.sub.2-mediated endothelial cell
proliferation is not suppressed in the presence of the APJ receptor
antagonist apelin-13(F13A). Thus, salcut-NH.sub.2 mediates its
effects through a different receptor or receptor complex than
APJ.
[0211] Recent findings have demonstrated that crypto expression is
up-regulated by hypoxia and that apelin/APJ functions downstream of
cripto during cardiomyocyte differentiation from embryonic cells
(Bianco et al., Am. J. Path, 175:2146-2158, 2009; D'Aniello et al.,
Circ. Res., 105:231-238, 2009) and may function with cripto in
regulating tumor neovascularization. Thus, in addition to playing a
role in breast carcinogenesis, salcut-NH.sub.2 could play a
modulatory role in nodal/cripto regulation of embryogenesis and
melanoma progression (Strizzi et al., Breast Dis., 29:91-103, 2008)
and cripto control of cardiomyocyte differentiation (D'Aniello et
al., Circ. Res., 105:231-238, 2009).
[0212] An increase or decrease in cell growth or proliferation can
be quantified using any method known to those of skill in the art.
An increase or decrease in cell growth can be expressed as a
statistically significant change in the number or percentage of
cells in a cell population in the presence of salcut (for example,
salcut-NH.sub.2 or salcut-OH), compared to the same cell population
in the absence of salcut. In specific, non-limiting examples, an
increase in cell growth or proliferation can be a 10%, 20%, 30%,
50%, 70%, 80%, 90%, 100%, 200%, or more increase in cell growth or
proliferation. In other specific, non-limiting examples, a decrease
in cell growth or proliferation can be a 10%, 20%, 30%, 50%, 70%,
80%, 90%, or 100% decrease in cell growth or proliferation.
[0213] Salcut and its modified forms modulate the growth of cells
derived from any species, for example human, bovine, dog, rat,
mouse, rhesus monkey, or opossum cells. In addition,
salcut-NH.sub.2 and salcut-OH modulate cell growth either in vitro
or in vivo.
[0214] A. Apelin-36 (42-57) (Salcut) and Derivatives Thereof as
Mitogens
[0215] The current disclosure demonstrates that apelin-36 (42-57)
(salcut) is a mitogen and can modulate cell growth. The amidated
form of apelin-36 (42-57) (salcut-NH.sub.2) is a potent mitogen and
is capable of stimulating or enhancing the growth of cells,
compared to unmodified apelin-36 (42-57), or other derivatives of
apelin-36 (42-77). In one embodiment, salcut-NH.sub.2 stimulates or
enhances the growth of endothelial cells. Specific, non-limiting
examples of endothelial cells include endothelial cells of the
blood or lymphatic systems. In other embodiments, salcut-NH.sub.2
stimulates or enhances the growth of inflammatory cells (for
example, mast cells, granulocytes, lymphocytes, macrophages) or
tumor cells (for example, breast cancer or gastric cancer cells).
In particular embodiments, the free-acid derived form of salcut
(salcut-OH) is a less potent mitogen than salcut-NH.sub.2.
[0216] The cell growth enhancing or stimulating activity of salcut
occurs in a dose-dependent fashion. In one embodiment,
salcut-NH.sub.2 is growth enhancing in the 10 pM-100 nM
concentration range. In other embodiments, salcut-NH.sub.2 has
growth enhancing activity in the 100 pM-100 nM, 100 pM-10 nM, 10
pM-10 nM, 10 pM-1.0 nM, the 100 pM-1.0 nM, or the 1.0 nM-10 nM
concentration range. The glycine-extended form of apelin-36 (42-57)
(salcut-Gly) is substantially devoid of cell growth modulating
activity.
[0217] B. Apelin-36 (42-57) (Salcut) and Derivatives Thereof as
Suppressors of Proliferation
[0218] It has also been surprisingly demonstrated that apelin-36
(42-57) (salcut) also has growth suppression activity. In
particular embodiments, the amidated form of apelin-36 (42-57)
(salcut-NH.sub.2) has cell growth inhibitory activity at higher
concentrations (for example, at 100 nM or higher concentration of
salcut-NH.sub.2) in addition to mitogenic activity at lower
concentrations (for example, at 100 nM or lower concentration of
salcut-NH.sub.2). In one specific embodiment, salcut-NH.sub.2
demonstrates a growth inhibitory effect of endothelial cells (for
example, cells of the blood or lymphatic systems). In other
embodiments, salcut-NH.sub.2 inhibits the growth of inflammatory
cells (for example, mast cells) or tumor cells (for example, lung
cancer cells).
[0219] The cell growth inhibiting activity of salcut-NH.sub.2
occurs in a dose-dependent fashion. In another embodiment,
salcut-NH.sub.2 has growth inhibiting activity in the 10 nM-10
.mu.M concentration range. In other embodiments, salcut-NH.sub.2 is
growth inhibiting in the 500 nM-1.0 .mu.M, 1.0 .mu.M-10 .mu.M, 100
nM-10 .mu.M, 100 nM-1 .mu.M concentration range, or at
concentrations of salcut-NH.sub.2 greater than 10 .mu.M.
VIII. Production of an Antibody to Apelin-36 (42-57), and Apelin-36
(42-57) Variants and Derivatives
[0220] Monoclonal or polyclonal antibodies may be produced to
either the normal apelin-36 (42-57) protein or variant or mutant
forms of this protein. In one embodiment, antibodies raised against
the apelin-36 (42-57) protein would specifically detect the
apelin-36 (42-57) protein. That is, such antibodies would recognize
and bind the apelin-36 (42-57) protein, or fragments thereof, and
would not substantially recognize or bind to other apelin proteins
found in cells from the same species. In particular embodiments,
such antibodies would recognize an epitope including the amidated
glycine of apelin-36 (42-57) and could thereby distinguish the
amidated apelin-36 (42-57) protein from the unmodified apelin-36
(42-57), the glycine-extended apelin-36 (42-58) form, or the larger
apelin-36 (42-77) proteins from the same species. In some
embodiments, antibodies against the human apelin-36 (42-57) protein
may recognize apelin-36 (42-57) from other species (e.g., dog
apelin-36 (42-57)), and vice versa.
[0221] Monoclonal antibodies directed against apelin-36 (42-57) can
modify (either enhance or suppress) the activity of apelin-36
(42-57) when bound to the peptide. For example, such a monoclonal
antibody can inhibit (or neutralize) the activity of apelin-36
(42-57) by blocking an epitope on apelin-36 (42-57) that is
required for receptor binding (inhibitory or neutralizing
antibody). In other embodiments, a monoclonal antibody can inhibit
(or neutralize) the activity of apelin-36 (42-57) by blocking an
epitope on the apelin-36 (42-57) receptor. Alternatively,
monoclonal antibodies can be activating, for example a monoclonal
antibody directed against apelin-36 (42-57) can block a proteolytic
site on apelin-36 (42-57) and augment the half-life of the peptide,
without affecting receptor binding, thereby enhancing the activity
of apelin-36 (42-57).
[0222] Monoclonal or polyclonal antibodies to the protein can be
prepared as follows:
[0223] A. Monoclonal Antibody Production by Hybridoma Fusion
[0224] Monoclonal antibody to epitopes of the apelin-36 (42-57)
protein identified and isolated as described can be prepared from
murine hybridomas according to the classical method of Kohler and
Milstein (Nature 256:495-497, 1975) or derivative methods thereof.
In one specific, non-limiting embodiment, a mouse is repetitively
inoculated with a few micrograms of the selected protein over a
period of a few weeks. The mouse is then sacrificed, and the
antibody-producing cells of the spleen isolated. The spleen cells
are fused with mouse myeloma cells using polyethylene glycol, and
the excess, non-fused, cells destroyed by growth of the system on
selective media comprising aminopterin (HAT media). Successfully
fused cells are diluted and aliquots of the dilution placed in
wells of a microtiter plate, where growth of the culture is
continued. Antibody-producing clones are identified by detection of
antibody in the supernatant fluid of the wells by immunoassay
procedures, such as ELISA, as originally described by Engvall
(Enzymol. 70(A):419-439, 1980), and derivative methods thereof.
Selected positive clones can be expanded and their monoclonal
antibody product harvested for use. Detailed procedures for
monoclonal antibody production are described in Harlow and Lane
(Antibodies, A Laboratory Manual, CSHL, New York, 1988). A
monoclonal antibody is further intended to include humanized
monoclonal molecules that specifically bind the target antigen.
Methods of humanizing monoclonal antibodies are well known in the
art.
[0225] B. Polyclonal Antibody Production by Immunization
[0226] Polyclonal antiserum containing antibodies to heterogeneous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein (for instance, expressed using a
method described herein), which, in one specific, non-limiting
embodiment, can be modified to enhance immunogenicity. Effective
polyclonal antibody production is affected by many factors related
both to the antigen and the host species. In one embodiment, small
molecules may tend to be less immunogenic than others and may
require the use of carriers and adjuvant, examples of which are
known. In another embodiment, host animals may vary in response to
site of inoculations and dose, with either inadequate or excessive
doses of antigen resulting in low titer antisera. In one specific,
non-limiting embodiment, a series of small doses (ng level) of
antigen administered at multiple intradermal sites may be most
reliable. An effective immunization protocol for rabbits can be
found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab.
33:988-991, 1971).
[0227] In one embodiment, booster injections will be given at
regular intervals, and antiserum harvested when antibody titer
thereof begins to fall, as determined semi-quantitatively (for
example, by double immunodiffusion in agar against known
concentrations of the antigen). See, for example, Ouchterlony et
al. (In Handbook of Experimental Immunology, Wier, D. (ed.) chapter
19. Blackwell, 1973). In one specific, non-limiting embodiment the
plateau concentration of antibody is usually in the range of about
0.1 to 0.2 mg/ml of serum (about 12 .mu.M). Affinity of the
antisera for the antigen is determined by preparing competitive
binding curves, as described, for example, by Fisher (Manual of
Clinical Immunology, Ch. 42, 1980).
[0228] C. Antibodies Raised Against Synthetic Peptides
[0229] A third approach to raising antibodies against the apelin-36
(42-57) protein is to use synthetic peptides synthesized on a
commercially available peptide synthesizer based upon the sequence
of the apelin-36 (42-57) protein. Polyclonal antibodies can be
generated by injecting such peptides into, for instance, rabbits
(Example 3, for instance).
