Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED POLYPEPTIDE SPECIES (FRAGMENTS FROM CHITOTRIOSIDASE)
REDUCED IN CARDIOVASCULAR DISORDERS
FIELD OF THE INVENTION
The invention relates to secreted polypeptide species absent from the plasma
of individuals
with cardiovascular disorders, but present in the plasma of individuals free
from the disease, isolated
polynucleotides encoding such polypeptides, polyrnorphic variants thereof, and
the use of said
nucleic acids and polypeptides or compositions thereof in detection assays,
for cardiovascular
disorder diagnosis, for. cardiovascular disorder treatment and for drug
development.
BACKGROUND
Cardiovascular disease is a major health risk throughout the industrialized
world. Coronary
Artery Disease (CAD) is characterized by atherosclerosis or hardening of the
arteries.
Atherosclerosis is the most prevalent of cardiovascular diseases, is the
principal cause of heart
attack, stroke, and gangrene of the extremities, and thereby the principle
cause of death in the United
States. Atherosclerosis is a complex disease involving many cell types and
molecular factors
(described in, for example, Ross, 1993, Nature 362: 801-809). In normal
circumstances a protective
response to insults to the endothelium and smooth muscle cells (SMCs) of the
wall of the artery
consists of the formation of fibrofatty and fibrous lesions or plaques,
preceded.and accompanied by
inflammation. The advanced lesions of atherosclerosis may occlude the artery
concerned, and result
from an excessive inflammatory-fibroproliferative response to numerous
difFerent forms of insult.
Injury or dysfunction of the vascular endothelium is a common feature of many
conditions that
predispose an individual to accelerated development of atherosclerotic
cardiovascular disease.
Atherosclerotic plaques occlude the blood vessel concerned and restrict the
flow of blood,
resulting in ischemia. Ischemia is a condition characterized by a lack of
oxygen supply in tissues of
organs dlxe to inadequate perfusion. Such. inadequate perfusion can have a
number of natural,causes,
including atherosclerotic or restenotic lesions, anemia, or stroke. The most
cornrnon cause of
ischemia in the heart is atherosclerotic disease of epicardial coronary
arteries. By reducing the lumen
of these vessels, atherosclerosis causes an absolute decrease in myocardial
perfusion in the basal
state or limits appropriate increases in perfusion when the demand for flow is
augmented. Coronary
blood flow can also be limited by arterial thrombi, spasm, and,' raxely,
coronary emboli, as well as by
ostial narrowing due to luetic aortitis. Congenital abnormalities, such as
anomalous origin of the left
anterior descending coronary artery from the pulmonary artery, may cause
myocardial ischemia and
infarction in infancy, but this cause is very rare in adults.
Myocardial ischemia can also occur if myocardial oxygen demands are abnormally
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increased, as in severe ventricular hypertrophy due to hypertension or aortic
stenosis. The latter can
be present with angina that is indistinguishable from that caused by coronary
atherosclerosis. A
reduction in the oxygen-carrying capacity of the blood, as in extremely severe
anemia or in the
presence of carboxy-hemoglobin, is a rare cause of myocardial ischemia. Not
infrequently, two or
more causes of ischemia will coexist, such as an increase in oxygen demand due
to left ventricular
hypertrophy and a reduction in oxygen supply secondary to coronary
atherosclerosis.
Extensiye_cline.cal_studies_have~identifed_factors.that increase the risk of
cardiovascular
disorders. Some of these risk factors, such as age, gender, and family history
cannot be changed.
Other risk factors include the following: smoking, high blood pressure, high
fat and high cholesterol
diet, diabetes, lack of exercise, obesity, and stress.
Fortunately, many contributing factors are controllable through lifestyle
changes. The risk
of cardiovascular disorders for smokers is more than twice that of non-
smokers. When a person
stops smoking, regardless of how much he or she may have smoked in the past,
their risk of
developing a disorder rapidly~declines. Serum cholesterol level is directly
related to prevalence of
cardiovascular disorder and hypertension or high blood pressure is an
important risk factor.
Physical activity has been postulated to reduce the risk of developing a
cardiovascular disorder
through various mechanisms: it increases myocardial oxygen supply, decreases
oxygen demand, and
improves myocardial contraction and its electrical impulse stability. Reduced
oxygen demand and
myocardial work are reflected in lowered heart rate and blood pressure at
rest. Physical activity also
increases the diameter and dilatory capacity of coronary arteries, increases
collateral artery
formation, and reduces rates of progression of coronary artery
atherosclerosis. Obesity and the
serum fatty acids are reduced by activity.
There may be no noticeable symptoms of a cardiovascular disorder at rest, but
symptoms
such as chest pressure may occur with increased activity or stress. Other
first signs that can appear
are heartburn, nausea, vomiting, numbness, shortness of breath, heavy cold
sweating, unexplained
fatigue, and feelings of anxiety. The more severe symptoms of cardiovascular
disorders are chest
_ pain (angina pectoris), rhythm disturbances (arrhythmias), stroke, or heart
attack (myocardial
infarction). Strokes and heart attacks result from a blocked artery in the
brain and heart tissue,
respectively. Because symptoms vary, the tests and treatments chosen can be
very different from one
patient to another.
Diagnostic tests useful in determining the extent and severity of
cardiovascular disorder
include: electrocardiogram (EKG), stress test, nuclear scanning, coronary
angiography, resting EKG,
EKG Multiphase Information Diagnosis Indexes, Holter monitor, late potentials,
EKG mapping,
echocardiogram, Thallium scan, PET, MRI, CT, angiogram and IVLJS. Additional
risk factor
measures and useful diagnostics are comnnon and best applied by one of skill
in the art of medicine.
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There are many different therapeutic approaches, depending on the seriousness
of the disease. For
many people, cardiovascular disorders are managed with lifestyle changes and
medications. More
severe diagnoses may indicate a need for surgery.
Surgical approaches to the treatment of ischemic atherosclerosis include
bypass grafting,
coronary angioplasty, laser angioplasty, atherectomy, endaxterectomy, and
percutaneous
translumenal angioplasty (PCTA). The failure rate after these approaches due
to restenosis, in which
the occlusions recur and often become even worse, is extraordinarily high (30-
50%). It appears that
much of the restenosis is due to further inflammation, smooth muscle
accumulation, and thrombosis.
Additional therapeutic approaches to cardiovascular disease have included
treatments that
encouraged angiogenesis and tissue remodeling in such conditions as ischemic
heart and limb
disease.
The non-specific nature of most CAD and cardiovascular disorder symptoms makes
definitive diagnosis difficult. More quantitative diagnostic methods suffer
from variability, both
between individuals and between readings on a single individual. Thus,
diagnostic measures must be
standardized and applied to individuals with well-documented and extensive
medical histories.
Further, current diagnostic methods often do not reveal the underlying cause
for a given observation
or reading. Therefore, a therapeutic strategy based on a particular positive
result likely will not
address the causative problem and may even be harmful to the individual.
Methods of diagnosis that rely on nucleotide detection include genetic
approaches and
expression profiling. For example, genes that are known to be involved in
cardiovascular disorders
may be screened for mutations using common genotyping techniques such as
sequencing,
hybridization-based techniques, or PCR. In another example, expression from a
known gene may be
tracked by standard techniques including RTPCR, various hybridization-based
techniques, and
sequencing. These strategies often do not enable a practitioner to detect
differences in mRNA
processing and splicing, translation rate, mRNA stability, and
posttranslational modifications such
as proteolytic processing, phosphorylation, glycosylation, and amidation.
To address the current weaknesses in the diagnostic state of the art for
cardiovascular
disorders, the invention provides polypeptides relevant to cardiovascular
disorders. The invention
provides the identity of polypeptide species that axe undetectable in plasma
from individuals with
Coronary Artery Disease but present in control plasma. By providing the actual
polypeptide species,
differences in mRNA processing and splicing, translation rate, mRNA stability,
and posttranslational
modifications such as proteolytic processing, phosphorylation, glycosylation,
and amidation are
revealed. The polypeptides of the invention are thus described as
"Cardiovascular disorder Plasma
Polypeptides" or CPPs. These polypeptide sequences are described as SEQ ID
NOs:3-4, and those
comprising at least one of the amino acid sequences selected from the tryptic
peptide of Table 1.
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The present invention discloses "Cardiovascular disorder Plasma Polypeptides"
(CPPs),
fragments, and post-translationally modified species of CPPs that are reduced
in the plasma obtained
from individuals with Coronary Artery Disease (CAD). Preferred fragments of
the invention are
those described as SEQ ID NOs:3-4. Thus, the CPPs of the invention represent
an important
diagnostic tool for determining the risk of CAD, coronary heart disease (CHD),
peripheral vascular
disease, cerebral ischemia (stroke), congestive heart failure,
atherosclerosis, hypertension, and other
cardiovascular diseases. CPPs are secreted factors and as~such, are ideal
candidates for protein-
based therapies. For dosage modulation in a clinical setting, protein therapy
is preferable to genetic
therapy, which is hampered by the lack of finely regulable expression.
Further, as secreted factors,
the polypeptide species of the invention are easy to target, e.g., with a
small molecule or protein
modulator. Thus, the polypeptide species of the invention are useful for drug
development and design
of therapeutic strategies to prevent and treat cardiovascular disease.
SUMMARY OF THE INVENTION
The present invention is directed to compositions related to secreted, active
polypeptide
species that, while present in normal human plasma, are undetectable in plasma
from individuals
with a cardiovascular disorder. These polypeptide species are designated
herein "Cardiovascular
disorder Plasma Polypeptides," or CPPs. In particular, preferred CPPs of the
invention are
fragments, described as SEQ ID N0:3-4, and designated herein CPP 6 and CPP 7.
Compositions
include CPP precursors, antibodies specific for CPPs, including monoclonal
antibodies and other
binding compositions derived therefrom. Further included are methods of making
and using these
compositions. Precursors of the invention include unmodified precursors,
proteolytic precursors of
SEQ ID NOs:l-5, and intermediates resulting from alternative proteolytic sites
in the amino acid
sequences of SEQ ID NOs:l-5.
A preferred embodiment of the invention includes CPPs having a
posttranslational
modification, such as a phosphorylation, glycosylation, acetylation,
amidation, or a C-, N- or O-
linked carbohydrate group. Additionally preferred are CPPs with intra- or
inter-molecular
interactions, e.g., disulfide and hydrogen bonds, that result in higher order
structures. Also preferred
are CPPs that result from differential mRNA processing or splicing.
Preferably, the CPPs represent
posttranslationally modified species, structural variants, or splice variants
that are differentially
reduced in the plasma from individuals having or at risk of developing a
cardiovascular disorder.
In another aspect, the invention includes CPPs comprising a sequence which is
at least 95
percent identical to a SEQ ID NOs:3-5. Preferably, the invention includes
polypeptides comprising
at least 97 percent, and more preferably at least 98 percent, and still more
preferably at least 99
percent, identity with any one of the sequences selected from SEQ ID NOs:3-5.
Most preferably, the
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invention includes polypeptides comprising a sequence at least 99 percent
identical to a SEQ ID
NOs:3-5.
In another aspect, the invention includes natural variants of CPPs having a
frequency in a
selected population of at least two percent. More preferably, such natural
variant has a frequency in
a selected population of at least five percent, and still more preferably, at
least ten percent. Most
preferably, such natural variant has a frequency in a selected population of
at least twenty percent.
The selected population may be any recognized population of study in the field
of population
genetics. Preferably, the selected population is Caucasian, Negroid, or Asian.
More preferably, the
selected population is French, German, English, Spanish, Swiss, Japanese;
Chinese, Irish, Korean,
Singaporean, Icelandic, North American, Israeli, Arab, Turkish, Greek,
Italian, Polish, Pacific
Islander, Finnish, Norwegian, Swedish, Estonian, Austrian, or Indian. More
preferably, the selected
population is Icelandic, Saami, Finnish, French of Caucasian ancestry, Swiss,
Singaporean of
Chinese ancestry, Korean, Japanese, Quebecian, North American Pima Indians,
Pennsylvanian
Amish and Amish Mennonite, Newfoundlander, or Polynesian.
A preferred aspect of the invention provides a composition comprising an
isolated CPP, i.e.,
a CPP free from proteins or protein isoforms having a significantly different
isoelectric point or a
significantly different apparent molecular weight from the CPP. The
isoelectric point and molecular
weight of a CPP may be indicated by affinity and size-based separation
chromatography, 2-
dimensional gel analysis, and mass spectrometry.
1n a preferred aspect, the invention provides particular polypeptide species
that comprise an
amino acid sequence selected from the group consisting of SEQ ID NOs:3-5.
Preferably, the
particular polypeptide species further comprises contiguous amino acid
sequence from the
appropriate full length sequence selected from the group consisting of SEQ ID
NOs: l and 2.
Preferred species are polypeptides that i) comprise an amino acid sequence
selected from the group
consisting of SEQ ID NOs:3-5; ii) appear at a higher level in plasma from
control individuals
without a cardiovascular disorder; and iii) result from proteolytic processing
of a full length
polypeptide sequence selected from the group consisting of SEQ ID NOs:l and 2.
Particularly
preferred polypeptides are the CPP 6 (SEQ ID N0:3) and CPP 7 (SEQ ID N0:4).
In an additional aspect, the invention includes modified CPPs. Such
modifications include
protecting/blocking groups, linkage to an antibody molecule or other cellular
ligand, and detectable
labels, such as an enzymatic, fluorescent, isotopic or affinity label to allow
for detection and
isolation of the protein. Chemical modifications may be carried out by known
techniques, including
but not limited, to specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain,
V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, or
metabolic synthesis in the
presence of tunicamycin.
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.Also provided by the invention are chemically modified derivatives of the
polypeptides of the
invention which may provide additional advantages such as increased
solubility, stability and
circulating time of the polypeptide, or decreased immuriogenicity (e.g., water
soluble polymers such
as polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose,
dextran, polyvinyl alcohol). 'The CPPs are modified at random positions within
the molecule, or at
predetermined positions within the molecule and may include one, two, three or
more attached
chemical moieties.
In another embodiment, the invention provides a method of identifying a
modulator of at
least one CPP biological activity comprising the steps of: i) contacting a
test modulator of a CPP
biological activity with the polypeptide comprising the amino acid sequence
selected from the group
consisting of SEQ ID NOs: l-5; ii) detecting the level of said CPP biological
activity; and iii)
comparing the level of said CPP biological activity to that of a control
sample lacking said test
modulator. Where the difference in the level of CPP protein biological
activity is a decrease, the test
modulator is an inhibitor of at least one CPP biological activity. Where the
difference in the level of
CPP biological activity is an increase, the test substance is an activator of
at least one CPP
biological activity.
In another, aspect, the invention includes polynucleotides encoding a CPP of
the invention,
polynucleotides encoding a polypeptide.having an amino acid sequence selected
from the group
consisting of SEQ ID NOs: l-5, antisense oligonucleotides complementary to
such sequences,
oligonucleotides complementary to CPP gene sequences for diagnostic and
analytical assays (e.g.,
PCR, hybridization-based techniques), and vectors fox expressing CPPs.
In another aspect, the invention provides a vector comprising DNA encoding a
CPP. The
invention also includes host cells and transgenic nonhuman animals comprising
such a vector.
There is also provided a method of making a CPP or CPP precursor. One
preferred method
comprises the steps of (a) providing a host cell containing an expression
vector as disclosed above;
(b) culturing the host cell under conditions whereby the DNA segment is
expressed; and (c)
recovering the protein encoded by the DNA segment. Another preferred method
comprises the steps
of (a) providing a host cell capable of expressing a CPP; (b) culturing said
host cell under
conditions that allow expression of said CPP; and (c) recovering said CPP.
Within one embodiment
the expression vector further comprises a secretory signal sequence operably
linked to the DNA
segment, the cell secretes the protein into a culture medium, and the pxotein
is recovered from the
medium. An especially preferred method of making a CPP includes chemical
synthesis using
standard peptide synthesis techniques, as described in the section titled
"Chemical Manufacture of
CPP Compositions" and in Example 3.
In another aspect, the invention includes isolated antibodies specific for any
of the
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polypeptides, peptide fragments, or peptides described above. Preferably, the
antibodies of the
invention are monoclonal antibodies. Further preferred are antibodies that
bind to a CPP
exclusively, that is, antibodies that do not recognize other polypeptides,
e.g., those not derived from a
polypeptide selected from the group consisting of SEQ ID NOs: l-5. Anti-CPP
antibodies have
purification, diagnostic and therapeutic applications, particularly in
treating CPP-related disorders.
Preferred anti-CPP antibodies for purification and diagnosis are attached to a
label group. Preferred
CPP-related disorders for diagnosis include coronary artery disease (CAD),
coronary heart disease
(CHD), peripheral vascular disease, cerebral ischemia (stroke), congestive
heart failure,
atherosclerosis, hypertension, and other cardiovascular diseases. Treatment
and diagnostic methods
include, but are not limited to, those that employ antibodies or antibody-
derived compositions
specific for a CPP antigen. Diagnostic methods for detecting CPPs in specific
tissue samples and
biological fluids (preferably plasma), and for detecting levels of expression
of CPPs in tissues, also
form part of the invention. Compositions comprising one or more antibodies
described above,
together with a pharmaceutically acceptable carrier are also within the scope
of the invention.
The invention further provides methods for diagnosis of cardiovascular
disorders that
comprise detecting in a sample of body fluid, preferably blood plasma, the
presence or level of at
least one CPP disclosed herein or any combination thereof. These methods are
also suitable for
clinical screening, prognosis, monitoring the results of therapy, identifying
patients most likely to
respond to a particular therapeutic treatment, drug screening and development,
and identification of
new targets for drug treatment.
The invention provides kits that may be used in the above-recited methods and
that may
comprise single or multiple preparations, or antibodies, together with other
reagents, label groups,
substrates, if needed, and directions for use. The kits may be used for
diagnosis of disease, or may be
assays for the identification of new diagnostic andlor therapeutic agents.
In one embodiment, Coronary Artery Disease (CAD) is defined by the appearance
of at least
one symptom. Such symptoms become more serious as the disease progresses. CAD
is often
accompanied by reduced left ventricle capacity or output. Early CAD symptoms
include elevated
plasma levels of cholesterol and low-density lipoprotein (especially oxidized
forms), as well as
platelet-rich plasma aggregations. The vascular endothelium responds to
inflammation and thus
formation of plaques and levels of inflammatory and fibrinogenic factors
increase. .In addition, CAD,
or atherosclerosis, is characterized by vascular calcification and hardening
of the arteries. The
resulting partial occlusion of the blood vessels leads to hypertension and
ischemic heart disease.
Eventual complete vascular occlusion results in myocardial infarction, stroke,
or gangrene.
In a preferred embodiment, detection of reduced plasma levels of at least one
CPP of the
invention indicates an increased risk that an individual will develop CAD.
Preferably, said detection
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indicates that an individual has at least a 1.05-fold, 1.1-fold, 1.15-fold,
and more preferably at least
a 1.2-fold increased likelihood of developing CAD. Alternatively, detection of
reduced plasma levels
of at least one CPP of the invention indicates that an individual has CAD. The
amount of CPP
decrease observed in an individual plasma sample compared to a control sample
will correlate with
the certainty of the prediction or diagnosis of CAD. As individual plasma CPP
levels will vary
depending on family history and other risk factors, each will preferably be
examined on a case-by-
case basis. In preferred embodiments, CPP is detected in a human plasma sample
by the methods of
the invention. Especially preferred techniques are mass spectrometry and
immunodetection.
Preferably, a prediction or diagnosis of CAD is based on at least a 1. l-,
1.15-, 1.2-, 1.25-, and more
preferably a 1.5-fold decrease in the experimental CPP level as compared to
the control.
The invention further includes methods of using CPP compositions to treat
disorders
associated with aberrant expression or processing of CPPs of SEQ ID NOs:3-4 in
an individual.
Preferred CPP-related disorders include coronary artery disease (CAD),
coronary heart disease
(CHD), peripheral vascular disease, cerebral ischemia (stroke), congestive
heart failure,
atherosclerosis, hypertension, and other cardiovascular diseases. Further
included are methods of
using CPP compositions, including primers complementary to CPP genes and/or
messenger RNA
and anti- CPP antibodies, for detecting and measuring quantities of CPPs in
tissues and biological
fluids, preferably plasma. The invention further includes methods of screening
.for compounds that
increase the expression of CPPs, as well as methods of screening for compounds
that interact with
and/or increase the activity of CPPs. The invention further includes methods
of screening for
compounds that inhibit the expression of CPPs, as well as methods of screening
for compounds that .
interact with and/or inhibit the activity of CPPs. The invention further
encompasses compounds thus
identified, as well as compositions thereof
In still a further aspect, the invention includes pharmaceutical compositions
and
formulations comprising a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NOs:l-5, and a pharmaceutically acceptable Garner
compound.