[0230] D. Antibodies Raised by Injection of Apelin-36 (42-57)
Encoding Sequence
[0231] In one embodiment, antibodies may be raised against the
apelin-36 (42-57) protein by subcutaneous injection of a
recombinant DNA vector that expresses the apelin-36 (42-57) protein
into laboratory animals, such as mice. In one specific,
non-limiting embodiment, delivery of the recombinant vector into
the animals may be achieved using a hand-held form of the Biolistic
system (Sanford et al., Particulate Sci. Technol. 5:27-37, 1987),
as described by Tang et al. (Nature 356:152-154, 1992). In other
embodiments, expression vectors suitable for this purpose may
include those that express the apelin-36 (42-57) encoding sequence
under the transcriptional control of either the human .beta.-actin
promoter or the cytomegalovirus (CMV) promoter.
[0232] Antibody preparations prepared according to these protocols
are useful in quantitative immunoassays which determine
concentrations of antigen-bearing substances in biological samples;
they are also used semi-quantitatively or qualitatively to identify
the presence of antigen in a biological sample.
IX. Qualitative and Quantitative Detection of Apelin-36 (42-57)
Peptide
[0233] Antibodies can be used to assess the presence or absence of
apelin-36 (42-57) (amidated or free-acid forms) in cultured cells,
primary cells, or biological samples. The determination that an
antibody specifically detects the apelin-36 (42-57) is made by any
one of a number of standard immunoassay methods; for instance, the
Western blotting technique (Sambrook et al., In Molecular Cloning:
A Laboratory Manual, CSHL, New York, 1989). In one embodiment, it
is determined whether a given antibody preparation (such as one
produced in a mouse) specifically detects the apelin-36 (42-57)
peptide by Western blotting. In one specific, non-limiting
embodiment total cellular protein is extracted from normal human
cells (for example, endothelial cells or lymphocytes) and
electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. In
another embodiment, the cellular protein is extracted from a tumor.
The proteins are then transferred to a membrane (for example,
nitrocellulose or PVDF) by Western blotting, and the antibody
preparation is incubated with the membrane. After washing the
membrane to remove non-specifically bound antibodies, the presence
of specifically bound antibodies is detected by the use of (by way
of example) an anti-mouse antibody conjugated to an enzyme such as
alkaline phosphatase. Application of an alkaline phosphatase
substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue
tetrazolium results in the production of a dense blue compound by
immunolocalized alkaline phosphatase. Antibodies that specifically
detect the apelin-36 (42-57) peptide will, by this technique, be
shown to bind to the apelin-36 (42-57) protein band (which will be
localized at a given position on the gel determined by its
molecular weight). Alternatively, this peptide can be recognized on
a Western blot using an antibody that recognizes apelin or a
peptide overlapping salcut and noting the different
position/molecular weight of salcut. In particular embodiments,
such antibodies would recognize an epitope including the amidated
glycine of apelin-36 (42-57) and could thereby distinguish the
amidated apelin-36 (42-57) protein from other apelin proteins.
[0234] Non-specific binding of the antibody to other proteins may
occur and may be detectable as a weak signal on the Western blot.
The non-specific nature of this binding will be recognized by one
skilled in the art by the weak signal obtained on the Western blot
relative to the strong primary signal arising from the specific
antibody-apelin-36 (42-57) protein binding.
[0235] In other embodiments, antibodies against the apelin-36
(42-57) peptide (amidated or free-acid forms) are used to localize
apelin-36 (42-57) to specific cell types or to specific subcellular
locations in immunohistochemical or immunofluorescence assays. In
one embodiment, the cells are selected from a variety of cell
lines. In other embodiments, primary cells are isolated from a
tumor in a subject and are maintained in culture or the tumor is
biopsied and sectioned, and the sections are prepared directly for
immunohistochemistry or immunofluorescence. In one specific,
non-limiting embodiment, the cells are fixed, incubated in a
blocking medium, incubated with the antibody directed against
apelin-36 (42-57) followed by a second incubation with a secondary
antibody that is conjugated to a fluorescent probe or a
colorimetric agent. Cells that express an apelin-36 (42-57) peptide
that is recognized by the antibody exhibit a color or are
fluorescent when viewed under a light or fluorescence microscope,
respectively. Hence, uses of antibodies directed against apelin-36
(42-57) include diagnostic tests (for example, to assess apelin-36
(42-57) levels in tissue extracts and body fluids).
[0236] An alternative method of diagnosing apelin-36 (42-57)
deletion, reduction, amplification, or mutation is to quantitate
the level of the apelin-36 (42-57) peptide (including the amidated
or free-acid forms) in the cells of a subject. In one embodiment,
this diagnostic tool would be useful for detecting reduced levels
of the apelin-36 (42-57) peptide that result from, for example,
mutations in the promoter regions of the apelin gene or mutations
within the coding region of the apelin gene that produce truncated,
non-functional or unstable apelin peptides, as well as from
deletions of the entire apelin gene. In another embodiment,
duplications (or more copies) of the apelin gene may be detected as
an increase in the expression level of apelin-36 (42-57) peptide.
The determination of reduced or increased apelin-36 (42-57) peptide
levels would be an alternative or supplemental approach to the
direct determination of apelin gene deletion, duplication or
mutation status.
[0237] The availability of antibodies specific to the apelin-36
(42-57) peptide (amidated or free-acid forms) will facilitate the
quantitation of cellular apelin-36 (42-57) peptide by one of a
number of immunoassay methods (for example, an ELISA or
Enzyme-Linked ImmunoSorbent Assay), which are well known in the art
and are presented herein and in, for instance, Harlow and Lane
(Antibodies, A Laboratory Manual, CSHL, New York, 1988). Many
techniques are commonly known in the art for the detection and
quantification of antigen (for example, the apelin-36 (42-57)
peptide). In one specific, non-limiting embodiment, the purified
antigen will be bound to a substrate (for example, a multiwell
plate), the antibody of the sample will bind via its Fab portion to
this antigen, the substrate will then be washed and a second,
labeled antibody will then be added which will bind to the Fc
portion of the antibody that is the subject of the assay. The
second, labeled antibody will be species specific, i.e., if the
serum is from a rabbit, the second, labeled antibody will be
anti-rabbit-IgG antibody. The specimen will then be washed and the
amount of the second, labeled antibody that has been bound will be
detected and quantified by standard methods.
[0238] Examples of methods for the detection of antibodies in
biological samples, including methods employing dip strips or other
immobilized assay devices, are disclosed for instance in the
following patents: U.S. Pat. No. 5,965,356 (Herpes simplex virus
type specific seroassay); U.S. Pat. No. 6,114,179 (Method and test
kit for detection of antigens and/or antibodies); U.S. Pat. No.
6,077,681 (Diagnosis of motor neuropathy by detection of
antibodies); U.S. Pat. No. 6,057,097 (Marker for pathologies
comprising an auto-immune reaction and/or for inflammatory
diseases); and U.S. Pat. No. 5,552,285 (Immunoassay methods,
compositions and kits for antibodies to oxidized DNA bases).
[0239] In one embodiment, for the purposes of quantitating the
apelin-36 (42-57) peptide (amidated or free-acid forms), a
biological sample of the subject, which sample includes cellular
proteins, is used. Such a biological sample may be obtained from
body cells, such as those present in peripheral blood, urine,
saliva, tissue biopsy, bronchioalveolar lavage fluids,
amniocentesis samples, surgical specimens and autopsy material.
Biological samples can be obtained from normal, healthy subjects or
from subjects who are predisposed to or who are already suffering
from any one of a variety of tumors, such as, but not limited to,
tumors of the breast, lung, colon, pancreas, liver, brain, blood,
skin, prostate, testis, ovary, and stomach, or any disorder caused
by abnormal angiogenesis. In one embodiment, quantitation of the
apelin-36 (42-57) peptide is achieved by immunoassay and compared
to levels of the protein found in healthy cells (e.g., cells from a
subject known not to suffer from a tumor). In one embodiment, a
significant (e.g., 10% or greater, for instance, 20%, 25%, 30%,
50%, 75%, 90%, 95%, or more) reduction in the amount of apelin-36
(42-57) peptide in the cells of a subject compared to the amount of
apelin-36 (42-57) peptide found in normal cells from a subject of
the same species would be taken as an indication that the subject
may have deletions or mutations in the apelin gene locus. In some
embodiments, a 100% reduction in the amount of apelin-36 (42-57)
would be taken as an indication that the subject may have deletions
or mutations in the apelin gene locus. In another embodiment, a
significant (e.g., 10% or greater, for instance, 20%, 25%, 30%,
50%, 75%, 90%, 95%, 100%, or more) increase would indicate that a
duplication or enhancing mutation had occurred.
X. Pharmaceutical Compositions and Uses Thereof
[0240] The apelin-36 (42-57) peptides (amidated or free-acid forms)
of the disclosure can be used to treat any disorder in a subject,
especially mammals (e.g., humans), for which modulating cell growth
is beneficial. Such conditions include, but are not limited to
neoplasia, cardiovascular disease, peripheral vascular disease,
hypertension, preeclampsia syndrome, abnormal angiogenesis,
diabetes, ocular degeneration, idiopathic pulmonary fibrosis, wound
healing, altered mast cell migration, chronic obstructive pulmonary
disease, inflammatory diseases such as arthritis (juvenile and
rheumatoid) and inflammatory bowel disease, avascular or ischemic
insult, myocardial infarction, stroke, vasculititis/angiitis,
systemic or vascular sclerosis, gangrene, congelation (severe
frostbite), alopecia, eczema, ulcers, lymphedema (parasite induced,
for example elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, endometriosis, and the like.
[0241] Abnormal angiogenesis plays an active role in numerous
diseases and conditions. Thus, apelin-36 (42-57) peptides can be
used to stimulate angiogenesis in subjects experiencing the
following vessel suppressive disorders: avascular or ischemic
insult, cardiovascular disease, myocardial infarction, stroke,
vasculititis/angiitis, systemic or vascular sclerosis, gangrene,
congelation (severe frostbite), alopecia, eczema, ulcers,
lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced). Apelin-36 (42-57)
peptides also can be used to inhibit angiogenesis in subjects
experiencing the following vessel stimulatory disorders: neoplasia,
vascular hyperplasia, hemangioma, diabetic induced retinopathy,
macular degenerative disease, psoriasis, endometriosis, arthritis
(juvenile and rheumatoid), and the like.