Further aspects of the invention are also described in the specification and
in the claims.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID N0:1 represents the amino acid sequence of full-length Chitotriosidase
(SwissProt
Q 13231).
SEQ ID N0:2 represents the amino acid sequence of the mature Chitotriosidase
polypeptide.
SEQ ID NOs:3 and 4 are the amino acid sequences of plasma proteins that are
absent or
reduced in the plasma of individuals with coronary artery disease, which are
herein designated CPP 6
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and CPP 7, respectively.
SEQ ID N0:5 describes the amino acid sequence of the tryptic peptide
discovered by MS-
MS and/or MS-MALDI mass spectrometry in plasma samples of individuals without
coronary artery
disease.
BRIEF DESCRIPTION OF THE TABLE
Table 1 lists the tryptic peptide found in fractionated plasma of control
individuals without
Coronary Artery Disease (CAD) and absent in the plasma of patients with CAD.
The column
labelled CEX indicates in which of the 18 cation exchange fractions the
tryptic peptide was eluted,
and the column labelled Salt indicates the NaCI concentration for the elution
of these fractions,
according to the protocol described in Step 3 of Example 1 herein. RP 1 refers
to the reverse phase
fractions (fractions 1-30), and %B indicates the percentage of elution buffer
for these fractions,
according to the protocol described in Step 4 of Example 1 herein. The second
reverse phase fraction
(fractions 1-24) is indicated as the last two digits of the Run Number.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 shows the sequences of human Chitotriosidase preprotein (SEQ ID NO:
l); mature
Chitotriosidase polypeptide (SEQ ID N0:2); and Cardiovascular disorder Plasma
Polypeptides 6-7
(SEQ ID NOs:3-4). The tryptic peptide observed in control plasma samples by
tandem mass
spectrometry is in bold and illustrated as SEQ ID N0:5. The signal sequence is
underlined. Dibasic
proteolytic cleavage sites are denoted by italic and underlined letters. The
amino acid sequences of
the resulting processed peptides, CPP 6-7 (SEQ ID NOs:3-4), are listed below.
The putative active
site of the protein is shown in bold and underlined (Boot, R.G. et al., J.
Biol. Chem., (1995), 270,
p.26252-56).
DETAILED DESCRIPTION OF THE INVENTION
The present invention described in detail below provides methods,
compositions, and kits
useful for screening, diagnosis, and treatment of a cardiovascular disorder in
a mammalian
individual; for identifying individuals most likely to respond to a particular
therapeutic treatment; for
monitoring the results of cardiovascular disorder therapy; for screening CPP
modulators; and for
drug development. When the invention is used for drug development, e.g., to
determine the ability of
a CPP modulator or drug candidate to induce an anti-cardiovascular disorder
response, the body
fluid analyzed for the level of at least one CPP is preferably from a non-
human mammal. The non-
human mammal is preferably one in which the induction of an anti-
cardiovascular disorder response
by endogenous and/or exogenous agents is predictive of the induction of such a
response in a human.
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Rodents (mice, rats, etc.) and primates are particularly suitable for use in
this aspect of the
invention. The invention also encompasses the administration of therapeutic
compositions to a
mammalian individual to treat or prevent cardiovascular disorders. The
mammalian individual may
be a non-human mammal, but is preferably human, more preferably a human adult.
For clarity of
disclosure, and not by way of limitation, the invention will be described with
respect to the analysis
of blood plasma samples. However, as one skilled in the art will appreciate,
the assays and
techniques described below can be applied to other biological fluid samples
(e.g. cerebrospinal fluid,
lymph, bile, serum, saliva or urine) or tissue samples from an individual at
risk of having or
developing a cardiovascular disorder. The methods and compositions of the
present invention are
useful for screening, diagnosis and prognosis of a living individual, but may
also be used for
postmortem diagnosis in an individual, for example, to identify family members
who are at risk of
developing the same disorder.
Defini tiot2s
As used herein, the term "nucleic acids" and "nucleic acid molecule" is
intended to include
DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and
analogs of
the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule
can be single-
stranded or double-stranded, but preferably is double-stranded DNA. Throughout
the present
specification, the expression "nucleotide sequence" may be employed to
designate indifferently a
polynucleotide or a nucleic acid. More precisely, the expression "nucleotide
sequence" encompasses
the nucleic material itself and is thus not restricted to the sequence
information (i.e. the succession of
letters chosen among the four base letters) that biochemically characterizes a
specific DNA or RNA
molecule. Also, used interchangeably herein are terms "nucleic acids",
"oligonucleotides", and
"polynucleotides".
An "isolated" nucleic acid molecule is one which is separated from other
nucleic acid
molecules which are present in the natural source of the nucleic acid.
Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the nucleic acid
(i.e., sequences located at the
5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from
which the nucleic acid
is derived. For example, in various embodiments, the isolated CPP nucleic acid
molecule can contain
less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide
sequences which naturally
flank the nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free
of other cellular material, or culture medium when produced by recombinant
techniques, or
substantially free of chemical precursors or other chemicals when chemically
synthesized. A. nucleic
acid molecule of the present invention can be isolated using standard
molecular biology techniques
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and the sequence information provided herein. Using all or a portion of the
nucleic acid, as a
hybridization probe, CPP nucleic acid molecules can be isolated using standard
hybridization and
cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and
Maniatis, T. Molecular
Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
As used herein, the term "hybridizes to" is intended to describe conditions
for moderate
stringency or high stringency hybridization, preferably where the
hybridization and washing
conditions permit nucleotide sequences at least 60% homologous to each other
to remain hybridized
to each other. Preferably, the conditions are such that sequences at least
about 70%, more preferably
at least about 80%, even more preferably at least about 85%, 90%, 95% or 98%
homologous to each
other typically remain hybridized to each other. Stringent conditions are
known to those skilled in
the art and can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y.
(1989), 6.3.1-6.3.6. In a preferred, non-limiting example, stringent
hybridization conditions for
nucleic acid interactions are as follows: the hybridization step is realized
at 65°C in the presence of 6
x SSC buffer, 5 x Denhardt's solution, 0,5% SDS and 100~g/ml of salmon sperm
DNA. The
hybridization step is followed by four washing steps:
- two washings during 5 min, preferably at 65°C in a 2 x SSC and
0.1%SDS buffer;
- one washing during 30 min, preferably at 65°C in a 2 x SSC and 0.1%
SDS buffer,
- one washing during 10 min, preferably at 65°C in a 0.1 x SSC.and
0.1%SDS buffer,
these hybridization conditions being suitable for a nucleic acid molecule of
about 20 nucleotides in
length. It will be appreciated that the hybridization conditions described
above are to be adapted
according to the length of the desired nucleic acid, following techniques well
known to the one skilled
in the art, for example be adapted according to the teachings disclosed in
Hames B.D. and Higgins
S.J. (1985) Nucleic Acid Hybridization: A Practical Approach. Hames and
Higgins Ed., IRL Press,
Oxford; and Current Protocols in Molecular Biology (supra).
"Percent homology" is used herein to refer to both nucleic acid sequences and
amino acid
sequences. As used herein, amino acid or nucleic acid "identity" is equivalent
to amino acid or
nucleic acid "homology". To determine the percent homology of two amino acid
sequences or of two
nucleic acids, the sequences are aligned for optimal comparison purposes
(e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid sequence for
optimal alignment with a
second amino or nucleic acid sequence and non-homologous sequences can be
disregarded for
comparison purposes). The length of a reference sequence aligned for
comparison purposes is at
least 30%, preferably at least 40%, more preferably at least 50%, even more
preferably at least 60%,
and even more preferably at least 70%, 80%, 90% or 95% of the length of the
reference sequence.
The amino acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions
11
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are then compared. When a position in the first sequence is occupied by the
same amino acid residue
or nucleotide as the corresponding position in the second sequence, then the
molecules are
homologous at that position. The percent homology between the two sequences is
a function of the
number of identical positions shared by the sequences (i.e., % homology=# of
identical
positions/total # of positions 100).
The comparison of sequences and determination of percent homology between two
sequences
can be accomplished using a mathematical algorithm. A preferred, non-limiting
example of a
mathematical algorithm utilized for the comparison of sequences is the
algorithm of Karlin and
Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin
and Altschul (1993)
Proc. Natl. Acad. Sci. USA 90:5873-77, the disclosures of which are
incorporated herein by
reference in their entireties. Such an algorithm is incorporated into the
NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST nucleotide
searches can be performed with the NBLAST program, score=100, wordlength=12 to
obtain
nucleotide sequences homologous to the sequences of the invention. BLAST
protein searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences
homologous to the polypeptide sequences of the invention. To obtain gapped
alignments for
comparison purposes, Gapped BLAST can be utilized as described in Altschul et
al., (1997) Nucleic
Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the
default parameters of the respective programs (e.g., XBLAST and NBLAST) can be
used. See
http://www.ncbi.nlin.nih.gov, the disclosures of which are incorporated herein
by reference in their
entireties. Another preferred, non-limiting example of a mathematical
algorithim utilized for the
comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989),
the disclosures of
which are incorporated herein by reference in their entireties. Such an
algorithm is incorporated into
the ALIGN program (version 2.0) which is part of the GCG sequence alignment
software package.
When utilizing the ALIGN program for comparing amino acid sequences, a PAM120
weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The term "polypeptide" refers to a polymer of amino acids without regard to
the length of
the polymer; thus, peptides, oligopeptides, and proteins are included within
the definition of
polypeptide. This term also does not specify or exclude post-translational
modifications of
polypeptides, for example, polypeptides which include the covalent attachment
of glycosyl, acetyl,
phosphate, amide, lipid, carboxyl, acyl, or carbohydrate groups are expressly
encompassed by the
term polypeptide. Also included within the definition are polypeptides which
contain one or more
analogs of an amino acid (including, for example, non-naturally occurring
amino acids, amino acids
which only occur naturally in an unrelated biological system, modified amino
acids from mammalian
systems etc.), polypeptides with substituted linkages, as well as other
modifications known in the art,
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both naturally occurring and non-naturally occurring.
The term "protein" as used herein may be used synonymously with the term
"polypeptide" or
may refer to, in addition, a complex of two or more polypeptides which may be
linked by bonds other
than peptide bonds, for example, such polypeptides making up the protein may
be linked by disulfide
bonds. The term "protein" may also comprehend a family of polypeptides having
identical amino
acid sequences but different post-translational modifications, particularly as
may be added when
such proteins are expressed in eukaryotic hosts.
An "isolated" or "purified" protein or biologically active portion thereof is
substantially free
of cellular material or other contaminating proteins from the cell or tissue
source from which the
protein of the invention (i.e., CPP or biologically active fragment thereof)
is derived, or substantially
free from chemical precursors or other chemicals when chemically synthesized.
The language
"substantially free of cellular material" includes preparations of a protein
according to the invention
in which the protein is separated from cellular components of the cells from
which it is isolated or .
recombinantly produced. In one embodiment, the language "substantially free of
cellular material"
includes preparations of a protein according to the invention having less than
about 30% (by dry
weight) of protein otlier than the protein of the invention (also referred to
herein as a "contaminating
protein"), more preferably less than about 20% of protein other than the
protein according to the
invention, still more preferably less than about 10% of protein other than the
protein according to the
invention, and most preferably less than about 5% of protein other than the
protein according to the
invention. When the protein according to the invention or biologically active
portion thereof is
recombinantly produced, it is also preferably substantially free of culture
medium, i.e., culture
medium represents less than about 20%, more preferably less than about 10%,
and most preferably
less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of a protein of the invention in which the protein is separated
from chemical precursors
or other chemicals which are involved in the synthesis of the protein. In one
embodiment, the
language "substantially free of chemical precursors or other chemicals"
includes preparations of a
protein of the invention having less than about 30% (by dry weight) of
chemical precursors or non-
protein chemicals, more preferably less than about 20% chemical precursors or
non-protein
chemicals, still more preferably less than about 10% chemical precursors or
non-protein chemicals, .
and most preferably less than about 5% chemical precursors or non protein
chemicals.
The term "recombinant polypeptide" is used herein to refer to polypeptides
that have been
artificially designed and which comprise at least two polypeptide sequences
that are not found as
contiguous polypeptide sequences in their initial natural environment, or to
refer to polypeptides
which have been expressed from a recombinant polynucleotide.
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The term "Cardiovascular disorder Plasma Polypeptide" or "CPP" refers to a
polypeptide
comprising the sequence described by any one of SEQ ID NOs:3-5, preferably,
comprising the
polypeptide sequence of SEQ ID NO:S. Such polypeptide may be post-
translationally modified as
described herein. CPPs may also contain other structural or chemical
modifications such as disulfide
linkages or amino acid side chain interactions such as hydrogen and amide
bonds that result in
complex secondary or tertiary structures. CPPs also include mutant
polypeptides, such as deletion,
addition, swap, or truncation mutants, fusion polypeptides comprising such
polypeptides, and .
. polypeptide fragments of at least three, but preferably 8, 10, 12, 15, or 21
contiguous amino acids of
a sequence selected from the group consisting of SEQ ID NOs:3-5. Further
included are CPP
proteolytic precursors and intermediates of the sequence selected from the
group consisting of SEQ
ID NOs: l-5. The invention embodies polypeptides encoded by the nucleic acid
sequences of CPP
genes or CPP mRNA species, preferably human CPP genes and mRNA species,
including isolated
CPPs consisting of, consisting essentially of, or comprising the sequence of
any one of SEQ ID
NOs: l-5. Preferred CPPs have a sequence comprising the sequence of SEQ ID
NOs:3-5. Preferred
CPPs retain at least one biological activity of CPPs of SEQ ID NOs:3-4
The term "biological activity" as used herein refers to any fimction carned
out by a CPP.
These include but are not limited to: (1) protection from cardiovascular
disease; (2) circulating
through the bloodstream; (3) antigenicity, or the ability to bind an anti-CPP
specific antibody; (4)
immunogenicity, or the ability to generate an anti-CPP specific antibody; or
(5) interacting with a
CPP target molecule, preferably a protein.
As used herein, a "CPP modulator" or "CPP 6 modulator" or "CPP 7 modulator" is
a
molecule (e.g., polynucleotide, polypeptide, small molecule, or antibody) that
is capable of
modulating (i.e., increasing or decreasing) either the expression or the
biological activity of the CPPs
of the invention. A CPP modulator that enhances CPP expression or activity is
described as a CPP
activator or agonist. Conversely, a CPP modulator that represses CPP
expression or activity is
described as a CPP inhibitor or antagonist. Preferably, CPP modulators
increase/ decrease the
expression or activity by at least 5, 10, or 20%. CPP inhibitors include anti-
CPP antibodies,
fragments thereof, antisense polynucleotides, and molecules characterized by
screening assays, as
described herein. CPP agonists include polynucleotide expression vectors and
molecules
characterized by screening assays as described herein.
A "CPP-related disorder" or "CPP-related disease" describes a cardiovascular
disorder.
Preferred disorders include coronary artery disease (CAD), coronary heart
disease (CHD), peripheral
vascular disease, cerebral ischemia (stroke), congestive heart failure,
atherosclerosis, hypertension,
and other cardiovascular diseases. Preferably, the likelihood that an
individual will develop or
already has such a disorder is indicated by reduced plasma levels of at least
one CPP.
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Another aspect of the invention pertains to anti-CPP antibodies. The term
"antibody" as used
herein refers to immunoglobulin molecules and immunologically active portions
of immunoglobulin
molecules, i.e., molecules that contain an antigen-binding site which
specifically binds
(immunoreacts with) an antigen, such as CPP, or a biologically active fragment
or homologue
thereof. Preferred antibodies bind to one CPP selected from the group
consisting of SEQ ID NOs:3-4
exclusively and do not bind other polypeptide sequences with high affinity.
Examples of
immunologically active portions of immunoglobulin molecules include Flab) and
F(ab')z fragments
which can be generated by treating the antibody with an enzyme such as pepsin.
The invention
provides polyclonal and monoclonal antibodies that bind a CPP, or a
biologically active fragment or
homologue thereof. The term "monoclonal antibody" or "monoclonal antibody
composition", as used
herein, refers to a population of antibody molecules that contain only one
species of an antigen-
binding site capable of immunoreacting with a particular epitope of a CPP. A
monoclonal antibody
composition thus typically displays a single binding affinity for a particular
CPP with which it
immunoreacts. Preferred CPP antibodies are attached to a label group.
As used herein, a "label group" is any compound that, when attached to a
polynucleotide or
polypeptide (including antibodies), allows for detection or purification of
said polynucleotide or
polypeptide. Label groups may be detected or purified directly or indirectly
by a secondary
compound, including an antibody specific for said label group. Useful label
groups include
radioisotopes (e.g., 3zP; 3sS~ 3H~ Izsl)~ fluorescent compounds (e.g., 5-
bromodesoxyuridin,
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein,
dansyl chloride, phycoerythrin acetylaminofluorene, digoxigenin), luminescent
compounds (e.g.,
luminol, GFP, luciferin, aequorin), enzymes or enzyme co-factor detectable
labels (e.g., peroxidase,
luciferase, alkaline phosphatase, galactosidase, or acetylcholinesterase), or
compounds that are
recognized by a secondary factor such as strepavidin, GST, or biotin.
Preferably, a label group is
attached to a polynucleotide or polypeptide in such a way as to not interfere
with the biological
activity of the polynucleotide or polypeptide.
Radioisotopes may be detected by direct counting of radioemission, filin
exposure, or by
scintillation counting, for example. Enzymatic labels may be detected by
determination of
conversion of an appropriate substrate to product, usually causing a
fluorescent reaction.
Fluorescent and luminescent compounds and reactions may be detected by, e.g.,
radioemission,
fluorescent microscopy, fluorescent activated cell sorting, or a lurninometer.
As used herein with respect to antibodies, an antibody is said to "selectively
bind" or
"specifically bind" to a target if the antibody recognizes and binds the
target of interest but does not
substantially recognize and bind other molecules in a sample, e.g., a
biological sample, which
includes the target of interest.
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As used herein, the teen "vector" refers to a nucleic acid molecule capable of
transporting
another nucleic acid to which it has been linked. One type of vector is a
"plasmid", which refers to a
circular double stranded DNA loop into which additional DNA segments can be
ligated. Another
type of vector is a viral vector, wherein additional DNA segments can be
ligated into the viral
genome. Certain vectors are capable of autonomous replication in a host cell
into which they are
introduced (e.g., bacterial vectors having a bacterial origin of replication
and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) axe integrated
into the genome of a
host cell upon introduction into the host cell, and thereby are replicated
a,Iong with the host genome.
Moreover, certain vectors axe capable of directing the expression of genes to
which they are
operatively linked. Such vectors are referred to herein as "expression
vectors". In general, expression
vectors of utility in recombinant DNA techniques are often in the form of
plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably as the
plasmid is the most
commonly used form of vector. However, the invention is intended to include
such other forms of
expression vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
As used herein, "effective amount" describes the amount of an agent,
preferably a CPP or
CPP modulator of the invention, sufficient to have a desired effect. For
example, an
anticardiovascular disorder effective amount is the amount of an agent
required to reduce a symptom
of a cardiovascular disorder in an individual by at least I, 2, 5, 10, 15, or
preferably ZS%. The term
may also describe the amount of an agent required to ameliorate a
cardiovascular disorder-caused
symptom in an individual. Common symptoms of cardiovascular disorders include:
chest pressure,
heartburn, nausea, vomiting, numbness, shortness of breath, heavy cold
sweating, unexplained
fatigue, and feelings of anxiety. The more severe symptoms of cardiovascular
disorders are chest
pain (angina pectoris), rhythm disturbances (arrhythmias), stroke, or heart
attack. The effective
amount for a particular patient may vary depending on such factors as the
diagnostic method of the
symptom being measured, the state of the condition being treated, the overall
health of the patient,
method of administration, and the severity of side-effects.
CPPs of the i~ve~ction
The Cardiovascular disorder Plasma Polypeptides (CPPs) of the invention are
described in
the sequence listing as SEQ ID NOs:3-4. CPPs having an amino acid sequence
selected from the
group consisting of SEQ ID NOs:3-5 are secreted and circulate in blood plasma.
Preferably, such
CPPs also comprise additional amino acids from the full-length human protein
of SEQ ID NO: I or
2. Such additional amino acids are fused in frame with the selected sequence
to form contiguous
fragment of the full-length human protein..
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Interestingly, levels of the CPPs of the invention are decreased and
undetectable in the
plasma of individuals suffering from cardiovascular disorders. As such, the
CPPs of the invention
provide a useful diagnostic tool, wherein a decreased level of a CPP indicates
an increased risk of
developing, or the presence of, a cardiovascular disorder. Further, CPPs are
useful for drug design
and in therapeutic strategies for prevention and treatment of cardiovascular
disorders.
The sequence from SEQ ID N0:1 is that of the protein Chitotriosidase, from the
family of
chitinases. Chitinases catalyze the hydrolysis of the beta-1,4 N-acetyl-D-
glucosamine linkages in
chitin polymers. They have been found in a wide variety of non-vertebrate
species. A human
homolog of chitinase, chitotriosidase, has been identified. Chitotriosidase is
a member of
glycosylhydrolase family 18, characterized by a conserved glutamate involved
in the catalyzation
reaction (Boot RG, et al., JBC, (1998); 273: 25680-2S68S). It is primarily
expressed by activated
macrophages.
Chitotriosidase is predominantly secreted, but in part processed into a 39-kDa
form that
accumulates in Iysosomes. The C terminal extension of the SO-kDa
chitotriosidase contains sialic-
acid containing O-linked glycans. It was observed that, in macrophages,
alternative splicing
generates a distinct chitotriosidase mRNA species, encoding a40-kDa
chitotriosidase that is C-
terminally truncated. This enzyme is almost identical to the 39 kDa
chitotriosidase formed from the
50-kDaprecursor by proteolytic processing. The C terminus present in the SO-
kDa chitotriosidase,
but absent in the 39- kDa isoform, mediates tight binding to chitin. However,
39-kDa chitotriosidase
is still active against soluble substrates. In the bloodstream, the secretory
SO-kDa chitotriosidase
occurs predominantly, whilethe 39-kDa form is abundant in tissues.
Chitotriosidase secretion from the abnormal macrophages of Gaucher patients is
markedly
increased (Giraldo P, et al., Haematologica, Sep (2001); 86(9): 977-984).
Gaucher disease is caused
2S by an autosomal recessive inherited deficiency in the lysosomal hydrolase
beta-glucocerebrosidase
that results in accwmulation of the lipid in lysosomes of tissue macrophages.
The enzymatic defect
leads to the accumulation of the lipid glucocerebroside in liver, spleen and
bone marrow. In some
other inheritedlysosomal storage disorders, especially sphingolipidoses such
as Niemann Pick, GMl-
gangliosidosis, and Krabbe disease, which involve accumulation of different
lipids, more modest
elevations in plasma chitotriosidase have been noted. In addition, macrophages
present in
atherosclerotic plaques express higher levels of the enzyme. These
observations have lead to
speculation that chitotriosidase is involved in tissue remodeling.
We describe here the "Cardiovascular disorder Plasma Polypeptides" of the
invention that
circulate in normal human plasma. The polypeptide species of the invention
represent an important
and accurate diagnostic tool for determining the risk of coronary artery
disease (CAD), coronary
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heart disease (CHD), peripheral vascular disease, cexebral ischemia (stroke),
congestive heart
failure, atherosclerosis, hypertension, and other cardiovascular diseases. The
polypeptides of the
invention are small, secreted factors and as such, are ideal candidates for
protein-based therapies.
For dosage modulation in a clinical setting, protein therapy is preferable to
genetic therapy, which is
hampered by the lack of fuiely regulable expression. Further, as secxeted
factors, the polypeptide
species of the invention are easy to target, e.g., with a small molecule or
protein inhibitor. Thus, the
polypeptide species of the invention are useful for drug development and
design of therapeutic
strategies to prevent and treat cardiovascular disease.
The polypeptide species of the invention contain the putative active site of
chitotriosidase
and are described as SEQ 117 NOs:3-5.
The terms "Cardiovascular disorder Plasma Polypeptide" and "CPP" are used
herein to
embrace any and all of the peptides, polypeptides and proteins of the present
invention. Also
forming part of the invention are polypeptides encoded by the polynucleotides
of the invention, as
well as fusion polypeptides comprising such polypeptides. The invention
embodies CPPs from
humans, including isolated or purified CPPs consisting of, consisting
essentially of, or comprising an
amino acid sequence selected from the group consisting of SEQ ID NOs:3-5.
Further included are
CPP proteolytic precursors and intermediates of the sequence selected from the
group consisting of
SEQ ID NOs:l-2.
The present invention embodies isolated, purified, and recombinant
polypeptides comprising
a contiguous span of at least 3 amino acids, preferably at least 8 to 10 amino
acids, with a CPP
biological activity. In preferred embodiments the contiguous stretch of amino
acids comprises the
site of a mutation or functional mutation, including a deletion, addition,
swap or truncation of the
amino acids in the CPP sequence. The invention also concerns the polypeptide
encoded by the CPP
nucleotide sequences of the invention, or a complementary sequence thereof or
a fragment thereof.
, One aspect of the invention pertains to isolated CPPs, and biologically
active portions thereof,
as well as polypeptide fragments suitable for use as immunogens to raise anti-
CPP antibodies. In one
embodiment, native CPP peptides can be isolated from plasma, cells or tissue
sources by an
appropriate purification scheme using standard protein purification
techniques. In another
embodiment, CPPs are produced by recombinant DNA techniques. Alternative to
recombinant
expression, a CPP can be synthesized chemically using peptide synthesis
techniques, as described in
the section titled "Chemical Manufacture of CPP compositions" and in Example
3.
Typically, biologically active portions comprise a domain or matif with at
least one activity of
a CPP. A biologically active CPP may, for example, comprise at least 1, 2, 3,
or 5 amino acid
changes from the sequence selected from the group consisting of SEQ ID NOs:3-
5, or comprise at
least 1%, 2%, 3%, 5%, 8%, 10% or 1S% change in amino acids from the sequence
selected from the
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group consisting of SEQ ID NOs:3-5.
Characterization of CPPs
The polypeptides of the invention, CPPs, are defined by the tryptic peptide of
SEQ ID NO:
5, listed in Table 1. This peptide was identified by Mass Spectrometry
analysis of a species isolated
from the plasma of control individuals without Coronary Artery Disease (CAD)
and characterized
according to the MicroProt.TM method, as described in Example 1. This species
was not detectable
in plasma from patients affected with CAD .
The CPPs of the invention are all less than or around 20kD in molecular
weight, as the
plasma sample is first separated based on molecular weight. Higher molecular
weight polypeptide
species are separated and characterized by a different method. As described in
Example 1, the
plasma sample is subjected to a number of chromatography separations. Details
about these
chromatography methods are given in Example 1.
The first separation is on a cation exchange chromatography column, which is
eluted with
increasing salt concentration. Eighteen fractions are collected. The CEX
column in Table 1 lists
which fraction contained the tryptic peptide, as well as its elution
conditions. Separation by cation
exchange provides an indication of the overall positive charge of a
polypeptide species. Cation
exchange is followed by a reverse phase HPLC separation. The RP 1 column in
Table 1 lists in which
of the 30 fractions the tryptic peptide eluted, as well as their elution
conditions. Separation by
reverse phase provides an indication of the overall hydrophobicity of a
polypeptide species The last
two digits of the column labeled Run Number indicate which of the 24 eluted
fractions contained the
tryptic peptide from the second reverse phase HPLC separation (see Example 1).
The information provided in Table 1 reveals a number of characteristics of the
polypeptide
species present in plasma of individuals without CAD. For example, the same
Cryptic peptide is
found in more than one fraction of the RP 1 separation. Since those RP 1
fractions are widely spaced,
this indicates that the tryptic peptide results from different circulating
polypeptide species.
Therefore, the appearance in Table 1 of the same peptide sequence in fraction
16 and fraction 20 of
the RP 1 separation indicates that there exists two polypeptide species from
which the tryptic peptide
was derived, and which have different overall hydrophobicities.
The observed tryptic peptide of SEQ ID NO:S is derived from the polypeptides
of SEQ ID
NOs:3-4 after trypsin digestion as described in Example 1. The polypeptides of
SEQ ID NOs:3-4 are
arrived at by removal, on the propeptide of SEQ ID NO:1, of the predicted
signal sequence and by
successive proteolyses at dibasic sites. Given the separation according to
molecular weight
undergone by the detected species (described in Step 2 of Example 1), CPP 6
and 7 (SEQ ID NOs:3
and 4, respectively) represent the likely plasma polypeptides from which the
tryptic peptide of SEQ
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ID N0:5 is derived and which are observed in RPl fractions 16 and 20 (Table
1). As such, CPP 6
and 7 are especially valuable for diagnostic and therapeutic strategies
relating to cardiovascular
disorders.
CPP nucleic acids
One aspect of the invention pertains to purified or isolated nucleic acid
molecules that encode
CPPs or biologically active portions thereof as further described herein, as
well as nucleic acid
fragments thereof. Said nucleic acids may be used for example in therapeutic
and diagnostic
methods and in drug screening assays as further described herein.
An object of the invention is a purified, isolated, or recombinant nucleic
acid coding for a
CPP, complementary sequences thereto, and fragments thereof. The invention
also pertains to a
purified or isolated nucleic acid comprising a polynucleotide having at least
95% nucleotide identity
with a polynucleotide coding for a CPP, advantageously 99 % nucleotide
identity, preferably 99.5%
nucleotide identity and most preferably 99.8 % nucleotide identity with a
polynucleotide coding for a
CPP, or a sequence complementary thereto or a biologically active fragment
thereof. Another object
of the invention relates to purified, isolated or recombinant nucleic acids
comprising a polynucleotide
that hybridizes, under the stringent hybridization conditions defined herein,
with a polynucleotide
coding for a CPP, or a sequence complementary thereto or a variant thereof or
a biologically active
fragment thereof.
In another preferred aspect, the invention pertains to purified or isolated
nucleic acid
molecules that encode a portion or variant of a CPP, wherein the portion or
variant displays a CPP
biological activity of the invention. Preferably said portion or variant is a
portion or variant of a
naturally occurring CPP or precursor thereof.
Another object of the invention is a purified, isolated, or recombinant
nucleic acid encoding a
CPP comprising the amino acid sequence selected from the group of SEQ ID NOs:3-
5, or fragments
thereof, wherein the isolated nucleic acid molecule encodes one or more motifs
(such as a CPP target
binding region, a secretion signal peptide, or a dibasic cleavage site).
The nucleotide sequence determined from the cloning of the CPP-encoding gene
allows for
the generation of probes and primexs designed for use in identifying and/or
cloning other CPPs (e.g.
sharing the novel functional domains), as well as CPP homologues from other
species.
A nucleic acid fragment encoding a "biologically active portion of a CPP" can
be prepared
by isolating a portion of a nucleotide sequence coding for a CPP, which
encodes a polypeptide
having a CPP biological activity, expressing the encoded portion of the CPP
(e.g., by recombinant
expression in vitro or in vivo) and assessing the activity of the encoded
portion of the CPP.
The invention further encompasses nucleic acid molecules that differ from the
CPP
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nucleotide sequences of the invention due to degeneracy of the genetic code
and encode the same
CPPs of the invention.
In addition to the CPP nucleotide sequences described above, it will be
appreciated by those
skilled in the art that DNA sequence polymorphisms that lead to changes in the
amino acid sequences
of the CPPs may exist within a population (e.g., the human population). Such
genetic polymorphism
may exist among individuals within a population due to natural allelic
variation. Such natural allelic
variations can typically result in 1-5% variance in the nucleotide sequence of
a CPP-encoding gene
or nucleic acid sequence.
Nucleic acid molecules corresponding to natural allelic variants and
homologues of the CPP
nucleic acids of the invention can be isolated based on their homology to the
CPP nucleic acids
disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a
hybridization probe
according to standard hybridization techniques under stringent hybridization
conditions.
It will be appreciated that the invention comprises polypeptides having an
amino acid
sequence encoded by any of the polynucleotides of the invention.
Uses of CPP nucleic acids
Polynucleotide sequences (or the complements thereof) encoding CPPs have
various
applications, including uses as hybridization probes, in chromosome and gene
mapping, and in the
generation of antisense RNA and DNA. In addition, CPP-encoding nucleic acids
are useful as targets
for pharmaceutical intervention, e.g. for the development of DNA vaccines or
antisense
compositions, and for the preparation of CPPs by recombinant techniques, as
described herein. The
polynucleotides described herein, including sequence variants thereof, can be
used in diagnostic
assays. Accordingly, diagnostic methods based on detecting the presence of
such polynucleotides in
body fluids or tissue samples are a feature of the present invention. Examples
of nucleic acid based
diagnostic assays in accordance with the present invention include, but are
not limited to,
hybridization assays, e.g., in situ hybridization, and PCR-based assays.
Polynucleotides, including
extended length polynucleotides, sequence variants and fragments thereof, as
described herein, may
be used to generate hybridization probes or PCR primers for use in such
assays. Such probes and
primers will be capable of detecting polynucleotide sequences, including
genomic sequences that are
similar, or complementary to, the CPP polynucleotides described herein. v
The invention includes primer pairs for carrying out a PCR to amplify a
segment of a
polynucleotide of the invention. Each primer of a pair is an oligonucleotide
having a length of
between 15 and 30 nucleotides such that i) one primer of the pair forms a
perfectly matched duplex
with one strand of a polynucleotide of the invention and the other primer of
the pair form a perfectly
match duplex with the complementary strand of the same polynucleotide, and ii)
the primers of a pair
21
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
form such perfectly matched duplexes at sites on the polynucleotide that
separated by a distance of
between 10 and 2500 nucleotides. Preferably, the annealing temperature of each
primer of a pair to
its respective complementary sequence is substantially the same.
Hybridization probes derived from polynucleotides of the invention can be
used, for
example, in performing in situ hybridization on tissue samples, such as fixed
or frozen tissue sections
prepared on microscopic slides or suspended cells. Briefly, a labeled DNA or
RNA probe is allowed
to bind its DNA or RNA target sample in the tissue section on a prepared
microscopic, under
controlled conditions. Generally, dsDNA probes consisting of the DNA of
interest cloned into a
plasmid or bacteriophage DNA vector are used for this purpose, although ssDNA
or ssRNA probes
may also be used. Probes are generally oligonucleotides between about 15 and
40 nucleotides in
length. Alternatively, the probes can be polynucleotide probes generated by
PCR random priming
primer extension or in vitro transcription of RNA from plasmids (riboprobes).
These latter probes
are typically several hundred base pairs in length. The probes can be labeled
by any of a number of
label groups and the particular detection method will correspond to the type
of label utilized on the
probe (e.g., autoradiography, X-ray detection, fluorescent or visual
microscopic analysis, as
appropriate). The reaction can be further amplified in situ using
immunocytochemical techniques
directed against the label of the detector molecule used, such as an antibody
directed to a fluorescein
moiety present on a fluorescently labeled probe. Specific labeling and in situ
detection methods can
be found, for example, in Howard, G. C., Ed., Methods in Not~radioactive
Detection, Appleton &
Lange, Norwalk, Conn., (1993), herein incorporated by reference.
Hybridization probes and PCR primers may also be selected from the genomic
sequences
corresponding to the full-length proteins identified in accordance with the
present invention,
including promoter, enhancer elements and introns of the gene encoding the
naturally occurring
polypeptide. Nucleotide sequences encoding a CPP can also be used to construct
hybridization
probes for mapping the gene encoding that CPP and for the genetic analysis of
individuals.
Individuals carrying variations of, or mutations in the gene encoding a CPP of
the present invention
may be detected at the DNA level by a variety of techniques. Nucleic acids
used for diagnosis may
be obtained from a patient's cells, including, for example, tissue biopsy and
autopsy material.
Genomic DNA may be used directly for detection or may be amplified
enzyrnatically by using PCR
(Saiki, et al. Nature 324:163-166 (1986)) prior to analysis. RNA or cDNA rnay
also be used for the
same purpose. As an example, PCR primers complementary to the nucleic acid of
the present
invention can be used to identify and analyze mutations in the gene of the
present invention.
Deletions and insertions can be detected by a change in size of the amplified
product in comparison
to the normal genotype. Point mutations can be identified by hybridizing
amplified DNA to
radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA
sequences of the
22
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WO 2004/061096 PCT/EP2004/000027
invention. Sequence changes at specific locations may also be revealed by
nuclease protection
assays, such as RNase and Sl protection or the chemical cleavage method (e.g.
Cotton, et al., Proc.
Natl. Acad. Sci. USA 85:4397-4401 (1985)), or by differences in melting
temperatures. "Molecular
beacons" (Kostrikis L. G. et al., Science 279:1228-1229 (1998)), hairpin-
shaped, single-stranded
synthetic oligonucleotides containing probe sequences which are complementary
to the nucleic acid
of the present invention, may also be used to detect point mutations or other
sequence changes as
well as monitor expression levels of CPPs.
Oligonucleotide and Antisense Compounds
Oligonucleotides of the invention, including PCR primers and antisense
compounds, are
synthesized by conventional means on a commercially available automated DNA
synthesizer, e.g. an
Applied.Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA
synthesizer, or like
instrument. Preferably, phosphoramidite chemistry is employed, e.g. as
disclosed in the following
references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al,
U.S. patent
4,980,460; Koster et al, U.S. patent 4,725,677; Caruthers et al, U.S. patents
4,415,732; 4,458,066;
and 4,973,679; and the like. For therapeutic use, nuclease resistant backbones
are preferred. Many
types of modified oligonucleotides are available that confer nuclease
resistance, e.g.
phosphorothioate, phosphorodithioate, phosphoramidate, or the like, described
in many references,
e.g. phosphorothioates: Stec et al, U.S, patent 5,151,510; Hirschbein, U.S.
patent 5,166,387;
Bergot, U.S. patent 5,183,885; phosphoramidates: Froehler et al, International
application
PCT/US90/03138; and for a review of additional applicable chemistries: Uhlmann
and Peyman
(cited above). The length of the antisense oligonucleotides has to be
sufficiently large to ensure that
specific binding will take place only at the desired target polynucleotide and
not at other fortuitous
sites. The upper range of the length is determined by several factors,
including the inconvenience
and expense of synthesizing and purifying oligorners greater than about 30-40
nucleotides in length,
the greater tolerance of longer oligonuoleotides for mismatches than shorter
oligonucleotides, and the
like. Preferably, the antisense oligonucleotides of the invention have lengths
in the range of about 15
to 40 nucleotides. More preferably, the oligonucleotide moieties have lengths
in the range of about
18 to 25 nucleotides.
Primes and probes
Primers and probes of the invention can be prepared by any suitable method,
including, for
example, cloning and restriction of appropriate sequences and direct chemical
synthesis by a method
such as the phosphodiester method of Narang SA et al (Methods Enzymol
1979;68:90-98), the
phosphodiester method of Brown EL et al (Methods Enzymol 1979;68:109-151), the
23
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WO 2004/061096 PCT/EP2004/000027
diethylphosphoramidite method of Beaucage et al (Tetrahedron Lett 1981, 22:
1859-1862) and the
solid support method described in EP 0 707 592, the disclosures of which are
incorporated herein by
reference in their entireties.
Detection probes are generally nucleic acid sequences or uncharged nucleic
acid analogs
such as, for example peptide nucleic acids which are disclosed in
International Patent Application
WO 92/20702, morpholino analogs which are described in U.S. Patents Numbered
5,185,444;
5,034,506 and 5,142,047. If desired, the probe may be rendered "non-
extendable" in that additional
dNTPs cannot be added to the probe. In and of themselves analogs usually are
non-extendable and
nucleic acid probes can be rendered non-extendable by modifying the 3' end of
the probe such that
the hydroxyl group is no longer capable of participating in elongation. For
example, the 3' end of the
probe can be functionalized with the capture or detection label to thereby
consume or otherwise
block the hydroxyl group.
Any of the polynucleotides of the present invention can be labeled, if
desired, by incorporating
any label group known in the art to be detectable by spectroscopic,
photochemical, biochemical,
immunochemical, or chemical means. Additional examples include non-radioactive
labeling of
nucleic acid fragments as described in Urdea et al. (Nucleic Acids Research.
11:4937-4957, 1988) or
Sanchez-Pescador et al. (J. Clin. Microbiol. 26(10):1934-1938, 1988). In
addition, the probes
according to the present invention may have structural characteristics such
that they allow the signal
amplification, such structural characteristics being, for example, branched
DNA probes as those
described by Urdea et al (Nucleic Acids Symp. Ser. 24:197-200, 1991) or in the
European patent
No. EP 0225807 (Chiron).
A label can also be used to capture the primer, so as to facilitate the
immobilization of either
the primer or a_prirner extension product, such as am lp ified DNA _ on a
solid support. A capture __
label is attached to the primers or probes and can be a specific binding
member which forms a
binding pair with the solid's phase reagent's specific binding member (e.g.
biotin and streptavidin).
Therefore depending upon the type of label carried by a polynucleotide or a
probe, it may be
employed to capture or to detect the target DNA. Further, it will be
understood that the
polynucleotides, primers or probes provided herein, may, themselves, serve as
the capture label. For
example, in the case where a solid phase reagent's binding member is a nucleic
acid sequence, it may
be selected such that it binds a complementary portion of a primer or probe to
thereby immobilize he
primer or probe to the solid phase. In cases where a.polynucleotide probe
itself serves as the binding
member, those skilled in the art will recognize that the probe will contain a
sequence or "tail" that is
not complementary to the target. In the case where a polynucleotide primer
itself serves as the
capture label, at least a portion of the primer will be free to hybridize with
a nucleic acid on a solid
phase. DNA labeling techniques are well known to the skilled technician.