[0242] In particular embodiments, the peptides of the disclosure
can be used to modulate angiogenesis or inhibit tumorigenesis. In
one specific non-limiting example, the apelin-36 (42-57) peptides
(amidated or free-acid forms) can be used to inhibit endothelial
cell growth and reduce or inhibit angiogenesis. A reduction or
inhibition of angiogenesis can result in a reduction in size or
eradication of a tumor. In another specific, non-limiting example,
the apelin-36 (42-57) peptides can be used to inhibit tumor cell
growth.
[0243] The peptides described herein can be used alone or in
combination therapy with other cell growth modulating compositions
or drugs used to treat the foregoing conditions. Such combination
therapies include, but are not limited to simultaneous or
sequential administration of the drugs involved. For example, in
the treatment of tumorigenesis, the formulations comprising
apelin-36 (42-57) peptides can be administered with any one or more
of the tumor cell-growth inhibiting agents currently in use, for
example, cisplatin, HERCEPTIN.RTM., or tamoxifen.
[0244] In other embodiments, the apelin-36 (42-57) peptide
formulations can be administered with other cell growth modulating
compositions or drugs to prevent, reduce or inhibit cardiovascular
disease, peripheral vascular disease, hypertension, preeclampsia
syndrome, abnormal angiogenesis, diabetes, ocular degeneration,
idiopathic pulmonary fibrosis, wound healing, altered mast cell
migration, chronic obstructive pulmonary disease, or inflammatory
diseases such as arthritis (juvenile and rheumatoid) and
inflammatory bowel disease, avascular or ischemic insult,
myocardial infarction, stroke, vasculititis/angiitis, systemic or
vascular sclerosis, gangrene, congelation (severe frostbite),
alopecia, eczema, ulcers, lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, or endometriosis.
[0245] The present disclosure includes administering an inhibitor
of apelin-36 (42-57) (for instance an inhibitor that is specific
for this peptide or its receptor, such as a neutralizing monoclonal
antibody, a small molecule inhibitor, the free-acid form of
apelin-36 (42-57), or salcut-Gly) or a combination of an apelin-36
(42-57) inhibitor and one or more other pharmaceutical agents, to
the subject in a pharmaceutically compatible carrier and in an
amount effective to inhibit the condition, development or
progression of the disorder. For example, other pharmaceutical
agents may include one or more effective doses of another drug
recognized for treatment of abnormal cell growth (such as one or
more of those discussed at pages 260-269 of "Cecil Textbook of
Medicine" (1992) W. B. Saunders).
[0246] Although the treatments described herein can be used
prophylactically in any subject in a demographic group at
significant risk for the disclosed disorders, subjects can also be
selected using more specific criteria, such as a definitive
diagnosis of the condition. For example, treatment can be initiated
in a subject having signs and symptoms of a tumor or hypertension,
which are recognized by those of ordinary skill.
[0247] A. Administration of Peptides or Peptide Analogs
[0248] Apelin peptides (the modified or unmodified forms) and
inhibitors thereof can be administered directly to the subject. For
example, an apelin-36 (42-57) peptide (amidated or free-acid forms)
can be expressed in vitro, such as in an E. coli expression system,
as is well known in the art, and isolated in amounts useful for
therapeutic compositions.
[0249] In exemplary applications, therapeutic compositions are
administered to a subject suffering from a disorder, such as
neoplasia, cardiovascular disease, peripheral vascular disease,
hypertension, preeclampsia syndrome, abnormal angiogenesis,
diabetes, ocular degeneration, idiopathic pulmonary fibrosis, wound
healing, altered mast cell migration, chronic obstructive pulmonary
disease, inflammatory diseases such as arthritis (juvenile and
rheumatoid) and inflammatory bowel disease, avascular or ischemic
insult, myocardial infarction, stroke, vasculititis/angiitis,
systemic or vascular sclerosis, gangrene, congelation (severe
frostbite), alopecia, eczema, ulcers, lymphedema (parasite induced,
for example elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, or endometriosis, in an amount
sufficient to inhibit or treat the disorder. Amounts effective for
this use will depend upon the severity of the disorder and the
general state of the subject's health. A therapeutically effective
amount of the compound is that which provides either subjective
relief of a symptom(s) or an objectively identifiable improvement
as noted by the clinician or other qualified observer.
[0250] An apelin-36 (42-57) peptide (amidated or free-acid forms)
can be administered by any means known to one of skill in the art
(see, e.g., Banga, "Parenteral Controlled Delivery of Therapeutic
Peptides and Proteins," in Therapeutic Peptides and Proteins,
Technomic Publishing Co., Inc., Lancaster, Pa., 1995), such as by
intramuscular, subcutaneous, or intravenous injection, but even
oral, nasal, or anal administration is contemplated. In one
embodiment, administration is by subcutaneous or intramuscular
injection. To extend the time during which the apelin-36 (42-57)
peptide is available to inhibit or treat a disorder, the apelin-36
(42-57) peptide can be provided as an implant, an oily injection,
or as a particulate system. The particulate system can be a
microparticle, a microcapsule, a microsphere, a nanocapsule, or
similar particle (Banga, "Parenteral Controlled Delivery of
Therapeutic Peptides and Proteins," in Therapeutic Peptides and
Proteins, Technomic Publishing Co., Inc., Lancaster, Pa.,
1995).
[0251] In one specific, non-limiting example, an apelin-36 (42-57)
peptide is administered that includes one or more of the disclosed
amino acid sequences (for example, SEQ ID NOs: 19, 22, 25, 28, 31,
34, 37, or 42).
[0252] In another specific, non-limiting example, the apelin-36
(42-57) peptide (amidated or free-acid form) is administered in a
dosage that enhances angiogenesis, and optionally is administered
in combination with a mitogenic agent; such agent need not be
covalently linked, or even administered simultaneously with, the
peptide that promotes angiogenesis. Such agents need not be
covalently linked, or even administered simultaneously with, the
apelin-36 (42-57) peptide.
[0253] In a further specific, non-limiting example, the apelin-36
(42-57) peptide (amidated or free-acid form) is administered in a
dosage that inhibits angiogenesis, and optionally is administered
in combination with a cell growth inhibiting agent. Such agents
need not be covalently linked, or even administered simultaneously
with, the apelin-36 (42-57) peptide.
[0254] In yet another specific, non-limiting example, the apelin-36
(42-57) peptide (amidated or free-acid form) is administered in a
dosage that inhibits tumorigenesis, and optionally is administered
in combination with a cell growth inhibiting agent. Such agents
need not be covalently linked, or even administered simultaneously
with, the apelin-36 (42-57) peptide.
[0255] B. Administration of Nucleic Acid Molecules
[0256] In some embodiments, administration of the apelin-36 (42-57)
peptide (amidated or free-acid forms) can be achieved by an
appropriate nucleic acid expression vector (or combination of
vectors) which is administered so that it becomes intracellular,
for example, by use of a retroviral vector (see U.S. Pat. No.
4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, DuPont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci., 88:1864-1868, 1991). Alternatively, the nucleic acid
can be introduced intracellularly and incorporated within host cell
DNA for expression, for example, by homologous or non-homologous
recombination.
[0257] Use of a DNA expression vector (e.g., the vector pcDNA) is
an example of a method of introducing the foreign cDNA into a cell
under the control of a strong viral promoter (e.g.,
cytomegalovirus) to drive the expression. However, other vectors
can be used. Other retroviral vectors (such as pRETRO-ON, BD
Biosciences, Palo Alto, Calif.) also use this promoter but have the
advantages of entering cells without any transfection aid,
integrating into the genome of target cells only when the target
cell is dividing. It is also possible to turn on the expression of
a therapeutic nucleic acid by administering tetracycline when these
plasmids are used. Hence these plasmids can be allowed to transfect
the cells, then administer a course of tetracycline to achieve
regulated expression.
[0258] Other plasmid vectors, such as pMAM-neo (BD Biosciences,
Palo Alto, Calif.) or pMSG (Invitrogen, Carlsbad, Calif.) use the
MMTV-LTR promoter (which can be regulated with steroids) or the
SV10 late promoter (pSVL, Invitrogen, Carlsbad, Calif.) or
metallothionein-responsive promoter (pBPV, Invitrogen, Carlsbad,
Calif.) and other viral vectors, including retroviruses. Examples
of other viral vectors include adenovirus, AAV (adeno-associated
virus), recombinant HSV, poxviruses (vaccinia) and recombinant
lentivirus (such as HIV). All these vectors achieve the basic goal
of delivering into the target cell the cDNA sequence and control
elements needed for transcription.
[0259] Retroviruses have been considered a preferred vector for
gene therapy, with a high efficiency of infection and stable
integration and expression (Orkin et al., Prog. Med. Genet.
7:130-142, 1988). A nucleic acid encoding the apelin-36 (42-57)
peptide can be cloned into a retroviral vector and driven from
either its endogenous promoter (where applicable) or from the
retroviral LTR (long terminal repeat). Other viral transfection
systems may also be utilized for this type of approach, including
adenovirus, AAV (McLaughlin et al., J. Virol. 62:1963-1973, 1988),
vaccinia virus (Moss et al., Annu. Rev. Immunol. 5:305-324, 1987),
Bovine Papilloma virus (Rasmussen et al., Methods Enzymol.
139:642-654, 1987) or members of the herpesvirus group such as
Epstein-Barr virus (Margolskee et al., Mol. Cell. Biol.
8:2837-2847, 1988).
[0260] In addition to delivery of a nucleic acid encoding the
apelin-36 (42-57) peptide (amidated or free-acid forms) to cells
using viral vectors, it is possible to use non-infectious methods
of delivery. For instance, lipidic and liposome-mediated gene
delivery has recently been used successfully for transfection with
various genes (for reviews, see Templeton and Lasic, Mol.
Biotechnol., 11:175-180, 1999; Lee and Huang, Crit. Rev. Ther. Drug
Carrier Syst., 14:173-206, 1997; and Cooper, Semin. Oncol.,
23:172-187, 1996). For instance, cationic liposomes have been
analyzed for their ability to transfect monocytic leukemia cells,
and shown to be a viable alternative to using viral vectors (de
Lima et al., Mol. Membr. Biol., 16:103-109, 1999). Such cationic
liposomes can also be targeted to specific cells through the
inclusion of, for instance, monoclonal antibodies or other
appropriate targeting ligands (Kao et al., Cancer Gene Ther.,
3:250-256, 1996).