24
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WO 2004/061096 PCT/EP2004/000027
The probes of the present invention are useful for a number of purposes. They
can be notably
used in Southern hybridization to genomic DNA. The probes can also be used to
detect PCR
amplification products. They may also be used to detect mismatches in CPP-
encoding genes or
mRNA using other techniques.
Any of the nucleic acids, polynucleotides, primers and probes of the present
invention can be
conveniently immobilized on a solid support. Solid supports are known to those
skilled in the art and
include the walls of wells of a reaction tray, test tubes, polystyrene beads,
magnetic beads,
nitrocellulose strips, membranes, microparticles such as latex particles,
sheep (or other animal) red
blood cells, duracytes and others. The solid support is not critical and can
be selected by one skilled
in the art. Thus, latex particles, microparticles, magnetic or non-magnetic
beads, membranes, plastic
tubes, walls of microtiter wells, glass or silicon chips, sheep (or other
suitable animal's) red blood
cells and duracytes are all suitable examples. Suitable methods for
immobilizing nucleic acids on
solid phases include ionic, hydrophobic, covalent interactions and the like. A
solid support, as used
herein, refers to any material which is insoluble, or can be made insoluble by
a subsequent reaction.
The solid support can be chosen for its intrinsic ability to attract and
immobilize the capture reagent.
Alternatively, the solid phase can retain an additional receptor which has the
ability to attract and
immobilize the capture reagent. The additional receptor can include a charged
substance that is
oppositely charged with respect to the capture reagent itself or to a charged
substance conjugated to
the capture reagent. As yet another alternative, the receptor molecule can be
any specific binding
. member attached to the solid support and which has the ability to immobilize
the capture reagent
through a specific binding reaction. The receptor molecule enables the
indirect binding of the
capture reagent to a solid support material before the performance of the
assay or during the
performance of the assay. The solid phase thus can be a plastic, derivatized
plastic, magnetic or
non-magnetic metal, glass or silicon surface of a test tube, microtiter well,
sheet, bead, microparticle,
chip, sheep (or other suitable animal's) red blood cells, duracytes and other
configurations known to
those of ordinary skill in the art. The nucleic acids, polynucleotides,
primers and probes of the
invention can be attached to or immobilized on a solid support individually or
in groups of at least 2,
5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a
single solid support. In
addition, polynucleotides other than those of the invention may be attached to
the same solid support .
as one or more polynucleotides of the invention.
Any polynucleotide provided herein may be attached in overlapping areas or at
random
locations on a solid support. Alternatively the polynucleotides of the
invention may be attached in an
ordered array wherein each polynucleotide is attached to a distinct region of
the solid support which
does not overlap with the attachment site of any other polynucleotide.
Preferably, such an ordered
array of polynucleotides is designed to be "addressable" where the distinct
locations are recorded and
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
can be accessed as part of an assay procedure. Addressable polynucleotide
arrays typically
comprise a plurality of different oligonucleotide probes that are coupled to a
surface of a substrate in
different knovcm locations. The knowledge of the precise location of each
polynucleotides location
makes these "addressable" arrays particularly useful in hybridization assays.
Any addressable array
technology known in the art can be employed with the polynucleotides of the
invention. One
particular embodiment of these polynucleotide arrays is known as the
Genechips, and has been
generally described in US Patent 5,143,854; PCT publications WO 90/15070 and
92110092, the
disclosures of which are incorporated herein by reference in their entireties.
to Methods for obtaining variant nucleic acids and polypeptides
In addition to naturally-occurring allelic variants of the CPP sequences that
may exist in the
population, the skilled artisan will appreciate that changes can be introduced
by mutation into the
nucleotide sequences coding for CPPs, thereby leading to changes in the amino
acid sequence of the
encoded CPPs, with or without altering the functional ability of the CPPs.
15 Several types of variants are contemplated including 1) one in which.one or
more of the
amino acid residues are substituted with a conserved or non-conserved amino
acid residue and such
substituted amino acid residue may or may not be one encoded by the genetic
code, or 2) one in
which one or more of the amino acid residues includes a substituent group, or
3) one in which the
mutated CPP is fused with another compound, such as a compound to increase the
half life of the
20 polypeptide (for example, polyethylene glycol), or 4) one in which the
additional amino acids are
fused to the CPP, such as a leader, a signal or anchor sequence, a sequence
which is employed for
purification of the CPP, or sequence from a precursor protein. Such variants
are deemed to be within
the scope of those skilled in the art.
For example, nucleotide substitutions leading to amino acid substitutions can
be made in the
25 sequences that do not substantially change the biological activity of the
protein. An amino acid
residue-can be altered from the wild-type sequence encoding a CPP, or a
biologically active fragment
or homologue thereof without altering the biological activity. In general,
amino acid residues that are
shared among the CPPs of the present invention are predicted to be less
amenable to alteration.
In another aspect, the invention pertains to nucleic acid molecules encoding
CPPs that
30 contain changes in amino acid residues that result in increased biological
activity, or a modified
biological activity. In another aspect, the invention pertains to nucleic acid
molecules encoding
CPPs that contain changes in amino acid residues that are essential for a CPP
biological activity.
Such CPPs differ in amino acid sequence from SEQ ID NOs:3-4 and display
reduced activity, or
essentially lack one or more CPP biological activities.
35 Mutations, substitutions, additions, or deletions can be introduced into
any of SEQ ID
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WO 2004/061096 PCT/EP2004/000027
NOs:I-5, by standard techniques, such as site-directed mutagenesis and PCR-
mediated mutagenesis.
For example, conservative amino acid substitutions may be made at one or more
predicted non-
essential amino acid residues. A "conservative amino acid substitution" is one
in which the amino
acid residue is replaced with an amino acid residue having a similar side
chain. Families of amino
acid residues having similar side chains have been defined in the art. These
families include amino
acids with basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline,
phenylalanine, rnethionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine)
and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted
nonessential amino acid residue in a CPP, or a biologically active fragment or
homologue thereof
may be replaced with another amino 'acid residue from the same side chain
family. Alternatively, in
another embodiment, mutations can be introduced randomly along all or part of
a CPP coding
sequence, such as by saturation mutagenesis, and the resultant mutants can be
screened for CPP
biological activity to identify mutants that retain activity. Following
mutagenesis of the nucleotide
encoding one of SEQ ID NOs: l-5, the encoded protein can be expressed
recombinantly and the
activity of the protein can be determined in any suitable assay, for example,
as provided herein.
The invention also provides CPP chimeric or fusion proteins. As used herein, a
CPP
"chimeric protein" or "fusion protein" comprises a CPP of the invention or
fragment thereof,
operatively linked or fused in frame to a non-CPP polypeptide sequence. In a
preferred ernbodirnent,
a CPP fusion protein comprises at Least one biologically active portion of a
CPP. In another
preferred embodiment, a CPP fusion protein comprises at least two biologically
active portions of a
CPP. For example, in one embodiment, the fusion protein is a GST-CPP fusion
protein in which
CPP domain sequences are fused to the C terminus of the GST sequences. Such
fusion proteins can
facilitate the purification of recombinant CPPs. In another embodiment, the
fusion protein is a CPP
containing a heterologous signal sequence at its N-terminus, for example, to
allow for a desired
cellular localization in a certain host cell. In yet another embodiment, the
fusion is a CPP
biologically active fragment and an immunoglobulin molecule. Such fusion
proteins are useful, for
example, to increase the valency of CPP binding sites. For example, a bivalent
CPP binding site
may be formed by fusing biologically active CPP fragments to an IgG Fc
protein.
The CPP fusion proteins of the invention can be incorporated into
pharmaceutical
compositions and administered to a subject in vivo. Moreover, the CPP fusion
proteins of the
invention can be used as immunogens to produce anti-CPP antibodies in a
subject, to purify CPP or
CPP ligands and in screening assays to identify molecules which modulate the
interaction of CPP
with a CPP target molecule.
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WO 2004/061096 PCT/EP2004/000027
Furthermore, isolated fragments of CPPs can also be obtained by screening
peptides
recombinantly produced from the corresponding fragment of the nucleic acid
encoding such peptides.
In addition, fragments can be chemically synthesized using techniques known in
the art such as
conventional Merrifield solid phase f Moc or t-Boc chemistry. For example, a
CPP of the present
invention may be arbitrarily divided into fragments of desired length with no
overlap of the
fragments, or preferably divided into overlapping fragments of a desired
length. The fragments can
be produced (recombinantly or by chemical synthesis) and tested to identify
those peptidyl fragments
with a CPP biological activity, for example, by microinjection assays or in
vitro protein binding
assays. In an illustrative embodiment, peptidyl portions of a CPP, such as a
CPP target binding
region, can be tested for CPP activity by expression as thioredoxin fusion
proteins, each of which
contains a discrete fragment of the CPP (see, for example, U.S. Patents 5,
270,181 and 5,292,646;
and PCT publication W094/02502, the disclosures of which are incorporated
herein by reference).
In addition, libraries of fragments of a CPP coding sequence can be used to
generate a
variegated population of CPP fragments for screening and subsequent selection
of variants of a CPP.
In one embodiment, a library of coding sequence fragments can be generated by
treating a double
stranded PCR fragment of CPP coding sequence with a nuclease under conditions
wherein nicking
occurs only about once per molecule, denaturing the double stranded DNA,
renaturing the DNA to
form double stranded DNA which can include sense/antisense pairs from
different nicked products,
removing single stranded portions from reformed duplexes by treatment with S 1
nuclease, and
ligating the resulting fragment library into an expression vector. By this
method, an expression
library can be derived which encodes N-terminal, C terminal and internal
fragments of various sizes
of the CPP.
Modified CPPs can be used for such purposes as enhancing therapeutic or
prophylactic
efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic
degradation in vivo). Such
modified peptides, when designed to retain at least one activity of the
naturally occurring form of the
protein, are considered functional equivalents of the CPP described in more
detail herein. Such
modified peptide can be produced, for instance, by amino acid substitution,
deletion, or addition.
Whether a change in the amino acid sequence of a peptide results in a
functional CPP
homolog can be readily determined by assessing at least one CPP biological
activity of the variant
peptide. Peptides in which more than one replacement has taken place can
readily be tested in the
same manner.
This invention further contemplates a method of generating sets of
combinatorial mutants of
the presently disclosed CPPs, as well as truncation and fragmentation mutants,
and is especially
useful for identifying potential variant sequences which are functional in
binding to a CPP target
protein but differ from a wild-type form of the protein by, for example,
efficacy, potency and/or
28
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WO 2004/061096 PCT/EP2004/000027
intracellular half life. One purpose for screening such combinatorial
libraries is, for example, to
isolate novel CPP homologs with altered biological activity when compared with
the wild-type
protein, or alternatively, possessing novel activities all together. For
example, mutagenesis can give
rise to CPP homologs which have intracellular half lives dramatically
different than the
corresponding wild-type protein. The altered protein can be rendered either
more stable or less stable
to proteolytic degradation, or cellular processes which result in destruction
of, or otherwise
inactivation of, a CPP. Such CPP homologs, and the genes which encode them,
can be utilized to
alter the envelope of expression for a particular recombinant CPP by
modulating the half life of the
recombinant protein. For instance, a short half life can give rise to more
transient biological effects
associated with a particular recombinant CPP and, when part of an inducible
expression system, can
allow tighter control of recombinant protein levels within a cell and in
circulating plasma. As above,
such proteins, and particularly their recombinant nucleic acid constructs, can
be used in gene therapy
protocols.
In an illustrative embodiment of this method, the amino acid sequences for a
population of
CPP homologs or other related proteins are aligned, preferably to promote the
highest homology
possible. Such a population of variants can include, for example, CPP homologs
from one or more
species, or CPP homologs from the same species but which differ due to
mutation. Amino acids
which appear at each position of the aligned sequences are selected to create
a degenerate set of
combinatorial sequences. There are many ways by which the library of potential
CPP homologs can
be generated from a degenerate oligonucleotide sequence. Chemical synthesis of
a degenerate gene
sequence can be carried out in an automatic DNA synthesizer, and the synthetic
genes then be ligated
into an appropriate gene for expression. The purpose of a degenerate set of
genes is to provide, in
one mixture, all of the sequences encoding the desired set of potential CPP
sequences. The synthesis
of degenerate oligonucleotides is well known in the art (see for example.
Narang, SA (1983)
Tetrahedron 393; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland
Sympos.
Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp. 273-289; Itakura et al.
(1984) Annu.
Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.
(1983) Nucleic Acid Res.
11:477. Such techniques have been employed in the directed evolution of other
proteins (see, for
example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
89:2429-2433;
Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-
6382; as well as
U.S. Patents Nos: 5, 223,409, 5,198,346, and 5,096,815). The disclosures ofthe
above references
are incorporated herein by reference in their entireties.
Alternatively, other forms of mutagenesis can be utilized to generate a
combinatorial library,
particularly where no other naturally occurring homologs have yet been
sequenced. For example,
CPP hornologs (both agonist and antagonist forms) can be generated and
isolated from a library by
29
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WO 2004/061096 PCT/EP2004/000027
screening using, for example, alanine scanning mutagenesis and the like (Ruf
et al. ( 1994)
Biochemistry 33:1565-1572; Wang et al. (1994) J Biol. Chem. 269:3095-3099;
Balint et al. (1993)
Gene 137:109-118; Grodberg et al. (1993) Eur. J Biochem. 218:597-601;
Nagashima et al. (1993) J
Biol. Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838;
and Cunningham
et al. (1989) Science 244:1081-1085), by linker scanning mutagenesis (Dustin
et al. (1993) Virology
193:653-660; Brown et al. (1992) Mol. Cell Biol. 12:2644 2652; McKnight et al.
(1982) Science
232:316); by saturation mutagenesis (Meyers et al. (1986) Science 232:613); by
PCR mutagenesis
(Leung et al. (1989) Method Cell Mol Biol 1: 1-19); or by random mutagenesis
(Miller et al. (1992)
A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and
Greener et al.
(1994) Strategies in Mol Biol 7:32-34, the disclosures of which are
incorporated herein by reference
in their entireties).
A further method exploits automatic protein design to generate.protein
libraries for screening
and optimization of the sequence of a protein of the invention. See, for
example, U.S. Patent
6403312, disclosure of which is incorporated herein by reference. Briefly, a
primary library is
generated using computational processing based on the sequence and structural
characteristics of the
CPP. Generally speaking, the goal of the computational processing is to
determine a set of optimized
protein sequences that result in the lowest energy conformation of any
possible sequence. However, a
plurality of sequences that are not the global minimum may have low energies
and be useful. Thus,
a primary library comprising a rank ordered list of sequences, generally in
terms of theoretical
quantitative stability, is generated. These sequences may be used to
synthesize or express peptides
displaying an extended half life or stabilized interactions with CPP binding
compounds and proteins.
A wide range of techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations, as well as for screening cDNA
libraries for gene
products having a certain property. Such techniques will be generally
adaptable for rapid screening
of the gene libraries generated by the combinatorial mutagenesis of CPPs. The
most widely used
techniques for screening large gene libraries typically comprises cloning the
gene library into
replicable expression vectors, transforming appropriate cells with the
resulting library of vectors,
and expressing the combinatorial genes under conditions in which detection of
a desired activity
facilitates relatively easy isolation of the vector encoding the gene whose
product was detected.
Each of the illustrative assays described below are amenable to high
throughput analysis as
necessary to screen large numbers of degenerate CPP sequences created by
combinatorial
mutagenesis techniques. In one screening assay, the candidate gene products
are displayed on the
surface of a cell or viral particle, and the ability of particular cells or
viral particles to bind a CPP
target molecule (for example a modified peptide substrate) via this gene
product is detected in a
"panning assay". For instance, the gene library can be cloned into the gene
for a surface membrane
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
protein of a bacterial cell, and the resulting fusion protein detected by
panning (Ladner et al., WO
88/06630; Fuchs et al. (1991) BioTechnology 9:1370-1371, and Goward et al.
(1992) TIBS 18:136
140). In a similar fashion, ffuorescently labeled CPP target can be used to
score for potentially
functional CPP homologs. Cells can be visually inspected and separated under a
fluorescence
microscope, or, where the morphology of the cell permits, separated by a
fluorescence- activated cell
sorter.
In an alternate embodiment, the gene library is expressed as a fusion protein
on the surface
of a viral particle. For instance, in the.filamentous phage system, foreign
peptide sequences can be
expressed on the surface of infectious phages, thereby conferring two
significant benefits. First, since
these phages can be applied to affinity matrices at very high concentrations,
a large number of phage
can be screened at one time. Second, since each infectious phage displays the
combinatorial gene
product on its surface, if a particular phage is recovered from an affinity
matrix in low yield, the
phage can be amplified by another round of infection. The group of almost
identical E. coli
filamentous phages M13, fd, and fl are most often used in phage display
libraries, as either of the
phage gill or gVIII coat proteins can be used to generate fusion proteins
without disrupting the
ultimate packaging of the viral particle (Ladner et al. PCT publication WO
90/02909; Garrard et al.,
PCT publication WO 92/09690; Marks et al. (1992) J Biol. Chem. 267:16007-
16010; Griffiths et al.
(1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and
Barbas et al. (1992)
PNAS 89:4457 4461, the disclosures of which are incorporated herein by
reference in their
entireties). In an illustrative embodiment, the recombinant phage antibody
system (RPAS, Pharmacia
Catalog number 27-9400-O1) can be easily modified~for use in expressing CPP
combinatorial
libraries, and the CPP phage library can be panned on immobilized CPP target
molecule (glutathione
immobilized CPP target-GST fusion proteins or immobilized DNA). Successive
rounds of phage
amplification and panning can greatly enrich for CPP homologs which retain an
ability to bind a
CPP target and which can subsequently be screened further for biological
activities in automated
assays, in order to distinguish between agonists and antagonists.
The invention also provides for identification and reduction to functional
minimal size of the
CPP functional domains, to generate mimetics, e.g. peptide or non-peptide
agents, which are able to
disrupt binding of a polypeptide of the present invention with a CPP target
molecule. Thus, such
mutagenic techniques as described above are also useful to map the
determinants of CPPs
participating in protein-protein interactions involved in, for example,
binding to a CPP target protein.
To illustrate, the critical residues of a CPP involved in molecular
recognition of the CPP target can-
be determined and used to generate CPP target-13P-derived peptidomimetics that
competitively
inhibit binding of the CPP to the CPP target. For instance, non hydrolysable
peptide analogs of such
residues can be generated using retro-inverse peptides (e.g., see U.S. Patents
5,116,947 and
31
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
5,219,089; and Pallai et al. (1983) Int J Pept Protein Res 21:84-92),
benzodiazepine (e.g., see
Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM
Publisher: Leiden,
Netherlands, 1988), azepine (e.g., see Huffinan et al. in Peptides. Chemistry
and Biology, G.R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma
lactam rings
(Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM
Publisher: Leiden,
Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med
Chem 29:295; and
Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th
American Peptide
Symposium) Pierce Chemical Co. Rockland, IL, 1985), P-turn dipeptide cores
(Nagai et al. (1985)
Tetrahedron Left 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1: 123
1), and P-
aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and
Dann et al.
(1986) Biochem Biophys Res Commun 134:71, the disclosures of which are
incorporated herein by
reference in their entireties).
Chemical Ma~eufacture of CPP Compositions
Peptides of the invention are synthesized by standard techniques (e.g. Stewart
and Young,
Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, IL,
1984).
Preferably, a commercial peptide synthesizer is used, e.g. Applied Biosystems,
Inc. (Foster City,
CA) model 430A, and polypeptides of the invention may be assembled from
multiple, separately
synthesized and purified, peptide in a convergent synthesis approach, e.g.
Kent et al, U.S. patent
6,184,344 and Dawson and Kent, Annu. Rev. Biochem., 69: 923-960 (2000).
Peptides of the
invention may be assembled by solid phase synthesis on a cross-linked
polystyrene support starting
from the carboxyl terminal residue and adding amino acids in a stepwise
fashion until the entire
peptide has been formed. The following references are guides to the chemistry
employed during
synthesis: Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992);
Merrifield, J. Amer.
Chem. Soc., Vol. 85, pg. 2149 (1963); Kent et al., pg 185, in Peptides 1984,
Ragnarsson, Ed.
(Alinquist and Weksell, Stockholin, 1984); Kent et al., pg. 217 in Peptide
Chemistry 84, Izumiya,
Ed. (Protein Research Foundation, B.H. Osaka, 1985); Mernfield, Science, Vol.
232, pgs. 341-347
(1986); Kent, Ann. Rev. Biochem, Vol. 57, pgs. 957-989 (1988), and references
cited in these latter
two references.
. Preferably, chemical synthesis of polypeptides of the invention is carned
out by the assembly
of peptide fragments by native chemical ligation, as described by Dawson et
al, Science, 266: 776-
779 (1994) and Kent el al, U.S. patent 6,184,344. Briefly, in the approach a
first peptide fragment
is provided with an N terminal cysteine having an unoxidized sulfhydryl side
chain, and a second
peptide fragment is provided with a C terminal thioester. The unoxidized
sulfhydryl side chain of the
N terminal cysteine .is then condensed with the C terminal thioester to
produce an intermediate
32
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
peptide fragment which links the first and second peptide fragments with a (3-
aminothioester bond.
The J3-aminothioester bond of the intermediate peptide fragment then undergoes
an intramolecular
rearrangement to produce the peptide fragment product which links the first
and second peptide
fragments with an amide bond. Preferably, the N-terminal cysteine of the
internal fragments is
protected from undesired cyclization and/or concatenation reactions by a
cyclic thiazolidine
protecting group as described below. Preferably, such cyclic thiazolidine
protecting group is a
thioprolinyl group.
Peptide fragments having a C-terminal thioester may be produced as described
in the
following references, which are incorporated by reference: Kent et al, U.S.
patent 6,184,344; Tam et
al, Proc. Natl. Acad. Sci., 92: 12485-12489 (1995); Blake, Int. J. Peptide
Protein Res., 17: 273
(1981); Canne et al, Tetrahedron Letters, 36: 1217-1220 (1995); Hackeng et al,
Proc. Natl. Acad.
Sci., 94: 7845-7850 (1997); or Hackeng et al, Proc. Natl. Acad. Sci., 96:
10068-10073 (1999).
Preferably, the method described by Hackeng et al (1999) is employed. Briefly,
peptide fragments
are synthesized on a solid phase support (described below) typically on a 0.25
mmol scale by using
the in situ neutralization/HBTU activation procedure for Boc chemistry
disclosed by Schnolzer et al,
Int. J. Peptide Protein Res., 40: 180-193 (1992), which reference is
incorporated herein by reference.
(HBTU is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate and Boc is
tert-butoxycarbonyl). Each synthetic cycle consists of Na-Boc removal by a 1-
to 2- minute
treatment with neat TFA, a 1-minute DMF flow wash, a 10- to 20-minute coupling
time with 1.0
mmol of preactivated Boc-amino acid in the presence of DIEA, and a second DMF
flow wash.
(TFA is trifluoroacetic acid, DMF is N,N-dimethylformamide, and DIEA is N,N-
diisopropylethylamine). N"-Boc-amino acids (1.1 mmol) are preactivated for 3
minutes with 1.0
mmol of HBTU (0.5 M in DMF) in the presence of excess DIEA (3 mmol). After
each coupling
step, yields are determined by measuring residual free amine with a
conventional quantitative
ninhydrin assay, e.g. as disclosed in Sarin et al, Anal. Biochem., 117: 147-
157 (1981). After
coupling of Gln residues, a DCM flow wash is used before and after
deprotection by using TFA, to
prevent possible high-temperature (TFA/DMF)-catalyzed pyrrolidone formation.
After chain
assembly is completed, the peptide fragments are deprotected and cleaved from
the resin by treatment
with anhydrous HF for 1 hour at 0°C with 4%p-cresol as a scavenger. The
imidazole side-chain
2,4-dinitrophenyl (dnp) protecting groups remain on the His residues because
the dnp-removal
procedure is incompatible with C-terminal thioester groups. However, dnp is
gradually removed by
. thiols during the ligation reaction. After cleavage, peptide fragments are
precipitated with ice-cold
diethylether, dissolved in aqueous acetonitrile, and lyophilized.
Thioester peptide fragments described above are preferably synthesized on a
trityl-associated
mercaptopropionic acid-leucine (TAMPAL) resin, made as disclosed by Hackeng et
al (1999), or
33
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
comparable protocol. Briefly, N"-Boc-Leu (4 mmol) is activated with 3.6 rnrnol
of HBTU in the
presence of 6 mmol of DIEA and coupled for 16 minutes to 2 mmol of p-
methylbenzhydrylamine
(MBHA) resin, or the equivalent. Next, 3 mmol of S trityl mercaptopropionic
acid is activated with
2.7 mmol of HBTU in the presence of 6 mmol of DIEA and coupled for 16 minutes
to Leu-MBHA
resin. The resulting TAMPAL resin can be used as a starting resin for
polypeptide-chain assembly
after removal of the trityl protecting group with two 1-minute treatments with
3.5%
triisopropylsilane and 2.5% H20 in TFA. The thioester bond can be formed with
any desired amino
acid by using standard in situ-neutralization peptide coupling protocols for 1
hour, as disclosed in
Schnolzer et al (cited above). Treatment of the final peptide fragment with
anhydrous HF yields the
C terminal activated mercaptopropionic acid-leucine (MPAL) thioester peptide
fragments.
Preferably, thiazolidine-protected thioester peptide fragment intermediates
are used in native
chemical ligation under conditions as described by Hackeng et al (1999), or
like conditions. Briefly,
0.1 M phosphate buffer (pH 8.5) containing 6 M guanidine, 4% (vol/vol)
benzylinercaptan, and 4%
(vol/vol) thiophenol is added to dry peptides to be ligated, to give a final
peptide concentration of 1-3
mM at about pH 7, lowered because of the addition of thiols and TFA from the
lyophilized peptide.
Preferably, the ligation reaction is performed in a heating block at
37°C and is periodically vortexed
to equilibrate the thiol additives. The reaction may be monitored for degree
of completion by
MALDI-MS or HPLC and electrospray ionization MS.
After a native chemical ligation reaction is completed or stopped, the N-
terminal thiazolidine
ring of the product is opened by treatment with a cysteine deprotecting agent,
such as 0-
methylhydroxylamine (0.5 M) at pH 3.5-4.5 for 2 hours at 37° C, after
which a 10-fold excess of
Tris-(2-carboxyethyl)-phosphine is added to the reaction mixture to completely
reduce any oxidizing
reaction constituents prior to purification of the product by conventional
preparative HPLC.
Preferably, fractions containing the ligation product are identified by
electrospray MS, are pooled,
and lyophilized.
After the synthesis is completed and the final product purified, the final
polypeptide product
may be refolded by conventional techniques, e.g. Creighton, Meth. Enzymol.,
107: 305-329 (1984);
White, Meth. Enzymol., 11: 481-484 (1967); Wetlaufer, Meth. Enzymol., 107: 301-
304 (1984); and
the like. Preferably, a final product is refolded by air oxidation by the
following, or like: The
reduced lyophilized product is dissolved (at about 0.1 mg/mL) in 1 M guanidine
hydrochloride (or
like chaotropic agent) with 100 mM Tris, 10 mM rnethionine, at pH 8.6. After
gentle overnight
stirring, the re-folded product is isolated by reverse phase HPLC with
conventional protocols.
Recombinant Expressiotz Tlectors and Host Cells
The polynucleotide sequences described herein can be used in recombinant DNA
molecules
34
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
that direct the expression of the corresponding polypeptides in appropriate
host cells. Because of the
degeneracy in the genetic code, other DNA sequences may encode the equivalent
amino acid
sequence, and may be used to clone and express the CPPs. Codons preferred by a
particular host cell
may be selected and substituted into the naturally occurring nucleotide
sequences, to increase the rate
and/or efficiency of expression. The nucleic acid (e.g., cDNA or genomic DNA)
encoding the
desired CPP may be inserted into a replicable vector for cloning
(amplification of the DNA), or for
expression. The polypeptide can be expressed recombinantly in any of a number
of expression
systems according to methods known in the art (Ausubel, et al., editors,
Current Protocols in
Molecular Biology, John Wiley & Sons, New York, 1990). Appropriate host cells
include yeast,
bacteria, archebacteria, fixngi, and insect and animal cells, including
mammalian cells, for example
primary cells, including stem cells, including, but not limited to bone marrow
stem cells. More
specifically, these include, but are not limited to, microorganisms such as
bacteria transformed with
recombinant bacteriophage, plasmid or cosmid DNA expression vectors, and yeast
transformed with
yeast expression vectors. Also included, are insect cells infected with a
recombinant insect virus
(such as baculovirus), and mammalian expression systems. The nucleic acid
sequence to be
expressed may be inserted into the vector by a variety of procedures. In
general, DNA is inserted into
an appropriate restriction endonuclease site using techniques known in the
art. Vector components
generally include, but are not limited to, one or more of a signal sequence,
an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a transcription
termination
sequence. Construction of suitable vectors containing one or more of these
components employs
standard ligation techniques which are known to the skilled artisan.
The CPPs of the present invention are produced by culturing a host cell
transformed with an
expression vector containing a nucleic acid encoding a CPP, under the
appropriate conditions to
induce or cause expression of the protein. The conditions appropriate for CPP
expression will vary
with the choice of the expression vector and the host cell, as ascertained by
one skilled in the axt. For
example, the use of constitutive promoters in the expression vector may
require routine optimization
of host cell growth and proliferation, while the use of an inducible promoter
requires the appropriate
growth conditions for induction. In addition, in some embodiments, the timing
of the harvest is
important. For example, the baculoviral systems used in insect cell expression
are lytic viruses, and
thus harvest time selection can be crucial for product yield.
A host cell strain may be chosen for its ability to modulate the expression of
the inserted
sequences or to process the expressed protein in the desired fashion. Such
modifications of the
protein include, but are not limited to, glycosyl, acetyl, phosphate, amide,
lipid, carboxyl, acyl, or
carbohydrate groups. Post-translational processing, which cleaves a "prepro"
form of the protein,
may also be important for correct insertion, folding andlor fiznction. By way
of example, host cells
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
such as CHO, HeLa, BHK, MDCK, 293, W138, etc. have specific cellular machinery
and
characteristic mechanisms for such post translational activities and may be
chosen to ensure the
correct modification and processing of the introduced, foreign protein. Of
particular interest are
Drosophila melahogaster cells, Saccharomyces cerevisiae and other yeasts, E.
coli, Bacillus
subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and
HeLa cells,
fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid
cell lines, Jurkat
cells, human cells and other primary cells.
The nucleic acid encoding a CPP must be "operably linked" by placing it into a
functional
relationship with another nucleic acid sequence. For example, DNA for a
presequence or secretory
leader is operably linked to DNA for a polypeptide if it is expressed as a
preprotein that participates
in the secretion of the polypeptide; a promoter or enhancer is operably linked
to a coding sequence if
it afFects the transcription of the sequence; or a ribosome binding site is
operably linked to a coding
sequence if it is positioned so as to facilitate translation. Generally,
"operably linked" DNA
sequences axe contiguous, and, iri the case of a secretory leader or other
polypeptide sequence,
contiguous and in reading phase. However, enhancers do not have to be
contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such sites do not
exist, the synthetic
oligonucleotide adaptors or linkers are used in accordance with conventional
practice. Promoter
sequences encode either constitutive or inducible promoters. The promoters may
be either naturally
occurring promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than
one promoter, are also known in the art, and are useful in the present
invention. The expression
vector may comprise additional elements, for example, the expression vector
may have two
replication systems, thus allowing it to be maintained in two organisms, for
example in mammalian
or insect cells for expression and in a procaryotic host for cloning and
amplification. Both expression
and cloning vectors contain a nucleic acid sequence that enables the vector to
replicate in one or
more selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses.
The origin of replication from the plasmid pBR322 is suitable for most Gram-
negative bacteria, the
2: plasmid origin is suitable for yeast, and various viral origins (SV40,
polyoma, adenovirus, VSV
or BPV) are useful for cloning vectors in mammalian cells. Further, for
integrating expression
vectors, the expression vector contains at least one sequence homologous to
the host cell genome, and
preferably, two homologous sequences which flank the expression construct. The
integrating vector
may be directed to a specific locus in the host cell by selecting the
appropriate homologous sequence
for inclusion in the vector. Constructs for integrating vectors are well known
in the art. In an
additional embodiment, a heterologous expression control element may be
operably linked with the
endogenous gene in the host cell by homologous recombination (described in US
Patents 6410266
and 6361972, disclosures of which are hereby incorporated by reference in
their entireties). This
36
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
technique allows one to regulate expression to a desired level with a chosen
control element while
ensuring proper processing and modification of CPP endogenously expressed by
the host cell.
Useful heterologous expression control elements include but are not limited to
CMV immediate early
promoter, the HSV thymidine kinase promoter, the early and late SV40
promoters, the promoters of
retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV), and
metallothionein promoters.
Preferably, the expression vector contains a selectable marker gene to allow
the selection of
transformed host cells. Selection genes are well known in the art and will
vary with the host cell
used. Expression and cloning vectors will typically contain a selection gene,
also termed a selectable
marker. Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other
toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic
deficiencies, or (c) supply critical nutrients not available for from complex
media, e.g., the gene
encoding D-alanine racemase for Bacilli.
Host cells transformed with a nucleotide sequence encoding a CPP may be
cultured under
conditions suitable for the expression and recovery of the encoded protein
from cell culture. The
protein produced by a recombinant cell may be secreted, membrane-bound, or
contained
intracellularly depending on the sequence and/or the vector used. As will be
understood by those of
skill in the art, expression vectors containing polynucleotides encoding the
CPP can be designed with
signal sequences which direct secretion of the CPP through a prokaryotic or
eukaryotic cell
membrane. The desired CPP may be produced recombinantly not only directly, but
also as a fusion
polypeptide with a heterologous polypeptide, which may be a signal sequence or
other polypeptide
having a specific cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the
signal sequence may be a component of the vector, or it may be a part of the
CPP-encoding DNA .
that is inserted into the vector. The signal sequence may be a prokaryotic
signal sequence selected,
for example, from the group of the allcaline phosphatase, penicillinase, lpp,
or heat-stable enterotoxin
II leaders. For yeast secretion the signal sequence may be, e.g., the yeast
invertase leader, alpha
factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the
latter described in
U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C, albica~s
glucoamylase leader (EP
362,179 published Apr. 4, 1990), ~or the signal described in WO 90113646
published Nov. 15, 1990.
In mammalian cell expression, mammalian signal sequences may be used to direct
secretion of the
protein, such as signal sequences from secreted polypeptides of the same or
related species, as well
as viral secretory leaders. According to the expression system selected, the
coding sequence is
inserted into an appropriate vector, which in turn may require the presence of
certain characteristic
"control elements" or "regulatory sequences." Appropriate constructs are known
generally in the art
(Ausubel, et al., 1990) and, in many cases, are available from commercial
suppliers such as
Invitrogen (San Diego, Calif.), Stratagene (La Jolla, Cali~), Gibco BRL
(Rockville, Md.) or
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CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
Clontech (Palo Alto, Calif.).
Expression in Bacterial Systems
Transformation of bacterial cells may be achieved using an inducible promoter
such as the
hybrid lacZ promoter of the "BLUESCRIPT" Phagemid (Stratagene) or "pSPORTI"
(Gibco BRL).
In addition, a number of expression vectors may be selected for use in
bacterial cells to produce
cleavable fusion proteins that can be easily detected and/or purified,
including, but not limited to
"BLUESCRIPT" (a-galactosidase; Stratagene) or pGEX (glutathione S-transferase;
Promega,
Madison, Wis.). A suitable bacterial promoter is any nucleic acid sequence
capable of binding
bacterial RNA polymerase and initiating the downstream (3') transcription of
the coding sequence of
the CPP gene into mRNA. A bacterial promoter has a transcription initiation
region which is usually
placed proximal to the 5' end of the coding sequence. This transcription
initiation region typically
includes an RNA polymerase binding site and a transcription initiation site.
Sequences encoding
metabolic pathway enzymes provide particularly useful promoter sequences.
Examples include
promoter sequences derived from sugar metabolizing enzymes, such as galactose,
lactose and
maltose, and sequences derived from biosynthetic enzymes such as tryptophan.
Promoters from
bacteriophage may also be used and are known in the art. In addition,
synthetic promoters and hybrid
promoters are also useful; for example, the tat promoter is a hybrid of the
trp and lac promoter
sequences. Furthermore, a bacterial promoter can include naturally occurring
promoters of non-
bacterial origin that have the ability to bind bacterial RNA polymerase and
initiate transcription. An
efficient ribosome-binding site is also desirable. The expression vector may
also include a signal
peptide sequence that provides for secretion of the CPP in bacteria. The
signal sequence typically
encodes a signal peptide comprised of hydrophobic amino acids which direct the
secretion of the
protein from the cell, as is well known in the art. The protein is either
secreted into the growth media
(gram-positive bacteria) or into the periplasmic space, located between the
inner and outer membrane
of the cell (gram-negative bacteria). The bacterial expression vector may also
include a selectable
marker gene to allow for the selection of bacterial strains that have been
transformed. Suitable
selection genes include drug resistance genes such as ampicillin,
chloramphenicol, erythromycin,
kanamycin, neomycin and tetracycline. Selectable markers also include
biosynthetic genes, such as
those in the histidine, tryptophan and leucine biosynthetic pathways. When
large quantities of CPPs
are needed, e.g., for the induction of antibodies, vectors which direct high
level expression of fusion
proteins that are readily purified may be desirable. Such vectors include, but
are not limited to,
multifunctional E. coli cloning and expression vectors such as BLUESCRIPT
(Stratagene), in which
the CPP coding sequence may be ligated into the vector in-frame with sequences
for the amino-
terminal Met and the subsequent 7 residues of beta-galactosidase so that a
hybrid protein is
3~
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
produced; PIN vectors (Van Heeke & Schuster JBiol Chem 264:5503-5509 1989));
PET vectors
(Novagen, Madison Wis.); and the like. Expression vectors for bacteria include
the various
components set forth above, and are well known in the art. Examples include
vectors for Bacillus
subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividahs, among
others. Bacterial
expression vectors are transformed into bacterial host cells using techniques
well known in the art,
such as calcium chloride mediated transfection, electroporation, and others.
Expression in Yeast
Yeast expression systems are well known in the art, and include expression
vectors for
Saccharomyces cerevisiae, Cahdida albica~s and C. maltosa, Hahsenula
polymorpha,
Kluyveromyces fragilis and K. Zactis, Pichia guillermohdii and P pastoris,
Schizosaccharomyces
pombe, and Yarrowia lipolyiica. Examples of suitable promoters for use in
yeast hosts include the
promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.
255:2073 (1980)) or other
glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg. 7:149 (1968); Holland,
Biochemistry 17:4900
(1978)), such as enolase, glyceraldehyde-3- phosphate dehydrogenase,
hexokinase, pyruvate
decarboxylase, phosphofnzctokinase, glucose- 6-phosphate isomerase, 3-
phosphoglycerate mutase,
pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, alpha
factor, the
ADH2IGAPDH promoter, glucokinase alcohol oxidase, and PGH. See, for example,
Ausubel, et al.,
1990; Grant et al., Methods in Er~zymology 153:516-544, (1987). Other yeast
promoters, which are
inducible have the additional advantage of transcription controlled by growth
conditions, include the
promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative
enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-
phosphate
dehydrogenase, and enzymes responsible for maltose and galactose utilization.
Suitable vectors and
promoters for use in yeast expression are further described in EP 73,657.
Yeast selectable markers
include ADE2. HIS4. LEU2. TRPI. and ALG7, which confers resistance'to
tunicamycin; the
neomycin phosphotransferase gene, which confers resistance to 6418; and the
CUP 1 gene, which
allows yeast to grow in the presence of copper ions. Yeast expression vectors
can be constructed for
intracellular production or secretion of a CPP from the DNA encoding the CPP
of interest. For
example, a 'selected signal peptide and the appropriate constitutive or
inducible promoter may be
inserted into suitable restriction sites in the selected plasmid for direct
intracellular expression of the
CPP. For secretion of the CPP, DNA encoding the CPP can be cloned into the
selected plasmid,
together with DNA encoding the promoter, the yeast alpha-factor secretory
signal/leader sequence,
and linker sequences (as needed), for expression of the CPP. Yeast cells, can
then be transformed
with the expression plasmids described above, and cultured in an appropriate
fermentation media.
The protein produced by such transformed yeast can then be concentrated by
precipitation with 10%
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CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
trichloroacetic acid and analyzed following separation by SDS-PAGE and
staining of the gels with
Coomassie Blue stain. The recombinant CPP can subsequently be isolated and
purified from the
fermentation medium by techniques known to those of skill in the art.
Expression in Mammalian Systems
The CPP may be expressed in mammalian cells. Mammalian expression systems are
known
in the art, and include retroviral vector mediated expression systems.