[0261] C. Representative Methods of Administration, Formulations
and Dosage
[0262] The provided apelin-36 (42-57) peptides (amidated or
free-acid forms), constructs, or vectors encoding such peptides,
can be combined with a pharmaceutically acceptable carrier (e.g., a
phospholipid or other type of lipid) or vehicle for administration
to human or animal subjects. In some embodiments, more than one
apelin-36 (42-57) peptide can be combined to form a single
preparation. The apelin-36 (42-57) peptides can be conveniently
presented in unit dosage form and prepared using conventional
pharmaceutical techniques. Such techniques include the step of
bringing into association the active ingredient and the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers.
Formulations suitable for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of a sterile
liquid carrier, for example, water for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
may be prepared from sterile powders, granules and tablets commonly
used by one of ordinary skill in the art.
[0263] In certain embodiments, unit dosage formulations are those
containing a dose or unit, or an appropriate fraction thereof, of
the administered ingredient. It should be understood that in
addition to the ingredients particularly mentioned above,
formulations encompassed herein may include other agents commonly
used by one of ordinary skill in the art.
[0264] The pharmaceutical compositions provided herein, including
those for use in treating disorders such as neoplasia,
cardiovascular disease, peripheral vascular disease, hypertension,
preeclampsia syndrome, abnormal angiogenesis, diabetes, ocular
degeneration, idiopathic pulmonary fibrosis, wound healing, altered
mast cell migration, chronic obstructive pulmonary disease,
inflammatory diseases such as arthritis (juvenile and rheumatoid)
and inflammatory bowel disease, cardiovascular disease, avascular
or ischemic insult, myocardial infarction, stroke,
vasculititis/angiitis, systemic or vascular sclerosis, gangrene,
congelation (severe frostbite), alopecia, eczema, ulcers,
lymphedema (parasite induced, for example
elephantiasis/elephantitis, or tumor induced), vascular
hyperplasia, hemangioma, diabetic induced retinopathy, macular
degenerative disease, psoriasis, or endometriosis, may be
administered through different routes, such as oral, including
buccal and sublingual, rectal, parenteral, aerosol, nasal,
intramuscular, subcutaneous, intradermal, and topical. They may be
administered in different forms, including but not limited to
solutions, emulsions and suspensions, microspheres, particles,
microparticles, nanoparticles, and liposomes.
[0265] It may be desirable to administer the pharmaceutical
compositions locally to the area in need of treatment. This may be
achieved by, for example, and not by way of limitation, local or
regional infusion or perfusion during surgery, topical application
(e.g., wound dressing), injection, catheter, suppository, or
implant (e.g., implants formed from porous, non-porous, or
gelatinous materials, including membranes, such as sialastic
membranes or fibers), and the like.
[0266] In a specific embodiment, one or more of the disclosed
peptides capable of modulating cell growth may be associated either
by coating or impregnating an implant such as stent to treat a
vascular disorder. These peptides are prepared and purified as
described herein. In an example, the implant can be partially or
completely coated with the peptide. For instance, the luminal
surface of the implant may be coated with the peptide. The peptide
may be attached to the implant by any chemical or mechanical bond
or force, including linking agents. Alternatively, the coating may
be directly linked (tethered) to the first surface, such as through
silane groups. In other examples, the implant may be impregnated
with at least one peptide by methods known to those of skill in the
art so that multiple surfaces (such as the outer and inner
surfaces) of the implant include the peptide.
[0267] In an additional embodiment, the implant may be coated or
impregnated with materials in addition to the disclosed peptides to
further enhance their bio-utility. Examples of suitable coatings
are medicated coatings, drug-eluting coatings, hydrophilic
coatings, smoothing coatings.
[0268] In one embodiment, administration can be by direct injection
at the site (or former site) of a tissue that is to be treated,
such as the peripheral vasculature or a tumor. In another
embodiment, the pharmaceutical compositions are delivered in a
vesicle, in particular liposomes (see, e.g., Langer, Science
249:1527-1533, 1990; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, N.Y., pp. 353-365, 1989).
[0269] In yet another embodiment, the pharmaceutical compositions
can be delivered in a controlled release system. In one embodiment,
a pump can be used (see, e.g., Langer Science 249:1527-1533, 1990;
Sefton Crit. Rev. Biomed. Eng. 14:201-240, 1987; Buchwald et al.,
Surgery 88:507-516, 1980; Saudek et al., N. Engl. J. Med.
321:574-579, 1989). In another embodiment, polymeric materials can
be used (see, e.g., Ranger et al., Macromol. Sci. Rev. Macromol.
Chem. 23:61-64, 1983; Levy et al., Science 228:190-192, 1985;
During et al., Ann. Neurol. 25:351-356, 1989; and Howard et al., J.
Neurosurg. 71:105-112, 1989). Other controlled release systems,
such as those discussed in the review by Langer (Science
249:1527-1533, 1990), can also be used.
[0270] The amount of the pharmaceutical compositions that will be
effective depends on the nature of the disorder or condition to be
treated, as well as the stage of the disorder or condition.
Effective amounts can be determined by standard clinical
techniques. The precise dose to be employed in the formulation will
also depend on the route of administration, and should be decided
according to the judgment of the health care practitioner and each
subject's circumstances. For example, a therapeutically effective
amount of an active ingredient can vary from about 0.001 mg/kg body
weight to about 1 g/kg body weight. Another example of such a
dosage range is 0.1 to 200 mg/kg body weight in single or divided
doses. A further example of a dosage range is 1.0 to 100 mg/kg body
weight in single or divided doses. Alternatively, therapeutically
effective amounts can be calculated in moles, for instance from
about 0.5 nmol/kg to about 100 nmol/kg or more of an active
ingredient. It is recognized that salcut peptides, including
salcut-NH2, salcut-OH, or salcut-Gly, are administered such that
their circulating concentrations are in a physiologic range.
[0271] The specific dose level and frequency of dosage for any
particular subject may be varied and will depend upon a variety of
factors, including the activity of the specific compound, the
metabolic stability and length of action of that compound, the age,
body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, and severity
of the condition of the subject undergoing therapy.
[0272] The pharmaceutical compositions of the present disclosure
can be administered at about the same dose throughout a treatment
period, in an escalating dose regimen, or in a loading-dose regime
(e.g., in which the loading dose is about two to five times the
maintenance dose). In some embodiments, the dose is varied during
the course of a treatment based on the condition of the subject
being treated, the severity of the disease or condition, the
apparent response to the therapy, and/or other factors as judged by
one of ordinary skill in the art. The volume of administration will
vary depending on the route of administration. By way of example,
intramuscular injections may range from about 0.1 ml to about 1.0
ml. Those of ordinary skill in the art will know appropriate
volumes for different routes of administration.
XI. Methods of Screening for Inhibitors or Activators of Apelin-36
(42-57) Activity
[0273] In certain circumstances, it is desirable to reduce or
inhibit the activity of apelin-36 (42-57), for example, for the
treatment of neoplasia or abnormal angiogenesis. An effect can be
achieved by inhibiting apelin-36 (42-57) activity (amidated or
free-acid forms) in vitro or in vivo. In particular embodiments,
apelin-36 (42-57) activity is inhibited by administering an
apelin-36 (42-57) inhibitor to a subject. In other embodiments,
apelin-36 (42-57) activity is inhibited by administering an
apelin-36 (42-57) inhibitor to a cell in vitro. Such an inhibitor
can be identified in a screening assay for inhibitors of apelin-36
(42-57)-mediated angiogenesis or tumor cell growth.
[0274] In other embodiments, it is desirable to activate or augment
the activity of apelin-36 (42-57), for example, for the treatment
of abnormal angiogenesis. An effect can be achieved by increasing
apelin-36 (42-57) activity (amidated or free-acid forms) in vitro
or in vivo. In particular embodiments, apelin-36 (42-57) activity
is augmented by administering an apelin-36 (42-57) activator to a
subject. In other embodiments, apelin-36 (42-57) activity is
augmented by administering an apelin-36 (42-57) activator to a cell
in vitro. Such an activator can be identified in a screening assay
for activators of apelin-36 (42-57)-mediated angiogenesis.
[0275] In general, a screening assay is carried out by determining
whether a given test compound inhibits (or activates) apelin-36
(42-57)-mediated cell growth or suppression; inhibition of
apelin-36 (42-57)-mediated cell growth (at lower concentrations of
apelin-36 42-57)) or decreased apelin-36 (42-57)-mediated growth
suppression (at higher concentrations of apelin-36 (42-57))
indicates that the test compound is an apelin-36 (42-57) inhibitor.
Augmentation of apelin-36 (42-57)-mediated cell growth (at lower
concentrations of apelin-36 42-57)) or increased apelin-36
(42-57)-mediated growth suppression (at higher concentrations of
apelin-36 (42-57)) indicates that the test compound is an apelin-36
(42-57) activator. In some embodiments, this is accomplished by
contacting cells (for example, endothelial cells or tumor cells)
with apelin-36 (42-57) (amidated or free-acid forms) in the
presence and absence of the test compound.
[0276] A reduction of apelin-36 (42-57)-induced cell growth at
concentrations of 10 nM and below, for example as measured by
reduced relative luminescent units in a cell proliferation assay or
reduced endothelial cell tube formation, indicates that the test
compound is an inhibitor of the mitogenic effect of apelin-36
(42-57). A reduction of apelin-36 (42-57)-induced cell suppression
at concentrations of 10 nM and above, for example as measured by
increased relative luminescent units in a cell proliferation assay
or increased endothelial cell tube formation, indicates that the
test compound is an inhibitor of the cell growth suppressive effect
of apelin-36 (42-57). Similarly, an increase of apelin-36
(42-57)-induced cell growth at concentrations of 10 nM and below,
for example as measured by increased relative luminescent units in
a cell proliferation assay or endothelial cell tube formation,
indicates that the test compound is an activator of the mitogenic
effect of apelin-36 (42-57). An increase of apelin-36
(42-57)-induced cell suppression at concentrations of 10 nM and
above, for example as measured by decreased relative luminescent
units in a cell proliferation assay or decreased endothelial cell
tube formation, indicates that the test compound is an activator of
the cell growth suppressive effect of apelin-36 (42-57).
[0277] An apelin-36 (42-57) inhibitor can be any type of compound
that is capable of opposing (inhibiting or reducing) a cell growth
activity of apelin-36 (42-57), for example, an antibody (such as a
neutralizing monoclonal antibody), a small molecule inhibitor, a
receptor, a binding protein, or a peptide (for example, apelin-36
(42-58) or the free-acid form of apelin-36 (42-57)). An apelin-36
(42-57) activator can be any type of compound that is capable of
enhancing (increasing or stimulating) a cell growth activity of
apelin-36 (42-57), for example, an antibody (such as an activating
monoclonal antibody), a small molecule, a receptor, a binding
protein, or a peptide. Libraries of molecules useful for screening
for inhibitors or activators are well known to those of ordinary
skill in the art. See, for instance, published international
application PCT/US02/23172 (WO 03/008627; incorporated herein by
reference), which describes additional methods of screening for
interacting molecules and libraries adapted for such screens.
XII. Kits
[0278] The compounds disclosed herein, and in particular apelin-36
(42-57) (amidated or free-acid forms) or an inhibitor of apelin-36
(42-57), can be supplied in the form of kits for use in modulating
angiogenesis or inhibiting tumorigenesis, as well as in the
prevention and/or other treatment of a specific disorder, condition
or diseases (for example, cardiovascular disease, peripheral
vascular disease, hypertension, preeclampsia syndrome, diabetes,
ocular degeneration, idiopathic pulmonary fibrosis, wound healing,
chronic obstructive pulmonary disease). In such a kit, a clinically
effective amount of the active ingredient(s) is provided in one or
more containers. The active ingredient(s) may be provided suspended
in an aqueous solution or as a freeze-dried or lyophilized powder,
for instance. In certain embodiments, it will be provided in the
form of a pharmaceutical composition.
[0279] Kits according to certain embodiments of this disclosure can
also include instructions, usually written instructions, to assist
the user in treating a disorder, condition or disease with the
apelin-36 (42-57) peptide. Still other kits, particularly those in
which an inhibitor of apelin-36 (42-57) is provided, will include
instructions to assist the user in treating a disorder, condition
or disease with the apelin-36 (42-57) inhibitor. The instructions
in kits can be for use of the active ingredient for any of the
purposes described herein. Instructions can optionally be provided
on a computer readable medium.
[0280] The container(s) in which an active ingredient, optionally
with other compound(s), is supplied can be any conventional
container that is capable of holding the supplied form, for
instance, microfuge tubes, ampoules, or bottles. In some
applications, the therapeutic compound may be provided in
pre-measured single use amounts in individual, typically
disposable, tubes or equivalent containers.
[0281] The amount of active ingredient (for example, apelin-36
(42-57) or an inhibitor of apelin-36 (42-57)) supplied in the kit
can be any appropriate amount, depending for instance on the market
to which the product is directed. For instance, if the kit is
adapted for research or clinical use, the amount of vasoconstrictor
compound provided would likely be an amount sufficient for several
treatments.
[0282] Certain kits according to this disclosure will also include
one or more other agents useful in treating the conditions
disclosed herein. For example, such kits may include one or more
effective doses of other agents or other agents useful in the
treatment of particular conditions (such as an antibiotic in the
treatment of septic shock). Still other kits will also include one
or more effective doses of other drugs recognized for treatment of
hypertension (such as those discussed in "Cecil Textbook of
Medicine" (1992) W. B. Saunders, at pages 260-269 (incorporated
herein by reference) for instance), or other agents useful in the
treatment of particular conditions.
[0283] The subject matter of the present disclosure is further
illustrated by the following non-limiting Examples.
EXAMPLES
Example 1
Production of Amide, Free-Acid, and Glycine-Extended Forms of
Apelin-36 (42-57) (Salcut-NH.sub.2, Salcut-OH, and Salcut-Gly)
[0284] Apelin-36, apelin-13, (Pyr.sup.1)-apelin-13, and
(Ala.sup.13)-apelin-13 were obtained commercially from Bachem
(Switzerland). The amide, free-acid, and glycine extended forms of
apelin-36 (42-57) were synthesized using standard techniques on an
Applied Biosystems automated peptide synthesizer following
manufacturer's instructions.
Example 2
Effect of Salcut-NH.sub.2 on a Variety of Endothelial and
Epithelial Cell Lines
[0285] This example demonstrates the proliferative effect of
salcut-NH.sub.2 on a variety of cell types.
[0286] Proliferation response profiles of apelin, salcut-NH.sub.2,
salcut-Gly, and/or salcut-OH on a variety of endothelial and tumor
cell lines or primary endothelial cells were obtained using the
following method. Human blood vessel endothelial cells (HMEC-1),
human primary microvascular dermal EC (dB1), human lymphatic
endothelial cells (LEC), human mast cells (HMC-1), human breast
cancer cells (MCF-7; T47D), human gastric cancer cells (HTB 103),
human lung cancer cells (A549), monkey endothelial cells (CRL
1780), and porcine aortic endothelial cells (PAE) were seeded at
25,000 cells per well at 50 .mu.l volume in appropriate media
containing 0.5% serum in a 96 well plate. After overnight
incubation the peptide was added in serum free media and cells were
incubated for 3-5 days. To measure growth proliferation, the
adenosine triphosphate ATPLITE.TM. one-step assay (PerkinElmer),
which is based on firefly luciferase and has high sensitivity for
quantification of viable cells, was used. Data were expressed as
mean plus minus standard deviation. Statistical analyses were
performed using Student's t-test, p values less than 0.05 were
considered statistically significant.
[0287] FIGS. 3A-3C, 3E, and 3G-3O show that the cell proliferation
(as measured by relative luminescent units [RLU]) increases in the
presence of both apelin-13 (65-77) and the amidated form of
apelin-36 (42-57) (salcut-NH.sub.2), compared to cells cultured in
the absence of these compounds (0 Rx), and that the maximal
increase in proliferation occurs within the concentration range of
10 nM and 100 pM salcut-NH.sub.2. In contrast, FIG. 3D shows that
neither apelin-13 (65-77) nor salcut-NH.sub.2 affect proliferation
of the human lung cancer cell line A549 in the 10 nM to 100 pM
range. Side-by-side graphs of the effect of apelin-13 and
salcut-NH.sub.2 (FIG. 3E), as well as salcut-Gly and salcut-OH
(FIG. 3F), on human blood vessel endothelial (HMEC-1) cells
demonstrate that while apelin-13 (65-77) and salcut-NH.sub.2 are
potent stimulators of HMEC-1 proliferation, salcut-OH and
salcut-Gly have a minor effect on proliferation of these cells.
[0288] FIGS. 3A-3D, 3E, and 3G, 3I-3O show that salcut-NH2
concentrations of 10 nM and greater have a suppressive effect on
cell proliferation in these cells, whereas apelin-13 (65-77)
maintains its positive effect on proliferation. FIG. 3D shows that
although salcut-NH.sub.2 does not stimulate proliferation, it has a
cell growth suppressive effect at higher concentrations. FIG. 3O
shows that at high enough concentrations of apelin-13 (65-77) (for
example, at 10 .mu.M), a suppressive effect may be seen. This
biphasic (rise and fall) response is indicative of two different
receptors being involved in modulating the proliferative response:
(i) a high affinity receptor involved in cell growth
stimulation/proliferation at lower concentrations of
salcut-NH.sub.2 (10 nM and below) and (ii) a low affinity receptor
involved in cell growth inhibition or suppression at higher
concentrations of salcut-NH.sub.2 (10 nM and above).
[0289] FIG. 4 shows the biphasic (rise and fall) response of
salcut-NH.sub.2 on growth of the human breast cancer cell line
MCF-7, whereas the human lung cancer cell line A549 did not respond
in a statistically significant manner to higher concentrations of
salcut-NH.sub.2 by inhibiting proliferation, although there is a
downward trend in proliferation with increasing concentration of
salcut-NH.sub.2.
[0290] FIG. 5 shows the biphasic (rise and fall) response of
salcut-NH.sub.2 on growth of the human gastric cancer cell line
HTB-103, whereas the human mast cell line HMC-1 only responded to
lower concentrations of salcut-NH.sub.2 by stimulating
proliferation in a statistically significant manner.
[0291] FIG. 6 shows the biphasic (rise and fall) response of
salcut-NH.sub.2 on growth of the human blood endothelial cell line
HMEC-1, whereas the salcut-OH and salcut-Gly do not have a
statistically significant effect on cell growth.
Example 3
Tube Formation Assay
[0292] This example demonstrates the effect of salcut-NH.sub.2 on
endothelial cells using an in vitro tube formation assay.
[0293] Porcine aortic endothelial (PAE) cells were stably
transfected with Green Fluorescent Protein (GFP) and seeded at a
concentration of 18,000 cells per well of a 96-well culture plate.
The wells were coated with GELTREX.TM. basement membrane matrix
(Invitrogen, CA). Cells were resuspended in medium at a
concentration of 2.25.times.10.sup.5 to 2.5.times.10.sup.5
cells/ml. An 80 .mu.l cell suspension (18,000-20,000) cells were
added per well. Positive control cells were cultured with medium
supplemented with 1.0% fetal bovine serum (FBS). Negative control
cells were cultured with serum-free medium. Cells were incubated at
37.degree. C. with salcut-NH.sub.2. Salcut-NH.sub.2 was added to
each well to give the following concentrations: 0.1 pM, 1.0 pM, 10
pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 .mu.M, and 10 .mu.M. After three
or more hours of incubation, cells were assessed for tube formation
and were photographed. Assays were performed in triplicate.
[0294] Endothelial cell tubes began to form with as little as 1.0
pM to 10 pM of salcut-NH.sub.2 peptide, compared to the negative
control sample (FIG. 7). Maximum tube formation occurred between
100 pM and 10 nM of salcut-NH2 and then a suppressive effect was
observed at concentrations exceeding 10 nM salcut-NH.sub.2, with 10
.mu.M giving similar results as the negative control well. 1 nM and
10 nM concentrations of salcut-OH and salcut-Gly also gave similar
results as the negative control.