Mammalian host cells may be
transformed with any of a number of different viral-based expression systems,
such as adenovirus,
where the coding region can be ligated into an adenovirus
transcription/translation complex
consisting of the late promoter and tripartite leader sequence. Insertion in a
nonessential El or E3
region of the viral genome results in a viable virus capable of expression of
the polypeptide of
interest in infected host cells. A preferred expression vector system is a
retroviral vector system such
as is generally described in PCT/US97/01019 and PCT/US97/101048. Suitable
mammalian
expression vectors contain a mammalian promoter which is any DNA sequence
capable of binding
mammalian RNA polymerase and initiating the downstream (3') transcription of a
coding sequence
for CPP into mRNA. A promoter will have a transcription initiating region,
which is usually placed
proximal to the 5' end of the coding sequence, and a TATA box, using a located
25-30 base pairs
upstream of the transcription initiation site. The TATA box is thought to
direct RNA polymerase II
to begin RNA synthesis at the correct site. A mammalian promoter will also
contain an upstream
promoter element (enhancer element), typically located within 100 to 200 base
pairs upstream of the
TATA box. An upstream promoter element determines the rate at which
transcription is initiated and
can act in either orientation. Of particular use as mammalian promoters are
the promoters from
mammalian viral genes, since the viral genes are often highly expressed and
have a broad host range.
Examples include promoters obtained from the genomes of viruses such as
polyoma virus, fowlpox
virus (UI~ 2,211, 504 published Jul. 5,1989), adenovirus (such as Adenovirus
2), bovine papilloma
virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus
and Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin
promoter, and from heat-shock promoters, provided such promoters are
compatible with the host cell
systems. Transcription of DNA encoding a CPP by higher eukaryotes may be
increased by inserting
an enhancer sequence into the vector. Enhancers are cis-acting elements of
DNA, usually about from
10 to 300 bp, that act on a promoter to increase its transcription. Many
enhancer sequences are now
known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and
insulin). Typically,
however, one will use an enhancer from a eukaryotic cell virus. Examples
include the SV40
enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the
replication origin, and adenovirus enhancers. The enhancer is preferably
located at a site 5' from the
CA 02512629 2005-07-05
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promoter. In general, the transcription termination and polyadenylation
sequences recognized by
mammalian cells are regulatory regions located 3' to the translation stop
codon and thus, together
with the promoter elements, flank the coding sequence. The 3' terminus of the
mature mRNA is
formed by site-specific post translational cleavage and polyadenylation.
Examples of transcription
terminator and polyadenylation signals include those derived from SV40. Long
term, high-yield
production of recombinant proteins can be effected in a stable expression
system. Expression vectors
which contain viral origins of replication or endogenous expression elements
and a selectable marker
gene may be used for this purpose. Appropriate vectors containing selectable
markers for use in
mammalian cells axe readily available commercially and are known to persons
skilled in the art.
Examples of such selectable markers include, but are not limited to herpes
simplex virus thymidine
kinase and adenine phosphoribosyltransferase for use in tk- or hprt-cells,
respectively. The methods
of introducing exogenous nucleic acid into mammalian hosts, as well as other
hosts, is well known in
the art, and will vary with the host cell used. Techniques include dextran-
mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection, protoplast
fusion, electroporation,
viral infection, encapsulation of the polynucleotide(s) in liposomes, and
direct microinjection of the
DNA into nuclei.
CPPs can be purified from culture supernatants of mammalian cells transiently
transfected
or stably transformed by an expression vector carrying a CPP-encoding
sequence. Preferably, CPP
is purified from culture supernatants of COS 7 cells transiently transfected
by the pcD expression
vector. Transfection of COS 7 cells with pcD proceeds as follows: One day
prior to transfection,
approximately 106 COS 7 monkey cells are seeded onto individual 100 mm plates
in Dulbecco's
modified Eagle medium (DME) containing 10% fetal calf serum and 2 mM
glutamine. To perform
the transfection, the medium is aspirated from each plate and replaced with 4
ml of DME containing
50 mM Tris.HCl pH 7.4, 400 mg/ml DEAE-Dextran and 50 pg of plasmid DNA. The
plates are
incubated for four hours at 37oC, then the DNA-containing medium is removed,
and the plates are
washed twice with 5 ml of serum-free DME. DME is added back to the plates
which are then
incubated for an additional 3 hrs at 37oC. The plates are washed once with
DME, after which DME
containing 4% fetal calf serum, 2 rnM glutamine, penicillin (100 U/L) and
streptomycin (100 ~g/L)
at standard concentrations is added. The cells are then incubated for 72 hrs
at 37oC, after which the
growth medium is collected for purification of CPP. Plasmid DNA for the
transfections is obtained
by growing pcD(SRa), or like expression vector, containing the CPP-encoding
cDNA insert in E.
coli MC1061, described by Casadaban and Cohen, J. Mol. Biol., Vol. 138, pgs.
179-207 (1980), or
like organism. The plasmid DNA is isolated from the cultures by standard
techniques, e.g.
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold
Spring Harbor
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Laboratory, New York, 1989) or Ausubel et al (1990, cited above).
Expression in Insect Cells
CPPs may also be produced in insect cells. Expression vectors for the
transformation of
insect cells, and in particular, baculovirus-based expression vectors, are
well known in the art. In one
such system, the CPP-encoding DNA is fizsed upstream of an epitope tag
contained within a
baculovirus expression vector. Autographa califorrcica nuclear polyhedrosis
virus (AcNPV) is used .
as a vector to express foreign genes in Spodoptera frugiperda Sf9 cells or in
Trichoplusia larvae.
The CPP-encoding sequence is cloned into a nonessential region of the virus,
such as the polyhedrin
gene, and placed under control of the polyhedrin promoter. Successful
insertion of a CPP-encoding
sequence will render the polyhedrin gene inactive and produce recombinant
virus lacking coat protein
coat. The recombinant viruses are then used to infect S frugiperda cells or
Trichoplusia larvae in
which the CPP is expressed (Smith et al., J. Wol. 46:584 (1994); Engelhard E K
et al., Proc. Nat.
Acad. Sci. 91:3224-3227 (1994)). Suitable epitope tags for fusion to the CPP-
encoding DNA
include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A
variety of plasmids may
be employed, including commercially available plasmids such as pVL1393
(Novagen). Briefly, the
CPP-encoding DNA or the desired portion of the CPP-encoding DNA is amplified
by PCR with
primers complementary to the 5' and 3' regions. The 5' primer may incorporate
flanking restriction
sites. The PCR product is then digested with the selected restriction enzymes
and subcloned into an
expression vector. Recombinant baculovirus is generated by co-transfecting the
above plasmid and
BaculoGoldTM virus DNA (Pharmingen) into Spodoptera frugiperda ("S~") cells
(ATCC CRL
1711) using lipofectin (commercially available from GIBCO-BRL), or other
methods known to those
of skill in the art. Virus is produced by day 4-5 of culture in S~ cells at
28°C, and used for further
amplifications. Procedures are performed as further described in O'Reilley et
al., BACULOTIIRUS
EXPRESSION VECTORS: A LABORATORYMANZIAL, Oxford University Press (1994).
Extracts
may be prepared from recombinant virus-infected Sf9 cells as described in
Rupert et al., Nature
362:175-179 (1993). Alternatively, expressed epitope-tagged CPP can be
purified by affinity
chromatography, or for example, purification of an IgG tagged (or Fc tagged)
CPP can be performed
using chromatography techniques, including Protein A or protein G column
chromatography.
Evaluation of Gene Expression
Gene expression may be evaluated in a sample directly, for example, by
standard techniques .
known to those of skill in the art, e.g., Northern blotting to determine the
transcription of mRNA, dot
blotting (DNA or RNA), or in situ hybridization, using an appropriately
labeled probe, based on the
sequences provided herein. Alternatively, antibodies may be used in assays for
detection of
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polypeptides, nucleic acids, such as specific duplexes, including DNA
duplexes, RNA duplexes, and
DNA-RNA hybrid duplexes or DNA-protein duplexes. Such antibodies may be
labeled and the assay
carned out where the duplex is bound to a surface, so that upon the formation
of duplex on the
surface, the presence of antibody bound to the duplex can be detected. Gene
expression,
alternatively, may be measured by immunohistochemical staining of cells or
tissue sections and assay
of cell culture or body fluids, to directly evaluate the expression of a CPP
polypeptide or
polynucleotide. Antibodies useful for such imrnunological assays may be either
monoclonal or
polyclonal, and may be prepared against a native sequence CPP. Protein levels
may also be detected
by mass spectrometry. A further method of protein detection is with protein
chips.
Purifrcatioh of Expressed Protein
Expressed CPP may be purified or isolated after expression, using any of a
variety of
methods known to those skilled in the art. The appropriate technique will vary
depending upon what
other components are present in the sample. Contaminant components that are
removed by isolation
or purification are materials that would typically interfere with diagnostic
or therapeutic uses for the
polypeptide, and may include enzymes, hormones, and other solutes. The
purification steps) selected
will depend, for example, on the nature of the production process used and the
particular CPP
produced. As CPPs are secreted, they may be recovered from culture medium.
Alternatively, the
CPP may be recovered from host cell lysates. If membrane-bound, it can be
released from the
membrane using a suitable detergent solution (e.g. Triton-X 100) or by
enzymatic cleavage.
Alternatively, cells employed in expression of CPP can be disrupted by various
physical or chemical
means, such as freeze-thaw cycling, sonication, mechanical disruption, or by
use of cell lysing
agents. Exemplary purification methods include, but are not limited to, ion-
exchange column
chromatography; chromatography using silica gel or a cation-exchange resin
such as DEAF; gel
filtration using, for example, Sephadex G-75; protein A Sepharose columns to
remove contaminants
such as IgG; chromatography using metal chelating columns to bind epitope
tagged forms of the
CPP; ethanol precipitation; reverse phase HPLC; chromatofocusing; SDS-PAGE;
and ammonium
sulfate precipitation. Ordinarily, an isolated CPP will be prepared by at
least one purification step.
For example, the CPP may be purified using a standard anti-CPP antibody
column. Ultrafiltration
and dialysis techniques, in conjunction with protein concentration, are also
useful (see, for example,
Scopes, R., PROTEINPURIFICATION, Springer-Verlag, New York, N.Y., 1982). The
degree of
purification necessary will vary depending on the use of the CPP. In some
instances no purification
will be necessary. Once expressed and purified as needed, the CPPs and nucleic
acids of the present
invention are useful in a number of applications, as detailed herein.
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Tra~sgehic animals
The host cells of the invention can also be used to produce nonhuman
transgenic animals.
For example, in one embodiment, a host cell of the invention is a fertilized
oocyte or an embryonic
stem cell into which CPP-coding sequences have been introduced. Such host
cells can then be used to
create non-human transgenic animals in which exogenous CPP sequences have been
introduced into
their genome or homologous recombinant animals in which endogenous CPP
sequences have been
altered. Such animals are useful for studying the function andlor activity of
a CPP or fragment
thereof and for identifying and/or evaluating modulators of CPP biological
activity. As used herein, a
"transgenic animal" is a non-human animal, preferably a mammal, more
preferably a rodent such as
a rat or mouse, in which one or more of the cells of the animal include a
transgene. Other examples
of transgenic animals include non-human primates, sheep, dogs, cows, goats,
chickens, amphibians,
etc. A transgene is exogenous DNA which is integrated into the genome of a
cell from which a
transgenic animal develops and which remains in the genome of the mature
animal, thereby directing
the expression of an encoded gene product in one or more cell types or tissues
of the transgenic
animal. As used herein, a "homologous recombinant animal" is a non-human
animal, preferably a
mammal, more preferably a mouse, in which an endogenous gene has been altered
by homologous
recombination between the endogenous gene and an exogenous DNA molecule
introduced into a cell
of the animal, e.g., an embryonic cell of the animal, prior to development of
the animal.
A transgenic animal of the invention can be created by introducing a CPP-
encoding nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection
or retxoviral infection, and
allowing the oocyte to develop in a pseudopregnant female foster animal. The
CPP cDNA sequence
or a fragment thereof can be introduced as a transgene into the genome of a
non-human animal.
Alternatively, a nonhurnan homologue of a human CPP-encoding gene, such as
from mouse or rat,
can be used as a transgene. lntronic sequences and polyadenylation signals can
also be included in
the transgene to increase the efficiency of expression of the transgene. A
tissue-specific regulatory
sequences) can be operably linked to a CPP transgene to direct expression of a
CPP to particular
cells. Methods for generating transgenic animals via embryo manipulation and
microinjection,
particularly animals such as mice, have become conventional in the art and are
described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S.
Pat. No. 4,873,191
by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986, the disclosure of which is
incorporated herein by
reference in its entirety). Similar methods are used for production of other
transgenic animals. A
transgenic founder animal can be identified based upon the presence of a CPP
transgene in its
genome and/or expression of CPP mRNA in tissues or cells of the animals. A
transgenic founder
animal can then be used to breed additional animals harrying the transgene.
Moreover, transgenic
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animals carrying a transgene encoding a CPP can further be bred to other
transgenic animals
carrying other transgenes.
To create an animal in which a desired nucleic acid has been introduced into
the genome via
homologous recombination, a vector is prepared which contains at least a
portion of a CPP-encoding
sequence into which a deletion, addition or substitution has been introduced
to thereby alter, e.g.,
functionally disrupt, the CPP-encoding sequence. The CPP-encoding sequence can
be a human gene,
but more preferably, is a non-human homologue of a human CPP-encoding sequence
(e.g., a cDNA
isolated by stringent hybridization with a nucleotide sequence coding for a
CPP). For example, a
mouse CPP-encoding sequence can be used to construct a homologous
recombination vector suitable
for altering an endogenous gene in the mouse genome. In a preferred
embodiment, the vector is
designed such that, upon homologous recombination, the endogenous CPP-encoding
sequence is
functionally disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock out"
vector). Alternatively, the vector can be designed such that, upon homologous
recombination, the
endogenous CPP-encoding sequence is mutated or otherwise altered but still
encodes functional
protein (e.g., the upstream regulatory region can be altered to thereby alter
the expression of the
endogenous CPP-encoding sequence). In the homologous recombination vector, the
altered portion of
the CPP-encoding sequence is flanked at its 5' and 3' ends by additional
nucleic acid sequence of the
CPP gene to allow for homologous recombination to occur between the exogenous
sequence carned
by the vector and an endogenous gene in an embryonic stem cell. The additional
flanking nucleic acid
sequence is of sufficient length for successful homologous recombination with
the endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are
included in the vector
(see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503, the
disclosure of which is
incorporated herein by reference in its entirety, for a description of
homologous recombination
vectors). 'The vector is introduced into an embryonic stem cell line (e.g., by
electroporation) and cells
in which the introduced CPP-encoding sequence has homologously recombined with
the endogenous
gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915, the disclosure
of which is incorporated
herein by reference in its entirety). The selected cells are then injected
into a blastocyst of an animal
(e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in
Teratocarcinomas and
Embryonic Stem Cells. A Practical Approach, E. J. Robertson, ed. (IRL, Oxford,
1987) pp. 113-
152, the disclosure of which is incorporated herein by reference in its
entirety). A chirneric embryo
can then be implanted into a suitable pseudopregnant female foster animal and
the embryo brought to
term. Progeny harboring the hornologously recombined DNA in their germ cells
can be used to breed
animals in which all cells of the animal contain the homologously recombined
DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination vectors and
homologous recombinant animals are described further in Bradley, A. (1991)
Current Opinion in
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Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354
by Le Mouellec
et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO
93/04169 by Berns
et al., the disclosures of which are incorporated herein by reference in their
entireties.
In another embodiment, transgenic non-human animals can be produced which
contain
selected systems which allow for regulated expression of the transgene. One
example of such a
system is the crelloxP recombinase system of bacteriophage P 1. For a
description of the cre/loxP
recombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236, the
disclosure of which is
incorporated herein by reference in its entirety. Another example of a
recombinase system is the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science
251:1351-1355,
the disclosure of which is incorporated herein by reference in its entirety).
If a cre/loxP recombinase .
system is used to regulate expression of the transgene, animals containing
transgenes encoding both
the Cre recombinase and a selected protein are required. Such animals can be
provided through the
construction of "double" transgenic animals, e.g., by mating two transganic
animals, one containing a
transgene encoding a selected protein and the other containing a transgene
encoding a recombinase. w w
Assessing CPP activity
It will be appreciated that the invention further provides methods of testing
the activity of or
obtaining functional fragments and variants of CPPs and CPP sequences. Such
methods involve
providing a variant or modified CPP-encoding nucleic acid and assessing
whether the encoded
polypeptide displays a CPP biological activity. Encompassed is thus a method
of assessing the
function of a CPP comprising: (a) providing a CPP, or a biologically active
fragment or homologue
thereof; and (b) testing said CPP, or a biologically active fragment or
homologue thereof fox a CPP
biological activity. Any suitable format may be used, including cell free,
cell-based and in vivo .
formats. Fox example, said assay may comprise expressing a CPP nucleic acid in
a host cell, and
observing CPP activity in said cell and other affected cells. In another
example, a CPP, or a
biologically active fragment or homologue thereof is contacted with a cell,
and a CPP biological
activity is observed.
A CPP biological activity may be any activity as described herein, such as (1)
protection
from cardiovascular disease; (2) circulating through the bloodstream; (3)
antigenicity, or the ability
to bind an anti-CPP specific antibody; (4) immunogenicity, or the ability to
generate an anti-CPP
specific antibody; or (S) interacting with a CPP target molecule, preferably a
protein.
CPP biological activity can be assayed by any suitable method lrnown in the
art.
Antigenicity and immunogenicity may be detected, for example, as described in
the sections titled
"Anti CPP antibodies" and "Uses of CPP antibodies". Circulation in blood
plasma may be detected
as described in. "Diagnostic and Prognostic Uses". Interaction with a CPP
target molecule may be
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detected according to any of the methods described herein, for example, in the
section titled "Drug
Screening Assays".
Cardiovascular disorders may be diagnosed by any method determined appropriate
for an
individual by one of skill in the art. Further examples of symptoms and
diagnostics may be found in
the Background section, and are best determined appropriately by one of skill
in the art based on the
particular profile of a patient.
Intramolecular interactions may be detected by sequence-based structural
predictions. Such
predictions are generally based on X-ray crystallography or NMR structural
data for a polypeptide
with similar sequence. Detection of intramoleculax interactions may also be
accomplished using
SDS-PAGE. For the example of disulfide bonds, links formed between different
portions of a given
protein result in a more compacted protein, and thus, a reduced apparent
molecular weight.
Disulfide bonds may be disrupted by a reducing agent, for example,
dithiothreitol (DTT). A protein
sample that has been treated with a reducing agent may thus be compared to an
untreated control by
SDS-PAGE to detect a change in apparent molecular weight. Such methods are
common to the art.
Amidation may be detected by comparing the molecular weight of a sample
peptide to that of
an amidated form of the same peptide. The amidated form may be prepared
according to common
methods, for example, as disclosed in US Patent 4708934. Molecular weights are
easily compared
according to any method common to the art such as SDS-PAGE, gel
chromatography, or mass
spectrometry. Proteolysis may also be detected by comparing the molecular
weight of a sample
peptide to that of a peptide of known molecular weight. Preferably, the
molecular weight of a test
peptide is obtained by mass spectrometry and compared to a database comprising
molecular weights
of peptides with posttranslational modifications. Exemplary databases include
Genpept,
SWISSPROT, EMBL, and the Protein Sequence Database. Such techniques are
detailed further
herein.
Anti-CPP Atztibodies .
The present invention provides antibodies and binding compositions specific
for CPPs. Such
antibodies and binding compositions include polyclonal antibodies, monoclonal
antibodies, Fab and
single chain Fv fragments thereof, bispecific antibodies, heteroconjugates,
humanized antibodies, and
the like. Such antibodies and binding compositions may be produced in a
variety of ways, including
hybridoma cultures, recombinant expression in bacteria or mammalian cell
cultures, recombinant
expression in transgenic animals, and the like. There is abundant guidance in
the literature for
selecting a particular production methodology, e.g. Chadd and Chamow, Curr.
Opin. Biotechnol.,
12: 188-194 (2001).
The choice of manufacturing methodology depends on several factors including
the antibody
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structure desired, the importance of carbohydrate moieties on the antibodies,
ease of culturing and
purification, cost, and the like. Many different antibody structures may be
generated using standard
expression technology, including full-length antibodies, antibody fragments,
such as Fab and Fv
fragments, as well as chimeric antibodies comprising components from different
species. Antibody
fragments of small size, such as Fab and Fv fragments, having no effector
functions and limited
pharmokinetic activity may be generated in a bacterial expression system.
Single chain Fv fragments
are highly selective for in vivo tumors, show good tumor penetration and low
immunogenicity, and
are cleared rapidly from the blood, e.g. Freyre et al, J. Biotechnol., 76: 157-
163 (2000). Thus, such
molecules are desirable for radioimmunodetection and in situ radiotherapy.
Whenever
pharmacokinetic activity in the form of increased half life is required for
therapeutic purposes, then
full-length antibodies are preferable. For example, the immunoglobulin G (IgG)
molecule may be
one of four subclasses: yl, y2, ~y3, or y4. If a full-length antibody with
effector function is required,
then IgG subclasses yl or y3 are preferred, and IgG subclass yl is most
preferred. The yl and y3
subclasses exhibit potent effector function, complement activation, and
promote antibody-dependent
cell-mediated cytotoxicity through interaction with specific Fc receptors,
e.g. Raju et al,
Glycobiology, 10: 477-486 (2000); Lund et al, J. Immunol., 147: 2657-2662
(1991).