[0295] Formation of endothelial cell tubes in the presence of 1 nM
salcut-NH.sub.2 was not inhibited by the APJ receptor peptide
antagonist (Ala13)-apelin-13 (where the carboxy-terminal
phenylalanine is substituted with an alanine) (FIG. 8 and FIG. 13).
In contrast, (Ala13)-apelin-13 inhibited endothelial cell tube
formation in the presence of 1 nM apelin-13 (FIG. 8). As the effect
of salcut-NH.sub.2 is not altered by the APJ receptor antagonist,
these results indicate that salcut-NH.sub.2 does not act through
the APJ receptor. Instead, salcut-NH.sub.2 mediates its effect
through a different receptor or receptor complex. Thus,
salcut-NH.sub.2 and apelin-13 act via different mechanisms.
Example 4
Production of Polyclonal Antibody Directed Against Apelin-36
(42-57) Amide (Salcut-NH.sub.2)
[0296] Polyclonal serum directed against salcut-NH.sub.2 was
generated using standard protocols (see, for example, Harlow and
Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). This
example describes quantitation experiments which were used to
determine the sensitivity of the anti-salcut-NH.sub.2 polyclonal
antiserum at different dilutions. Also described are titration
assays of the anti-salcut-NH.sub.2 polyclonal antiserum using
ELISA.
Titration Protocol Binding to C-Salcut-NH.sub.2 or
Salcut-NH.sub.2
[0297] Solid phase cysteine-salcut-NH.sub.2 (C-salcut-NH.sub.2;
modified cross-linker immunogen; Rows A, B, C of a 96 well plate),
Apelin-36 (Rows D, E, F), and Apelin-13 (Rows G and H) were applied
to a 96 well plate at 100 ng/50 .mu.l/well and incubated for 2
hours at room temperature (or overnight at 4.degree. C.). The
solution containing unbound immunogen was then aspirated and the
wells were blocked with 1% bovine serum albumin (BSA) in phosphate
buffered saline (PBS) for 1 hour at room temperature. The blocking
solution was aspirated and the wells were washed three times with
PBS. Serial dilutions of 50 .mu.l of rabbit polyclonal
anti-salcut-NH.sub.2 (from a 1:100 to 1:204,800 dilution, columns
1-12 of the 96 well plate) previously absorbed three times with
solid phased (i) BSA (Rows A, D, and G), (ii) Apelin-36 (Rows B and
E), or (iii) C-salcut-NH.sub.2 (Rows C, F, and H) were applied to
the wells and were incubated at room temperature for 1.5 hours. The
anti-serum was aspirated and the wells washed three times with PBS.
Goat anti-rabbit IgG-horseradish peroxidase (HRP) secondary
antibody reagent (50 .mu.l) was added to the wells at a 1:500
dilution in 1% BSA in PBS. The wells were incubated for 1 hour at
room temperature, then aspirated and washed four times with PBS. In
order to visualize the antibody binding, 100 .mu.l of Stabilized
Chromogen (Biosource) was applied to the wells and the wells were
incubated in the dark for 30 minutes at room temperature. Stop
Solution (100 .mu.l) was added and the samples were immediately
read at 450 nm on a TECAN INFINITE.TM. M200 multi-reader
scanner.
[0298] The results of the ELISA titration curve, using different
adsorption peptides and different solid phased ligand targets, are
shown in FIG. 11. When the antiserum is preabsorbed with BSA or
apelin-36, the resulting cleared antiserum still effectively binds
to solid phased C-salcut-NH2, but when it is preabsorbed to
C-salcut-NH.sub.2 binding to C-salcut-NH.sub.2 is almost completely
blocked, even at the lowest dilution of antiserum, demonstrating
the specificity of the antiserum for salcut-NH.sub.2 and/or the
carboxy-terminal amidated glycine residue. Given the close
similarity of the primary amino acid sequence between the
amino-terminus of apelin-36 and C-salcut-NH.sub.2, the resulting
binding data would indicate that unique immune epitopes exist on
C-salcut-NH.sub.2 (original immunogen) that do not exist on
apelin-36, namely the carboxy-terminal amide. Antibodies to this
immune epitope would only be removed by absorption with
C-salcut-NH.sub.2. Furthermore, when the antiserum is preabsorbed
with BSA or C-salcut-NH.sub.2, the antiserum is still capable of
binding to solid phased apelin-36, indicating the presence of
residual antibodies that remain following C-salcut-NH.sub.2
absorption and recognize immune epitopes on apelin-36. Given the
large portion of antibodies that exist towards the amide, it is
presumed that during the absorption process, due to proportional
differences, weighted antibodies binding to the amide residue block
the attachment of glycine directed antibodies to C-salcut-NH.sub.2
via steric hindrance and thus remain free to bind to the regional
glycine residue on solid phased apelin-36. Finally, when the
antiserum is absorbed with apelin-36, binding to apelin-36 is
blocked.
Quantitative ELISA Assay for Salcut-NH.sub.2
[0299] Solid phase C-salcut-NH2 (immunogen) was applied to a 96
well plate either at 100 ng/50 .mu.l/well (Rows A and B), 50 ng/50
.mu.l/well (Rows C and D), 25 ng/50 .mu.l/well (Rows E and F), or
12.5 ng/50 .mu.l/well (Rows G and H) and incubated for one hour at
room temperature.
[0300] The solution containing unbound immunogen was then aspirated
and the wells were blocked with 1% BSA in PBS for 1 hour at room
temperature. The blocking solution was aspirated and the wells were
washed three times with PBS. 25 .mu.l of 1% BSA in PBS (columns 1
and 12) or free C-salcut-NH.sub.2 (10 pg, 50 pg, 100 pg, 500 pg, 1
ng, 5 ng, 10 ng, 50 ng, 100 ng, 500 ng; columns 2-11) in 1% BSA in
PBS was added to the wells, followed by 25 .mu.l of rabbit
polyclonal anti-salcut-NH.sub.2 (1:400 dilution). The wells were
incubated at room temperature for 1.5 hours. The anti-serum was
aspirated and the wells washed three times with PBS. Goat
anti-rabbit IgG-horseradish peroxidase (HRP) secondary antibody
reagent (50 .mu.l) was added to the wells at a 1:500 dilution in 1%
BSA in PBS. The wells were incubated for 1 hour at room
temperature, then aspirated and washed four times with PBS. In
order to visualize the antibody binding, 100 .mu.l of Stabilized
Chromogen (Biosource) was applied to the wells and the wells were
incubated in the dark for 30 minutes at room temperature. Stop
Solution (100 .mu.l) was added and the samples were immediately
read at 450 nm on a TECAN INFINITE.TM. M200 multi-reader
scanner.
[0301] The results of the quantitative ELISA are shown in FIG. 10
and demonstrate that sensitivity of the salcut-NH.sub.2 antiserum
was augmented by lowering the solid phase concentration of
C-salcut-NH.sub.2. Thus, as the concentration of the solid phased
C-salcut-NH.sub.2 is lowered, the resulting titration curve becomes
more linear and the sensitivity (detectable peptide) increases.
Example 5
Nude Mouse Xenograft Studies
[0302] Six week old female nude mice were used in this study; ten
mice were used per test group. A549 human bronchioloalveolar cancer
cells were cultured at the SAIC/Frederick facility.
1.times.10.sup.7 A549 cells were injected subcutaneously in the
hindquarter of each nude mouse. The following treatment regime was
started seven days following tumor injection: Group 1--PBS control;
Group 2--salcut-NH.sub.2 peptide (10 .mu.M). Appropriate Groups
were treated with 25 .mu.l injections at four corners around the
tumor, three times per week for five weeks. Mice were inspected,
weighed, and tumors measured (H.times.W.times.L) three times per
week with calipers. Following completion of the experiment, mice
were euthanized, tumors excised, and cut into four pieces. Two
segments were frozen and stored at -80.degree. C. and two segments
were paraffin embedded for pathology. Nude mice injected with the
A549 cell line showed a dramatic reduction in tumor growth when
treated with salcut-NH.sub.2, compared to treatment with PBS
alone.
[0303] In another experiment, six week old female nude mice were
used in this study; ten mice were used per test group. A549 human
bronchioloalveolar cancer cells were cultured at the SAIC/Frederick
facility. 1.times.10.sup.7 A549 cells were injected subcutaneously
in the hindquarter of each nude mouse. The following treatment
regime was started seven days following tumor injection: Group
1--PBS control; Group 2--salcut-NH.sub.2 peptide (10 pM); Group
3--salcut-NH.sub.2 peptide (10 nM); Group 4--salcut-NH.sub.2
peptide (10 .mu.M). Appropriate Groups were treated with 100 .mu.l
injections intraperitoneally, three times per week for four weeks.
Mice were inspected, weighed, and tumors measured
(H.times.W.times.L) three times per week with calipers. Following
completion of the experiment, mice were euthanized, tumors excised,
and cut into four pieces. Two segments were frozen and stored at
-80.degree. C. and two segments were paraffin embedded for
pathology. FIG. 12 demonstrates reduced tumor growth in the
presence of salcut-NH.sub.2, compared to PBS, a highly
statistically significant (<0.005) suppression of tumor growth
in the presence of 10 .mu.M salcut-NH.sub.2.
Example 6
Effect of Salcut-NH.sub.2 on Aortic Ring/Vessel Outcropping
[0304] This example measures the effect of various concentrations
of salcut-NH.sub.2 on a rat aortic ring assay.
[0305] On day 0, rats were euthanized and decapitated, and aortas
were harvested and transferred to a culture dish containing EGM-2
with growth factors (20 ml media; Clonetics). Fibroadipose tissue
and other non-aortic tissue were removed. Using dissecting
microscope, the aorta was sectioned into 1 mm-long rings. Aortic
rings were then rinsed 6-8.times. with EGM-2 with growth factors.
Aortic rings were each placed in the center of a matrix
(GELTREX.TM.; Invitrogen Corp)-coated culture well. Aortic tissue
was covered with 250 .mu.l GELTREX.TM. and each well was incubated
with 1 ml EGM-2 with growth factors supplemented for 24 hours at
37.degree. C., 5% CO.sub.2.
[0306] On day 1, media with growth factors was removed, 250 .mu.l
of media (without growth factors) with either 0.1 nM, 1.0 nM, or 10
nM of salcut-NH.sub.2 was added to each well and incubated at
37.degree. C., 5% CO.sub.2 for 5-7 days. FIG. 9 demonstrates that
increasing the concentration of salcut-NH.sub.2 generated an
increased number of vessels forming from the aortic ring. In
addition, at the 10 nM salcut-NH.sub.2 dose, vessels were beginning
to form within the aortic ring.
Example 7
Production of Neutralizing Monoclonal Antibody Directed Against
Apelin-36 (42-57) Amide (Salcut-NH.sub.2)
[0307] This example described the production of a neutralizing
monoclonal antibody against salcut-NH.sub.2.
[0308] Hybridomas expressing a neutralizing monoclonal antibody
against salcut-NH.sub.2 are generated as described in Harlow and
Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).
Hybridomas are screened for their ability to bind salcut-NH.sub.2
but not salcut-OH or salcut-Gly, thus selecting for antibodies that
exclusively target the carboxy-terminal amide. An antibody which
binds at or near the carboxy-terminal amide suppresses
peptide/receptor recognition and function as an antagonist. The
resulting anti-salcut-NH.sub.2 antibodies are evaluated in vitro
and in vivo assay systems to confirm the neutralizing antibody's
activity.
Example 8
Effect of APJ Antagonist on Salcut-NH.sub.2-Mediated Endothelial
Cell Proliferation
[0309] This example describes the effect of an APJ peptide
antagonist on salcut-NH.sub.2-mediated endothelial cell
proliferation.
[0310] Endothelial cell proliferation assays were performed as
described above with both spontaneously immortalized porcine aortic
endothelial (PAE) cells and SV40 immortalized human dermal
microvascular endothelial (HMEC-1). FIG. 13A (HMEC-1 cells) and
FIG. 13B (PAE cells) demonstrate that apelin-13(F13A) selectively
inhibits apelin-13 mediated proliferation. However, cell
proliferation regulated by Salcut-NH.sub.2 is not blocked by
apelin-13(F13A) (FIGS. 13A and 13B). These findings clearly
demonstrate that Salcut-NH.sub.2 does not initiate its biological
effects through the APJ receptor, as is done by apelin-13 and
instead mediates its effects through a different receptor or
receptor complex.
Example 9
Identification of Salcut-NH.sub.2 Receptor
[0311] This example describes the use of a biotinylated form of
salcut-NH.sub.2 to identify and isolate the salcut-NH.sub.2
receptor.
[0312] An N-terminal biotinylated salcut-NH.sub.2 ligand was
synthesized (a single biotin moiety was attached to the
amino-terminal leucine residue). Validity of the chemical
composition of the biotinylated salcut-NH.sub.2 was accomplished
using amino acid sequence analysis and MADLI-TOF mass spectrometry
characterization. Comparison studies on human breast cancer cell
line MDA-MB-435 show that salcut-NH.sub.2 produced by two different
manufacturers (FIGS. 14A and 14C) and biotinylated salcut (FIG.
14B) compounds are equipotent in proliferation assays (FIG.
14A-14C). In addition, the MDA-MB-435 cells demonstrate a large
change in response in the presence of different concentrations of
the salcut-NH.sub.2 or biotinylated salcut over a dose range of 1
nM to 1 uM (FIG. 14A-14C).
[0313] The biotinylated amino terminal derivative of
salcut-NH.sub.2 bound to cells demonstrates high intensity
fluorescent labeling. This binding can be inhibited by
administering unlabeled salcut-NH.sub.2 to the cells in the
presence of biotinylated salcut-NH.sub.2, indicating the
specificity of binding of the biotinylated salcut-NH.sub.2.
Salcut-NH.sub.2 is used in combination with ALEXA FLUOR.RTM.488
streptavidin to enrich MDA-MB-345 cells for high cognate receptor
expression via FACS sorting. Such high receptor expressing cells
serve as a source of solubilized biotin-Salcut-NH.sub.2/receptor
complexes. The enriched high-intensity salcut-NH.sub.2 receptor
expressing cells are isolated, and membranes isolated and purified
by sedimentation centrifugation. Isolated membranes are solubilized
with a non-ionic detergent and micelle biotin ligand/receptor
complex for salcut-NH.sub.2 is isolated on a streptavidin column.
The solid phased ligand/receptor complex is uncoupled using a mild
acid wash (0.1M glycine, pH 3.0) and the receptor protein eluate
rapidly neutralized (pH 7.0) with 1.0M ammonium hydroxide. The
sample with the receptor protein is further fractionated using
routine polyacrylamide gel electrophoresis and resulting protein
bands assessed by MALDI MS/MS for amino acid sequence
determination. Protein/molecular database analysis of resulting
amino acid sequences is used to identify known or orphan receptor
entities. Transfection studies with non-responsive cell lines (for
example A549) are performed to confirm functionality of the
receptor.
Example 10
Identification of Salcut-NH.sub.2 Signal Transduction Pathway
[0314] This example describes the identification of the
salcut-NH.sub.2 signal transduction pathway.
[0315] Studies with MDA-MB-435 using cholera or pertussis toxin
inhibitors determine if salcut-NH.sub.2 proliferative activity is
mediated through a G-coupled protein receptor (GCPR). In similar
studies, Chinese Hamster Ovary (CHO) target cells are transfected
with about 200 known and orphaned GCPRs linked to a
beta-galactosidase (beta-gal) reporter. When the transfected
receptor is activated with an appropriate ligand, the reported
cells are turned on to express beta-gal and are visually identified
by the addition of a color substrate. Hence, using this receptor
panning technique it is possible to identify a specific GCPR for
salcut-NH.sub.2 in the CHO cell library. The identified GCPR is
transfected into A549 cells (which are non-responsive in the
proliferation assays discussed above) to determine if growth
function is restored when the cells are exposed to salcut-NH.sub.2.
Many of the GCPRs have known signal transduction pathways and
inhibitors to these pathways will identify which signal
transduction avenue is required for salcut-NH.sub.2 induced cell
growth.
[0316] In view of the many possible embodiments to which the
principles of our invention may be applied, it should be recognized
that illustrated embodiments are only examples of the invention and
should not be considered a limitation on the scope of the
invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
Sequence CWU 1
1
431234DNAhomo sapiens 1atgaatctgc ggctctgcgt gcaggcgctc ctgctgctct
ggctctcctt gaccgcggtg 60tgtggagggt ccctgatgcc gcttcccgat gggaatgggc
tggaagacgg caatgtccgc 120cacctggtgc agcccagagg gtcaaggaat
gggccagggc cctggcaggg aggtcggagg 180aaattccgcc gccagcggcc
ccgcctctcc cataagggac ccatgccttt ctga 2342234DNACanis familiaris
2atgaatctgc ggcgctgcgt gcaggcgctc ctgctgctct ggctctctct gaccgcggcg
60tgtggagggc cgctgctgca gccttctgac ggcaaggcgc tggaggaagg caatatccgc
120cacctggtgc agcccagagg ctcgagaaac ggaccggggc cctggcaggg
cggtcggaag 180aaatttcgcc gtcagcggcc acgcctctcc cataagggcc
ccatgccttt ctga 2343234DNAbos taurus 3atgaatctgc ggcgctgcgt
gcaggcgctc ctgctgctct ggctctgcct gagcgcggtg 60tgcggaggac ccctgctgca
gacttctgac gggaaggaga tggaagaagg caccatccga 120tacctggtgc
agcccagggg gccgaggagc ggcccaggcc cctggcaggg aggtcggagg
180aagttccggc gccagcggcc acgcctctcc cacaagggtc ccatgccttt ctga
2344234DNArattus norvegicus 4atgaatctga gtttctgcgt gcaggcgctg
ctgctgctct ggctctcctt gactgccgtg 60tgtggagtgc cactgatgct gcctccagat
gggaaagggc tagaagaagg caacatgcgc 120tacctggtga agcccagaac
ttcgaggact ggaccagggg cctggcaggg aggcaggagg 180aaatttcgca
gacagcggcc ccgtctctcc cataagggac ccatgccttt ctaa 2345234DNAmus
musculus 5atgaatctga ggctctgcgt gcaggcgctg ctgctgctct ggctctcctt
gactgcagtt 60tgtggagtgc cactgatgtt gcctccagat ggaacaggac tagaagaagg
aagcatgcgc 120tacctggtga agcccagaac ttcgaggact ggaccaggag
cctggcaggg aggcaggagg 180aaatttcgca gacagcgccc ccggctctcc
cataagggcc ccatgccttt ctaa 2346231DNAMonodelphis domestica