Polyclo~al Antibodies
The anti-CPP antibodies of the present invention may be polyclonal antibodies.
Such
polyclonal antibodies can be produced in a mammal, for example, following one
or more injections
of an immunizing agent, and preferably, an adjuvant. Typically, the immunizing
agent and/or
adjuvant will be injected into the mammal by a series of subcutaneous or
intraperitoneal injections.
The immunizing agent may include CPPs or a fusion protein thereof. It may be
useful to conjugate
the antigen to a protein known to be immunogenic in the mammal being
immunized. Examples of
such imrnunogenic proteins include, but axe not limited to, keyhole limpet
hemocyanin (KI,H),
methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis
B surface
antigen, senun albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
Adjuvants include, for
example, Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid
A, synthetic
trehalose dicoryno-mycolate). The immunization protocol may be determined by
one skilled in the art
based on standard protocols or by routine experimentation.
Alternatively, a crude protein 'preparation which has been enriched for a CPP
or a portion
thereof can be used to generate antibodies. Such proteins, fragments or
preparations are introduced
into the non-human mammal in the presence of an appropriate adjuvant. If the
serum contains
polyclonal antibodies to undesired epitopes, the polyclonal antibodies are
purified by immunoaffinity
chromatography.
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Effective polyclonal antibody production is affected by many factors related
both to the
antigen and the host species. Also, host animals vary in response to site of
inoculations and dose,
with both inadequate and excessive doses of antigen resulting in low titer
antisera. Small doses (ng
level) of antigen administered at multiple intradermal sites appear to be most
reliable. Techniques for
producing and processing polyclonal antisera are known in the art, see for
example, Mayer and
Walker (1987), the disclosure of which is incorporated herein by reference in
its entirety. An
effective immunization protocol for rabbits can be found in Vaitukaitis, J. et
al. J. Clin. Endocrinol.
Metab. 33:988-991(1971), the disclosure of which is incorporated by reference
in its entirety.
Booster injections can be given at regular intervals, and antiserum harvested
when antibody titer
thereof, as determined semi-quantitatively, for example, by double
immunodiffusion in.agar against
known concentrations of the antigen, begins to fall. See, for example,
Ouchterlony, O. et al., Chap.
19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973).
Plateau concentration
of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12:
M). Affinity of the
antisera for the antigen is determined by preparing competitive binding
curves, as described, for
example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed.
(Rose and Friedman,
Eds.) Amer. Soc. For Microbiol., Washington, D. C. (1980).
Mohoclohal Antibodies
Alternatively, the anti-CPP antibodies may be monoclonal antibodies.
Monoclonal antibodies
may be produced by hybridomas, wherein a mouse, hamster, or other appropriate
host animal, is
immunized with an immunizing agent to elicit lymphocytes that produce or are
capable of producing
antibodies that will specifically bind to the immunizing agent, e.g. Kohler
and Milstein, Nature
256:495 (1975). The immunizing agent will typically include the CPP or a
fusion protein thereof and
optionally a Garner. Alternatively, the lymphocytes may be immunized in vitro.
Generally, spleen
cells or lymph node cells are used if non-human mammalian sources are desired,
or peripheral blood
lymphocytes ("PBLs") are used if cells of human origin are desired. The
lymphocytes are fused with
an immortalized cell line using a suitable fusing agent, such as polyethylene
glycol, to produce a
hybridoma cell, e.g. Goding, MONOCLONAL ANTIBODIES: PRINCIPLESAND PRACTICE,
Academic Press, pp. 59-103 (1986); Liddell and Cryer, A Practical Guide to
Monoclonal Antibodies
(John Wiley & Sons, New York, 1991); Malik and Lillenoj, Editors, Antibody
Techniques
(Academic Press, New York, 1994). In general, immortalized cell lines are
transformed mammalian
cells, for example, myeloma cells of rat, mouse, bovine or human origin. The
hybridoma cells are
cultured in a suitable culture medium that preferably contains one or more
substances that inhibit the '
growth or survival of unfused, immortalized cells. For example, if the
parental cells lack the enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT), the culture medium
for the hybridornas
49
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typically will include hypoxanthine, aminopterin, and thymidine (HAT),
substances which prevent
the growth of HGPRT-deficient cells. Preferred immortalized cell lines are
those that fuse efficiently,
support stable high level production of antibody, and are sensitive to a
medium such as HAT
medium. More preferred immortalized cell lines are murine or human myeloma
lines, which can be
obtained, for example, from the American Type Culture Collection (ATCC),
Rockville, MD. Human
myeloma and mouse-human heteromyeloma cell lines also have been described for
the production of
human monoclonal antibodies, e.g. Kozbor, J. Immunol. 133:3001 (1984); Brodeur
et al.,
Monoclonal Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New York, pp.
51-63 (1987).
The culture medium (supernatant) in which the hybridoma cells are cultured can
be assayed
for the presence of monoclonal antibodies directed against a CPP. Preferably,
the binding specificity
of monoclonal antibodies present in the hybridoma supernatant is determined by
immunoprecipitation
or by an in vitro binding assay, such as radio- immunoassay (RIA) or Enzyme-
Linked Immuno
Sorbent Assay (ELISA). Appropriate techniques and assays are known in the art.
The binding
affinity of the monoclonal antibody can, for example, be determined by the
Scatchard analysis of
Munson and Pollard, Ahal. Biochem. 107:220 (1980). After the desired antibody-
producing
hybridoma cells are identified, the cells may be cloned by limiting dilution
procedures and grown by
standard methods (Goding, 1986, supra). Suitable culture media for this
purpose include, for
example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the
hybridoma cells may be grown in vivo as ascites in a mammal. The monoclonal
antibodies secreted
by selected clones may be isolated or purified from the culture medium or
ascites fluid by
immunoglobulin purification procedures routinely used by those of skill in the
art such as, for
example, protein A-Sepharose, hydroxyl-apatite chromatography, gel
electrophoresis, dialysis, or
affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those
described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies
of the invention can
be isolated from the CPP-specific hybridoma cells and sequenced, e.g., by
using oligonucleotide
probes that are capable of binding specifically to genes encoding the heavy
and light chains of
murine antibodies. Once isolated, the DNA may be inserted into an expression
vector, which is then
transfected into host cells such as simian COS cells, Chinese hamster ovary
(CHO) cells, or
myeloma cells that do not otherwise produce immurioglobulin protein, to obtain
the synthesis of
monoclonal antibodies in the recombinant host cells. The DNA also may be
modified, for example,
by substituting the coding sequence for the murine heavy and light chain
constant domains for the
homologous human sequences (Mornson et al., Pfoc. Nat. Acad. Sci. 81:6851-6855
(1984);
Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454
(1985)), or by
CA 02512629 2005-07-05
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covalently joining to the immunoglobulin coding sequence all or part of the
coding sequence for a
non-immunoglobulin polypeptide. The non-immunoglobulin polypeptide can be
substituted for the
constant domains of an antibody of the invention, or can be substituted for
the variable domains of
one antigen-combining site of an antibody of the invention to create a
chimeric bivalent antibody.
The antibodies may also be monovalent antibodies. Methods for preparing
monovalent antibodies are
well known in the art. For example, in vitro methods are suitable for
preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be
accomplished using routine techniques known in the art.
Antibodies and antibody fragments characteristic of hybridomas of the
invention can also be
produced by recombinant means by extracting messenger RNA, constructing a cDNA
library, and
selecting clones which encode segments of the antibody molecule. The following
are exemplary
references disclosing recombinant techniques for producing antibodies: Wall et
al., Nucleic Acids
Research, Vol. 5, pgs. 3113-3128 (1978); Zakut et al., Nucleic Acids Research,
Vol. 8, pgs: 3591-
3601 (1980); Cabilly et al., Proc. Natl. Acad. Sci., Vol. 81, pgs. 3273-3277
(1984); Boss et al.,
Nucleic Acids Research, Vol. 12, pgs. 3791-3806 (1984); Amster et al., Nucleic
Acids Research,
Vol. 8, pgs. 2055-2065 (1980); Moore et al., U.S. Patent 4,642,334; Skerra et
al, Science, Vol. 240,
pgs. 1038-1041(1988); Huse et al, Science, Vol. 246, pgs. 1275-1281 (1989);
and U.S. patents
6,054,297; 5,530,101; 4,816,567; 5,750,105; and 5,648,237; which patents are
incorporated by
reference. ' In particular, such techniques can be used to produce
interspecific monoclonal antibodies,
wherein the binding region of one species is combined with non-binding region
of the antibody of
another species to reduce immunogenicity, e.g. Liu et al., Proc. Natl. Acad.
Sci., Vol. 84, pgs. 3439-
3443 (1987), and patents 6,054,297 and 5,530,101. Preferably, recombinantly
produced Fab and
Fv fragments are expressed in bacterial host systems. Preferably, full-length
antibodies are produced
by mammalian cell culture techniques. More preferably, full-length antibodies
are expressed in
Chinese Hamster Ovary (CHO) cells or NSO cells.
Both polyclonal and monoclonal antibodies can be screened by ELISA. As in
other solid
phase immunoassays, the test is based on the tendency of macromolecules to
adsorb nonspecifically
to plastic. The irreversibility of this reaction, without loss of
imrnunological activity, allows the
formation of antigen-antibody complexes with a simple separation of such
complexes from unbound
material. To titrate antipeptide serum, peptide conjugated to a Garner
different from that used in
immunization is adsorbed to the wells of a 96-well microtiter plate. The
adsorbed antigen is then
allowed to react in the wells with dilutions of anti-peptide serum. Unbound
antibody is washed
away, and the remaining antigen-antibody complexes are allowed to react with
an antibody specific
for the IgG of the immunized animal. This second antibody is conjugated to an
enzyme such as
alkaline phosphatase. A visible colored reaction produced when the enzyme
substrate is added
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WO 2004/061096 PCT/EP2004/000027
indicates which wells have bound antipeptide antibodies. The use of
spectrophotometer readings
allows better quantification of the amount of peptide-specific antibody bound.
Nigh-titer antisera
yield a linear titration curve between 10-3 and 10-5 dilutions.
CPP peptide carriers
The invention includes immunogens derived from CPPs, and immunogens comprising
conjugates between carriers and peptides of the invention. The term immunogen
as used herein
refers to a substance which is capable of causing an immune response. The term
carrier as used
herein refers to any substance which when chemically conjugated to a peptide
of the invention
permits a host organism immunized with the resulting conjugate to generate
antibodies specific for
the conjugated peptide. Garners include red blood cells, bacteriophages,
proteins, or synthetic
particles such as agarose beads. Preferably, Garners are proteins, such as
serum albumin, gamma-
globulin, keyhole limpet hemocyanin, thyroglobulin, ovalbumin, fibrinogen, or
the like.
The general technique of linking synthetic peptides to a carrier is described
in several
references, e.g. Walter and Doolittle, "Antibodies Against Synthetic
Peptides," in Setlow et al., eds.,
Genetic Engineering, Vol. 5, pgs. 61-91 (Plenum Press, N.Y., 1983); Green et
al. Cell, Vol. 28, pgs.
477-487 (1982); Lerner et al., Proc. Natl. Acad. Sci., Vol. 78, pgs. 3403-3407
(1981); Shimizu et
al., U.S. Patent 4,474,754; and Ganfield et al., U.S. Patent 4,311,639.
Accordingly, these references
are incorporated by reference. Also, techniques employed to link haptens to
carriers are essentially .
the same as the above-referenced techniques, e.g. chapter 20 in Tijssen,
Practice and Theory of
Enzyme Immunoassays (Elsevier, New York, 1985). The four most commonly used
schemes for
attaching a peptide to a carrier are (1) glutaraldehyde for amino coupling,
e.g. as disclosed by
Kagan and Glick, in Jaffe and Behrman, eds. Methods of Hormone
Ra,dioimmunoassay, pgs. 328-
329 (Academic Press, N.Y., 1979), and Walter et al. Proc. Natl. Acad. Sci.,
Vol. 77, pgs. 5197-
5200 (1980); (2) water-soluble carbodiimides for carboxyl to amino coupling,
e.g. as disclosed by
Hoare et al., J. Biol. Chem., Vol. 242, pgs. 2447-2453 (1967); (3) bis-
diazobenzidine (BDB) for
tyrosine to tyrosine sidechain coupling, e.g. as disclosed by Bassiri et al.,
pgs. 46-47, in Jaffe and
Behrman, eds. (cited above), and Walter et al. (cited above); and (4)
maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) for coupling cysteine (or other sulfhydryls) to
amino groups, e.g. as
disclosed by Kitagawa et al., J. Biochem. (Tokyo), Vol. 79, pgs. 233-239
(1976), and Lerner et al.
(cited above). A general rule for selecting an appropriate method for coupling
a given peptide~to a
protein carrier can be stated as follows: the group involved in attachment
should occur only once in
the sequence, preferably at the appropriate end of the segment. For example,
BDB should not be
used if a tyrosine residue occurs in the main part of a sequence chosen for
its potentially antigenic
character. Similarly, centrally located lysines rule out the glutaraldehyde
method, and the
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occurrences of aspartic and glutamic acids frequently exclude the carbodiimide
approach. On the
other hand, suitable residues can be positioned at either end of chosen
sequence segment as
attachment sites, whether or not they occur in the "native" protein sequence.
Internal segments,
unlike the amino and carboxy termini, will differ significantly at the
"unattached end" from the same
sequence as it is found in the native protein where the polypeptide backbone
is continuous. The
problem can be remedied, to a degree, by acetylating the a-amino group and
then attaching the
peptide by way of its carboxy terminus. The coupling efficiency to the carrier
protein is
conveniently measured by using a radioactively labeled peptide, prepared
either by using a
radioactive amino acid for one step of the synthesis or by labeling the
completed peptide by the
iodination of a tyrosine residue. The presence of tyrosine in the peptide also
allows one to set up a
sensitive radioimmune assay, if desirable. Therefore, tyrosine can be
introduced as a terminal
residue if it is not part of the peptide sequence defined by the native
polypeptide.
Preferred carriers are proteins, and preferred protein Garners include bovine
serum albumin,
myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH), or the like.
Peptides can be
linked to KLH through cysteines by MBS as disclosed by Liu et al.,
Biochemistry, Vol. 18, pgs.
690-697 (1979). The peptides are dissolved in phosphate-buffered saline (pH
7.5), 0.1 M sodium
borate buffer (pH 9.0) or 1.0 M sodium acetate buffer (pH 4.0). The pH for the
dissolution of the
peptide is chosen to optimize peptide solubility. The content of free cysteine
for soluble peptides is
determined by Ellinan's method, Ellinan, Arch. Biochem. Biophys., Vol. 82, pg.
7077 (1959). For
each peptide, 4 rng KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2)
is reacted with 0.7
mg MBS (dissolved in dimethyl formamide) and stirred for 30 min at room
temperature. The MBS
is added dropwise to ensure that the local concentration of formamide is not
too high, as KLH is
insoluble in >30% formamide. The reaction product, KLH-MBS, is then passed
through Sephadex
G-25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0) to remove free
MBS, KLH
recovery from peak fractions of the column eluate (monitored by OD280) is
estimated to be
approximately 80%. I~LH-MBS is then reacted with 5 mg peptide dissolved in 1
ml of the chosen
buffer. The pH is adjusted to 7-7.5 and the reaction is stirred for 3 hr at
room temperature.
Coupling efficiency is monitored with radioactive peptide by dialysis of a
sample of the conjugate
against phosphate-buffered saline, and may range from 8% to 60%. Once the
peptide-carrier
conjugate is available, polyclonal or monoclonal antibodies are produced by
standard techniques, e.g.
as disclosed by Campbell, Monoclonal Antibody Technology (Elsevier, New York,
1984); Hurrell,
ed. Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press,
Boca Raton, FL,
1982); Schreier et al. Hybridoma Techniques (Cold Spring Harbor Laboratory,
New York, 1980);
U.S. Patent 4,562,003; or the like. In particular, U.S. Patent 4,562,003 is
incorporated by reference.
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Humanized A~etibodies
'The anti-CPP antibodies of the invention may further comprise humanized
antibodies or
human antibodies. The term "humanized antibody" refers to humanized forms of
non-human (e.g.,
murine) antibodies that are chimeric antibodies, immunoglobulin chains or
fragments thereof (such
as Fv, Fab, Fab', F(ab'), or other antigen-binding partial sequences of
antibodies) which contain
some portion of the sequence derived from non-human antibody. Humanized
antibodies include
human immunoglobulins in which residues from a complementary determining
region (CDR) of the
human immunoglobulin are replaced by residues from a CDR of a non-human
species such as
mouse, rat or rabbit having the desired binding specificity, affinity and
capacity. In general, the
humanized antibody will comprise substantially all of at least one, and
generally two, variable
domains, in which all or substantially all of the CDR regions correspond to
those of a non-human
immunoglobulin and all or substantially all of the FR regions are those of a
human immunoglobulin
consensus sequence. The humanized antibody optimally also will comprise at
least a portion of an
immunoglobulin constant region (Fc), typically that of a human immunoglobulin
(Jones et al.,
Nature 321:522-525 (1986) and Presta, Curr. Op. Struct. Biol. 2:593-596
(1992)). Methods for
humanizing non-human antibodies are well known in the art. Generally, a
humanized antibody has
one or more amino acids introduced into it from a source which is non-human in
order to more
closely resemble a human antibody, while still retaining the original binding
activity of the antibody.
Methods for humanization of antibodies are further detailed in Jones et al.,
Nature 321:522-525
(1986); Riechmann et al., Nature 332:323-327 (1988); and Verhoeyen et al.,
Science 239:1534-
1536 (1988). Such "humanized" antibodies are chimeric antibodies in that
substantially less than an
intact human variable domain has been substituted by the corresponding
sequence from a non-human
species.
Heteroconjugate Antibodies
Heteroconjugate antibodies which comprise two covalently joined antibodies,
are also within
the scope of the present invention. Heteroconjugate antibodies may be prepared
in vitro using known
methods in synthetic protein chemistry, including those involving crosslinking
agents. For example,
immunotoxins may be prepared using a disulfide exchange reaction or by forming
a thioether bond.
Bispecific Antibodies
Bispecific antibodies have binding specificities for at least two different
antigens. Such
antibodies are monoclonal, and preferably human or humanized. One of the
binding specificities of a
bispecific antibody of the present invention is for a CPP, and the other one
is preferably for a cell-
surface protein or receptor or receptor subunit. Methods for making bispecific
antibodies are known
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WO 2004/061096 PCT/EP2004/000027
in the art, and in general, the recombinant production of bispecific
antibodies is based on the co-
expression of two imrnunoglobulin heavy-chain/light-chain pairs in hybridoma
cells, where the two
heavy chains have different specificities, e.g. Milstein and Cuello, Nature
305:537-539 (1983).
Given that the random assortment of immunoglobulin heavy and light chains
results in production of .
potentially ten different antibody molecules by the hybridomas, purification
of the correct molecule
usually requires some sort of affinity purification, e.g. affinity
chromatography.
Uses of CPP ahtibodies
CPP antibodies may be used as functional antagonists. Preferably, such
antibodies are
specific for CPPs. More preferably, the antagonists of the invention comprise
fragments or binding
compositions specific for CPPs. As used herein, the term "heavy chain variable
region" means a
polypeptide (1) which is from 110 to 125 amino acids in length, and (2) whose
amino acid sequence
corresponds to that of a heavy chain of an antibody of the invention, starting
from the heavy chain's
N-terminal amino acid. Likewise, the term "light chain variable region" means
a polypeptide (1)
which is from 95 to 115 amino acids in length, and (2) whose amino acid
sequence corresponds to
that of a light chain of an antibody of the invention, starting from the light
chain's N-terminal amino
acid. As used herein the term "monoclonal antibody" refers to homogeneous
populations of
immunoglobulins which are capable of specifically binding to CPPs. Preferably,
antagonists of the
invention are derived from monoclonal antibodies specific for CPPs. Monoclonal
antibodies capable
of blocking, or neutralizing, CPPs are selected by their ability to inhibit a
biological activity of
CPPs.
The use of antibody fragments is also well known, e.g. Fab fragments: Tijssen,
Practice and
Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and Fv fragments:
Hochman et al.
Biochemistry, Vol. 12, pgs. 1130-1135 (1973), Sharon et al., Biochemistry,
Vol. 15, pgs. 1591-1594
(1976) and Ehrlich et al., U.S. Patent 4,355,023; and antibody half molecules:
Auditore-
Hargreaves, U.S. Patent 4,470,925.