6atgaatttgc ggcgctgcct gcaggcgctg ctcctgctct ggctctccct ggcttcggtt
60tgcggagggc ccctggtgga gccatcagac aggaaggagc tggaggaagg gaacattcga
120accctggtgc agcccaaagg agcaagagtt ggaggaccct ggccaggtgg
taggaggaag 180ttccgaaggc agcgtccccg tctctcccac aaaggcccca
tgcctttctg a 2317234DNADanio rerio 7atgaatgtga agatcttgac
gctggtgatt gtgctggtgg tttctctgct gtgttcagcc 60agtgctggtc caatggcctc
caccgagcat agcaaagaga tcgaggaggt gggaagcatg 120aggactcctt
tgcggcagaa tcccgctcga gctggccgga gccaaagacc cgctggctgg
180aggaggagac gccctcgacc ccgcctctcc cataaggggc ccatgccatt ctag
234877PRThomo sapiens 8Met Asn Leu Arg Leu Cys Val Gln Ala Leu Leu
Leu Leu Trp Leu Ser 1 5 10 15 Leu Thr Ala Val Cys Gly Gly Ser Leu
Met Pro Leu Pro Asp Gly Asn 20 25 30 Gly Leu Glu Asp Gly Asn Val
Arg His Leu Val Gln Pro Arg Gly Ser 35 40 45 Arg Asn Gly Pro Gly
Pro Trp Gln Gly Gly Arg Arg Lys Phe Arg Arg 50 55 60 Gln Arg Pro
Arg Leu Ser His Lys Gly Pro Met Pro Phe 65 70 75 977PRTCanis
familiaris 9Met Asn Leu Arg Arg Cys Val Gln Ala Leu Leu Leu Leu Trp
Leu Ser 1 5 10 15 Leu Thr Ala Ala Cys Gly Gly Pro Leu Leu Gln Pro
Ser Asp Gly Lys 20 25 30 Ala Leu Glu Glu Gly Asn Ile Arg His Leu
Val Gln Pro Arg Gly Ser 35 40 45 Arg Asn Gly Pro Gly Pro Trp Gln
Gly Gly Arg Lys Lys Phe Arg Arg 50 55 60 Gln Arg Pro Arg Leu Ser
His Lys Gly Pro Met Pro Phe 65 70 75 1077PRTbos taurus 10Met Asn
Leu Arg Arg Cys Val Gln Ala Leu Leu Leu Leu Trp Leu Cys 1 5 10 15
Leu Ser Ala Val Cys Gly Gly Pro Leu Leu Gln Thr Ser Asp Gly Lys 20
25 30 Glu Met Glu Glu Gly Thr Ile Arg Tyr Leu Val Gln Pro Arg Gly
Pro 35 40 45 Arg Ser Gly Pro Gly Pro Trp Gln Gly Gly Arg Arg Lys
Phe Arg Arg 50 55 60 Gln Arg Pro Arg Leu Ser His Lys Gly Pro Met
Pro Phe 65 70 75 1177PRTrattus norvegicus 11Met Asn Leu Ser Phe Cys
Val Gln Ala Leu Leu Leu Leu Trp Leu Ser 1 5 10 15 Leu Thr Ala Val
Cys Gly Val Pro Leu Met Leu Pro Pro Asp Gly Lys 20 25 30 Gly Leu
Glu Glu Gly Asn Met Arg Tyr Leu Val Lys Pro Arg Thr Ser 35 40 45
Arg Thr Gly Pro Gly Ala Trp Gln Gly Gly Arg Arg Lys Phe Arg Arg 50
55 60 Gln Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe 65 70 75
1277PRTmus musculus 12Met Asn Leu Arg Leu Cys Val Gln Ala Leu Leu
Leu Leu Trp Leu Ser 1 5 10 15 Leu Thr Ala Val Cys Gly Val Pro Leu
Met Leu Pro Pro Asp Gly Thr 20 25 30 Gly Leu Glu Glu Gly Ser Met
Arg Tyr Leu Val Lys Pro Arg Thr Ser 35 40 45 Arg Thr Gly Pro Gly
Ala Trp Gln Gly Gly Arg Arg Lys Phe Arg Arg 50 55 60 Gln Arg Pro
Arg Leu Ser His Lys Gly Pro Met Pro Phe 65 70 75 1376PRTMonodelphis
domesticaulus 13Met Asn Leu Arg Arg Cys Leu Gln Ala Leu Leu Leu Leu
Trp Leu Ser 1 5 10 15 Leu Ala Ser Val Cys Gly Gly Pro Leu Val Glu
Pro Ser Asp Arg Lys 20 25 30 Glu Leu Glu Glu Gly Asn Ile Arg Thr
Leu Val Gln Pro Lys Gly Ala 35 40 45 Arg Val Gly Gly Pro Trp Pro
Gly Gly Arg Arg Lys Phe Arg Arg Gln 50 55 60 Arg Pro Arg Leu Ser
His Lys Gly Pro Met Pro Phe 65 70 75 1477PRTDanio rerio 14Met Asn
Val Lys Ile Leu Thr Leu Val Ile Val Leu Val Val Ser Leu 1 5 10 15
Leu Cys Ser Ala Ser Ala Gly Pro Met Ala Ser Thr Glu His Ser Lys 20
25 30 Glu Ile Glu Glu Val Gly Ser Met Arg Thr Pro Leu Arg Gln Asn
Pro 35 40 45 Ala Arg Ala Gly Arg Ser Gln Arg Pro Ala Gly Trp Arg
Arg Arg Arg 50 55 60 Pro Arg Pro Arg Leu Ser His Lys Gly Pro Met
Pro Phe 65 70 75 1536PRThomo sapiens 15Leu Val Gln Pro Arg Gly Ser
Arg Asn Gly Pro Gly Pro Trp Gln Gly 1 5 10 15 Gly Arg Arg Lys Phe
Arg Arg Gln Arg Pro Arg Leu Ser His Lys Gly 20 25 30 Pro Met Pro
Phe 35 1617PRThomo sapiens 16Lys Phe Arg Arg Gln Arg Pro Arg Leu
Ser His Lys Gly Pro Met Pro 1 5 10 15 Phe 1713PRThomo sapiens 17Gln
Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe 1 5 10 1816PRThomo
sapiens 18Leu Val Gln Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp
Gln Gly 1 5 10 15 1916PRThomo sapiensMOD_RES(16)..(16)AMIDATION
19Leu Val Gln Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gln Gly 1
5 10 15 2017PRThomo sapiens 20Leu Val Gln Pro Arg Gly Ser Arg Asn
Gly Pro Gly Pro Trp Gln Gly 1 5 10 15 Gly 2116PRTCanis familiaris
21Leu Val Gln Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gln Gly 1
5 10 15 2216PRTCanis familiarisMOD_RES(16)..(16)AMIDATION 22Leu Val
Gln Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gln Gly 1 5 10 15
2317PRTCanis familiaris 23Leu Val Gln Pro Arg Gly Ser Arg Asn Gly
Pro Gly Pro Trp Gln Gly 1 5 10 15 Gly 2416PRTbos taurus 24Leu Val
Gln Pro Arg Gly Pro Arg Ser Gly Pro Gly Pro Trp Gln Gly 1 5 10 15
2516PRTbos taurusMOD_RES(16)..(16)AMIDATION 25Leu Val Gln Pro Arg
Gly Pro Arg Ser Gly Pro Gly Pro Trp Gln Gly 1 5 10 15 2617PRTbos
taurus 26Leu Val Gln Pro Arg Gly Pro Arg Ser Gly Pro Gly Pro Trp
Gln Gly 1 5 10 15 Gly 2716PRTrattus norvegicus 27Leu Val Lys Pro
Arg Thr Ser Arg Thr Gly Pro Gly Ala Trp Gln Gly 1 5 10 15
2816PRTrattus norvegicusMOD_RES(16)..(16)AMIDATION 28Leu Val Lys
Pro Arg Thr Ser Arg Thr Gly Pro Gly Ala Trp Gln Gly 1 5 10 15
2917PRTrattus norvegicusMOD_RES(16)..(16)AMIDATION 29Leu Val Lys
Pro Arg Thr Ser Arg Thr Gly Pro Gly Ala Trp Gln Gly 1 5 10 15 Gly
3016PRTmus musculus 30Leu Val Lys Pro Arg Thr Ser Arg Thr Gly Pro
Gly Ala Trp Gln Gly 1 5 10 15 3116PRTmus
musculusMOD_RES(16)..(16)AMIDATION 31Leu Val Lys Pro Arg Thr Ser
Arg Thr Gly Pro Gly Ala Trp Gln Gly 1 5 10 15 3217PRTmus musculus
32Leu Val Lys Pro Arg Thr Ser Arg Thr Gly Pro Gly Ala Trp Gln Gly 1
5 10 15 Gly 3315PRTMonodelphis domesticaulus 33Leu Val Gln Pro Lys
Gly Ala Arg Val Gly Gly Pro Trp Pro Gly 1 5 10 15
3415PRTMonodelphis domesticaulusMOD_RES(15)..(15)AMIDATION 34Leu
Val Gln Pro Lys Gly Ala Arg Val Gly Gly Pro Trp Pro Gly 1 5 10 15
3516PRTMonodelphis domesticaulus 35Leu Val Gln Pro Lys Gly Ala Arg
Val Gly Gly Pro Trp Pro Gly Gly 1 5 10 15 36231DNAXenopus laevis
36atgaatctca gactttgggc actggcgctt ctgctcttca ttttaacctt gacttcagca
60tttggagctc cactggctga aggctcagat aggaatgacg aagaacagaa tatccggaca
120ctggtgaacc ccaaaatggt tcgtaactct gcacctcaac ggcaagcaaa
ccgaagaaaa 180ctcatacgtc aaagaccccg tctttcacac aagggcccaa
tgcccttcta a 2313776PRTXenopus laevis 37Met Asn Leu Arg Leu Trp Ala
Leu Ala Leu Leu Leu Phe Ile Leu Thr 1 5 10 15 Leu Thr Ser Ala Phe
Gly Ala Pro Leu Ala Glu Gly Ser Asp Arg Asn 20 25 30 Asp Glu Glu
Gln Asn Ile Arg Thr Leu Val Asn Pro Lys Met Val Arg 35 40 45 Asn
Ser Ala Pro Gln Arg Gln Ala Asn Arg Arg Lys Leu Ile Arg Gln 50 55
60 Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe 65 70 75
3817PRThomo sapiens 38Cys Leu Val Gln Pro Arg Gly Ser Arg Asn Gly
Pro Gly Pro Trp Gln 1 5 10 15 Gly 39234DNAMacaca mulatta
39atgaatctgc ggctctgcgt gcaggcgctc ctgctgctct ggctctcctt gaccgcggtg
60tgtggagggc ccctgatgca gcttccctat gggaatgggc tggaagaggg caatgtccgc
120cacctggtgc agcccagagg gtcgaggaac gggccagggc cctggcaggg
aggtcgaagg 180aaattccgcc gccagcggcc ccgcctctcc cataagggac
ccatgccttt ctga 2344077PRTMacaca mulatta 40Met Asn Leu Arg Leu Cys
Val Gln Ala Leu Leu Leu Leu Trp Leu Ser 1 5 10 15 Leu Thr Ala Val
Cys Gly Gly Pro Leu Met Gln Leu Pro Tyr Gly Asn 20 25 30 Gly Leu
Glu Glu Gly Asn Val Arg His Leu Val Gln Pro Arg Gly Ser 35 40 45
Arg Asn Gly Pro Gly Pro Trp Gln Gly Gly Arg Arg Lys Phe Arg Arg 50
55 60 Gln Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe 65 70 75
4116PRTMacaca mulatta 41Leu Val Gln Pro Arg Gly Ser Arg Asn Gly Pro
Gly Pro Trp Gln Gly 1 5 10 15 4216PRTMacaca
mulattaMOD_RES(16)..(16)AMIDATION 42Leu Val Gln Pro Arg Gly Ser Arg
Asn Gly Pro Gly Pro Trp Gln Gly 1 5 10 15 4317PRTMacaca mulatta
43Leu Val Gln Pro Arg Gly Ser Arg Asn Gly Pro Gly Pro Trp Gln Gly 1
5 10 15 Gly
* * * * *