Preferably, monoclonal antibodies, Fv fragments, Fab fragments, or other
binding
compositions derived from monoclonal antibodies of the invention have a high
affinity to CPPs. The
affinity of monoclonal antibodies and related molecules to CPPs may be
measured by conventional
techniques including plasmon resonance, ELISA, or equilibrium dialysis.
Affinity measurement by
plasmon resonance techniques may be caxried out, for example, using a BIAcore
2000 instrument
(Biacore AB, Uppsala, Sweden) in accordance with the manufacturer's
recommended protocol.
Preferably, affinity is measured by ELISA, for example, as described in U.S.
patent 6,235,883, or
Like reference. Preferably, the dissociation constant between CPPs and
monoclonal antibodies of the
invention is less than 10-5 molar. More preferably, such dissociation constant
is less than 10-g molar;
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still more preferably, such dissociation constant is less than 10-9 molar; and
most preferably, such
dissociation constant is in the range of 10-9 to 10-'1 molar.
In addition, the antibodies of the present invention are useful for detecting
CPPs. Such
detection methods are advantageously applied to diagnosis of cardiovascular
disorders, in particular,
coronary artery disease. The antibodies of the invention may be used in most
assays involving
antigen-antibody reactions. The assays may be homogeneous or heterogeneous. In
a homogeneous
assay approach, the sample can be a biological sample or fluid such as serum,
urine, whole blood,
lymphatic fluid, plasma, saliva, cells, tissue, and material secreted by cells
or tissues cultured in
vitro. The sample can be pretreated if necessary to remove unwanted materials.
The immunological
reaction usually involves the specific antibody, a labeled analyte, and the
sample suspected of
containing the antigen. The antigen can be directly labeled with any label
group described herein.
The signal arising from the label is modified, directly or indirectly, upon
the binding of the antibody
to the labeled analyte. Both immunological reaction and detection of the
extent thereof are carned out
in a homogeneous solution. Immunochemical labels which may be employed include
free radicals,
fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
In a heterogeneous assay approach, the reagents are usually the sample, the
specific
antibody, and means for producing a detectable signal. The specimen is
generally placed on a
support, such as a plate or a slide, and contacted with the antibody in a
liquid phase. The support is
then separated from the liquid phase and either the support phase or the
liquid phase is examined for
a detectable signal employing means for producing such signal or signal
producing system. The
signal is related to the presence of the antigen in the sample. Means for
producing a detectable signal
includes the use of radioactive labels, fluorescent compounds, enzymes, and so
forth. Exemplary
heterogeneous immunoassays are the radioimmunoassay, immunofluorescence
methods, enzyme-
linked immunoassays, and the like.
For a more detailed discussion of the above immunoassay techniques, see
"Enzyme-
Immunoassay," by Edward T. Maggio, CRC Press, Inc., Boca Raton, Fla., 1980.
See also, for
example, U.S. Pat. Nos. 3,690,834; 3,791,932; 3,817,837; 3,850,578; 3,853,987;
3,867,517;
3,901,654; 3,935,074; 3,984,533; 3,966,345; and 4,098,876, which listing is
not intended to be
exhaustive. Methods for conjugating labels to antibodies and antibody
fragments are well known in
the art. Such methods may be found in U.S. Pat. Nos. 4,220,450; 4,235,869;
3,935,974; and
3,966,345. Another example of a technique in which the antibodies of the
invention may be employed
is immunoperoxidase labeling. (Sternberger, Immunocytochemistry (1979) pp. 104-
169).
Alternatively, the antibodies may be bound to a radioactive material or to a
drug to form a
radiopharmaceutical or pharmaceutical, respectively. (Carrasquillo, et al.,
Cancer Treatment Reports
(1984) 68:317-328).
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One embodiment of an assay employing an antibody of the present invention
involves the use
of a surface to which the monoclonal antibody of the invention is attached.
The underlying structure
of the surface may take different forms, have different compositions and may
be a mixture of
compositions or laminates or combinations thereof. The surface may assume a
variety of shapes and
-forms and may have varied dimensions, depending on the manner of use and
measurement.
Illustrative surfaces may be pads, beads, discs, or strips which may be flat,
concave or convex.
Thickness is not critical, generally being from about 0.1 to 2 mm thick and of
any convenient
diameter or other dimensions. The surface typically will be supported on a
rod, tube, capillary,
fiber, strip, disc, plate, cuvette and will typically be porous and
polyfunctional or capable of being
polyfunctionalized so as to permit covalent binding of an antibody and permit
bonding of other
compounds which form a part of a means for producing a detectable signal. A
wide variety of
organic and inorganic polymers, both natural and synthetic, and combinations
thereof, may be
employed as the rriaterial for the solid surface. Illustrative polymers
include polyethylene,
polypropylene, poly(4-methylbutene), polystyrene, polymethracrylate,
polyethylene terephthalate),
rayon, nylon, polyvinyl butyrate), silicones, polyformaldehyde, cellulose,
cellulose acetate,
nitrocellulose, and latex. Other surfaces include paper, glasses, cerariiics,
metals, metaloids,
semiconductor materials, cements, silicates or the like. Also included are
substrates that form gels,
gelatins, lipopolysaccharides, silicates, agarose and polyacrylamides or
polymers which form several
aqueous phases such as dextrans, polyalkylene glycols (alkylene of 2 to 3
carbon atoms) or
surfactants such as phospholipids. The binding of the antibody to the surface
may be accomplished
by well known techniques, commonly available in the literature (see, for
example, "Immobilized
Enzymes," Ichiro Chibata, Press, New York (1978) and Cuatrecasas, J. Bio.
Chem., 245: 3059
(1970)). In carrying out the assay in accordance with this aspect of the
invention, the sample is
mixed with aqueous meditum and the medium is contacted with the surface having
an antibody bound
thereto. Labels may be included in the aqueous medium, either concurrently or
added subsequently
so as to provide a detectable signal associated with the surface. The means
for producing the
detectable signal can involve the incorporation of a labeled analyte or it may
involve the use of a
second monoclonal antibody having a label conjugated thereto. Separation and
washing steps will be
carried out as needed. The signal detected is related to the presence of CPP
in the sample. It is within
the scope of the present invention to include a calibration on the same
support. A particular
embodiment of an assay in accordance with the present invention, by way of
illustration and not
limitation, involves the use of a support such as a slide or a well of a petri
dish. The technique
involves fixing the sample to be analyzed on the support with an appropriate
fixing material and
incubating the sample on the slide with a monoclonal antibody. After washing
with an appropriate
buffer such as, for example, phosphate buffered saline, the support is
contacted with a labeled
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WO 2004/061096 PCT/EP2004/000027
specific binding partner for the antibody. After incubation as desired, the
slide is washed a second
time with an aqueous buffer and the determination is made of the binding of
the labeled monoclonal
antibody to the antigen. If the label is fluorescent, the slide may be covered
with a fluorescent
antibody mounting fluid on a cover slip and then examined with a fluorescent
microscope to
determine the extent of binding. On the other hand, the label can be an enzyme
conjugated to the
monoclonal antibody and the extent of binding can be determined by examining
the slide for the
presence of enzyme activity, which may be indicated by the formation of a
precipitate, color, etc. A
particular example of an assay utilizing the present antibodies is a double
determinant ELISA assay.
A support such as, e.g., a glass or vinyl plate, is coated with an antibody
specific for CPP by
conventional techniques. The support is contacted with the sample suspected of
containing CPP,
usually in aqueous medium. After an incubation period from 30 seconds to 12
hours, the support is
separated from the medium, washed to remove unbound CPP with, for example,
water or an aqueous
buffered medium, and contacted with an antibody specific for CPP, again
usually in aqueous
medium. The antibody is labeled with an enzyme directly or indirectly such as,
e.g., horseradish
peroxidase or alkaline phosphatase. After incubation, the support is separated
from the medium, and
washed as above. The enzyme activity of the support or the aqueous medium is
determined. This
enzyme activity is related to the amount of CPP in the sample.
The invention also includes kits, e.g., diagnostic assay kits, for carrying
out the methods
disclosed above. In one embodiment, the kit comprises in packaged combination
(a) a monoclonal
antibody more specifically defined above and (b) a conjugate of a specific
binding partner for the
above monoclonal antibody and a label capable of producing a detectable
signal. The reagents may
also include ancillary agents such as buffering agents and protein stabilizing
agents, e.g.,
polysaccharides and the like. The kit may further include, where necessary,
other members of the
signal producing system of which system the label is a member, agents for
reducing background
interference in a test, control reagents, apparatus for conducting a test, and
the like. In another
embodiment, the diagnostic kit comprises a conjugate of monoclonal antibody of
the invention and a
label capable of producing a detectable signal. Ancillary agents as mentioned
above may also be
present.
Further, an anti-CPP antibody (e.g., monoclonal antibody) can be used to
isolate CPPs by
standard techniques, such as affinity chromatography or immunoprecipitation.
For example, an anti-
CPP antibody can facilitate the purification of natural CPPs from cells and of
recombinantly
produced CPP expressed in host cells. Moreover, an anti-CPP antibody can be
used to isolate CPP to
aid in detection of low concentrations of CPP (e.g., in plasma, cellular
lysate or cell supernatant) or
in order to evaluate the abundance and pattern of expression of the CPP. Anti-
CPP antibodies can be
used diagnostically to monitor protein levels in tissue as part of a clinical
testing procedure, e.g., to
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determine the efficacy of a given treatment regimen. Detection can be
facilitated by coupling (i.e.,
physically linking) the antibody to a label group.
Pr oteirz Arrays
Detection, purification, and screening of the polypeptides of the invention
may be
accomplished using retentate chromatography (preferably, protein arrays or
chips), as described by
U.S. Patent 6225027 and U.S. Patent Application 20010014461, disclosures of
which are herein
incorporated by reference in their entireties. Briefly, retentate
chromatography describes methods in
which polypeptides (and/ or other sample components) are retained on an
adsorbent (e.g., array or
chip) and subsequently detected. Such methods involve (1) selectively
adsorbing polypeptides from a
sample to a substrate under a plurality of different adsorbent/eluant
combinations ("selectivity
conditions") and (2) detecting the retention of adsorbed polypeptides by
desorption spectrometry
(e.g., by mass spectrometry). In conventional chromatographic methods,
polypeptides are eluted off
of the adsorbent prior to detection. The coupling of adsorption chromatography
with detection by
desorption spectrometry provides extraordinary sensitivity, the ability to
rapidly analyze retained
components with a variety of different selectivity conditions, and parallel
processing of components
adsorbed to different sites (i.e., "affinity sites" or "spots") on the array
under different elution
conditions.
These methods are useful for: combinatorial, biochemical separation and
purification of the
CPPs; study of differential gene expression; detection of differences in
protein levels (e.g., for
diagnosis); and detection of molecular recognition events (e.g., for screening
and drug discovery).
Thus, this invention provides a molecular discovery and diagnostic device that
is characterized by the
inclusion of both parallel and multiplex polypeptide processing capabilities.
Polypeptides of the
invention and CPP-binding substances are preferably attached to a label group,
and thus directly
detected, enabling simultaneous transmission of two or more signals from the
same "circuit" (i.e.,
addressable "chip" location) during a single unit operation.
Detection of CPPs by mass spectrometry
In accordance with the present invention, any instrument, method, process,
etc. can be
utilized to determine the identity and abundance of proteins in a sample. A
preferred method of
obtaining identity is by mass spectrometry, where protein molecules in a
sample are ionized and then
the resultant mass and charge of the protein ions are detected and determined.
To use mass spectrometry to analyze proteins, it is preferred that the protein
be converted to
a gas-ion phase. Various methods of protein ionization are useful, including,
e.g., fast ion
bombardment (FAB), plasma desorption, laser desorption, thermal desorption,
preferably,
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electrospray ionization (ESI) and matrix-assisted laser desorption/ionization
(MALDI). Many
different mass analyzers are available for peptide and protein analysis,
including, but not limited to,
Time-of Flight (TOF), ion trap (ITMS), Fourier transform ion cyclotron (FTMS),
quadrupole ion
trap, and sector (electric and/or magnetic) spectrometers. See, e.g., U.S.
Pat. No. 5,572,025 for an
ion-trap MS. Mass analyzers can be used alone, or in combination with other
mass analyzers in
tandem mass spectrometers. In the latter case, a first mass analyzer can be
use to separate the protein
ions (precursor ion) from each other and determine the molecular weights of
the various protein
constituents in the sample. A second mass analyzer can be used to analyze each
separated
constituents, e.g., by fragmenting the precursor ions into product ions by
using, e.g. an inert gas.
Any desired combination of mass analyzers can be used, including, e.g., triple
quadrupoles, tandem
time-of flights, ion traps, and/or combinations thereof.
Different kinds of detectors can be used to detect the protein ions. For
example, destructive
detectors can be utilized, such as ion electron multipliers or cryogenic
detectors (e.g., U.S. Pat. No.
5,640,010). Additionally, non-destructive detectors can be used, such as ion
traps which are used as
ion current pick-up devices in quadrupole ion trap mass analyzers or FTMS.
For MALDI-TOF, a number of sample preparation methods can be utilized
including, dried
droplet (Karasand Hillenkamp, Anal. Chem., 60:2299-2301, 1988), vacuum-drying
(Winberger et
al., In Proceedings of the 41st ASMS Conference on Mass Spectrometry and
Allied Topics, San
Francisco, May 31-June 4, 1993, pp. 775a-b), crush crystals (Xiang et al.,
Rapid Comm. Mass
Spectrom., 8:199-204,1994), slow crystal growing (Xiang et al., Org. Mass
Spectrom, 28:1424-
1429, 1993); active film (Mock et al., Rapid Comm. Mass Spectrom.,6:233-238,
1992; Bai et al.,
Anal. Chem., 66:3423-3430, 1994), pneumatic spray (Kochling et al.,
Proceedings of the 43rd
ASMS Conference on Mass Spectrometry and Allied Topics; Atlanta, GA, May 21-
26, 1995,
p 1225); electrospray (Hensel et al., Proceedings of the 43rd ASMS Conference
on Mass
Spectrometry and Allied Topics; Atlanta, GA, May 21 -26, 1995, p947); fast
solvent evaporation
(Vorm et al., Anal. Chem., 66:3281-3287, 1994); sandwich (Li et al., J. Am.
Chem. Soc., 11
8:11662-11663,1996); and two-layer methods (Dal et al., Anal. Chem., 71:1087-
1091, 1999). See
also, e.g., Liang et al., Rapid Common. Mass Spectrom., 10: 1219-1226, 1996;
van Adrichemet al.,
Anal. Chem., 70:923-930, 1998.
For MALDI analysis, samples are prepared as solid-state co-crystals or thin
filins by mixing
them with an energy absorbing compound or colloid (the matrix) in the liquid
phase, and ultimately
drying the solution to the solid state upon the surface of an inert probe. In.
some cases an energy
absorbing molecule (EAM) is an integral component of the sample presenting
surface. Regardless of
EAM application strategy, the probe contents are allowed to dry to the solid
state prior to
introduction into the laser desorption/ionization time-of flight mass
spectrometer (LDIMS).
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Ion detection in TOF mass spectrometry is typically achieved with the use of
electro-
emissive detectors such as electron multipliers (EMP) or microchannel plates
(MCP). Both of these
devices function by converting primary incident charged particles into a
cascade of secondary,
tertiary, quaternary, etc. electrons. The probability of secondary electrons
being generated by the
impact of a single incident charged particle can be taken to be the ion to-
electron conversion
efficiency of this charged particle (or more simply, the conversion
efficiency). The total electron
yield for cascading events when compared to the total number of incident
charged particles is
typically described as the detector gain. Because generally the overall
response time of MCPs is far
superior to that of EMPs, MCPs are the preferred electro-emissive detector for
enhancing
mass/charge resolving power. However, EMPs function well for detecting ion
populations of
disbursed kinetic energies, where rapid response time and broad frequency
bandwidth are not
necessary.
In a preferred aspect, for the analysis of digested proteins, a liquid-
chromatography tandem
mass spectrometer (LC-TMS) is used. This system provides an additional stage
of sample separation
via use of a liquid chromatograph followed by tandem mass spectrometry.
In preferred aspects, a protein eluted from a column according to the system
described in
Example 1 is analyzed using both MS and MS-MS analysis. For example, a small
portion of intact
proteins eluting from RP2 may be diverted to online detection using LC-ESI MS.
The proteins are
aliquoted on a number of plates allowing digestion or not with trypsin,
preparation for MALDI-MS
as well as for ESI-MS, as well as preparation of the MALDI plates with
different matrices. The
methods thus allow, in addition to information on intact mass, to conduct an
analysis by both peptide
mass fingerprinting and MS-MS techniques.
The methods described herein of separating and fractionating proteins provide
individual
proteins or fractions containing small numbers of distinct proteins. These
proteins can be identified
by mass spectral determination of the molecular masses of the protein and
peptides resulting from the
fragmentation thereof. Making use of available information in protein sequence
databases, a
comparison can be made between proteolytic peptide mass patterns generated in
silico, and
experimentally observed peptide masses. A "hit-list" can be compiled, ranking
candidate proteins in
the database, based on (among other criteria) the number of matches between
the theoretical and
experimental proteolytic fragments. Several Web sites are accessible that
provide software for
protein identification on-line, based on peptide mapping and sequence database
search strategies
(e.g., http:l/www.expasy.ch). Methods of peptide mapping and sequencing using
MS are described in
WO 95/252819, U.S. Pat. No. 5,538,897, U.S. Pat. No. 5,869,240, U.S. Pat. No.
5,572,259, and
U.S. Pat. No. 5,696,376. See, also, Yates, J. Mass Spec., 33:1 (1998).
Data collected from a mass spectrometer typically comprises the intensity and
mass to
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charge ratio for each detected event. Spectral data can be recorded in any
suitable form, including,
e.g., in graphical, numerical, or electronic formats, either in digital or
analog form. Spectra are
preferably recorded in a storage medium, including, e.g., magnetic, such as
floppy disk, tape, or hard
disk; optical, such as CD-ROM or laser-disc; or, ROM-CHIPS.
The mass spectrum of a given sample typically provides information on protein
intensity,
mass to charge ratio, and molecular weight. In preferred embodiments of the
invention, the molecular.
weights of proteins in the sample are used as a matching criterion to query a
database. The molecular
weights are calculated conventionally, e.g., by subtracting the mass of the
ionizing proton for singly-
charged protonated molecular ions, by multiplying the measured mass/charge
ratio by the number of
charges for multiply-charged ions and subtracting the number of ionizing
protons.
Various databases are useful in accordance with the present invention. Useful
databases
include, databases containing genomic sequences, expressed gene sequences,
and/or expressed
protein sequences. Preferred databases contain nucleotide sequence-derived
molecular masses of
proteins present in a known organism, organ, tissue, or cell-type. There are a
number of algorithms
to identify open reading frames (ORF) and convert nucleotide sequences into
protein sequence and
molecular weight information. Several publicly accessible databases are
available, including, the
SwissPROT/TrEMBL database (http://www.expasy.ch).
Typically, a mass spectrometer is equipped with commercial software that
identifies peaks
above a certain threshold level, calculates mass, charge, and intensity of
detected ions. Correlating
molecular weight with a given output peak can be accomplished directly from
the spectral data, i.e.,
where the charge on an ion is one and the molecular weight is therefore equal
to the numerator value
minus the mass of the ionizing proton. However, protein ions can be complexed
with various
counter-ions and adducts, such as N, C, and K'. In such a case, it would be
expected that a given
protein ion would exhibit multiple peaks, such as a triplet, representing
different ionic states (or
species) of the same protein. Thus, it may be necessary to analyze and process
spectral data to
determine families of peaks arising from the same protein. This analysis can
be carried out
conventionally, e.g., as described by Mann et al., anal. Chem., 61:1702-1708
(1989).
In matching a molecular mass calculated from a mass spectrometer to a
molecular mass
predicted from a database, such as a genomic or expressed gene database, post-
translation
processing may have to be considered. There are various processing events
which modify protein
structure, including, proteolytic processing, removal of N terminal
methionine, acetylation,
methylation, glycosylation, phosphorylation, etc.
A database can be queried for a range of proteins matching the molecular mass
of the
unknown. The.range window can be determined by the accuracy of the instrument,
the method by
which the sample was prepared, etc. Based on the number of hits (where a hit
is match) in the
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spectrum, the unknown protein or peptide is identified or classified.
Methods of identifying one or more CPP by mass spectrometry are useful for
diagnosis and
prognosis of cardiovascular disorders. Preferably, such methods are used to
detect one or more CPP
present in human plasma. Exemplary techniques are described in U.S. Patent
Applications
02/0060290, 02/0137106, 02/0138208, 02!0142343, 02/0155509, disclosures of
which are
incorporated by reference in their entireties.
Diagnostic aid Prognostic Uses
The nucleic acid molecules, proteins, protein homologues, and antibodies
described herein
can be used in one or more of the following methods: diagnostic assays,
prognostic assays,
monitoring clinical trials, screening assays, and pharmacogenetics as further
described herein.
The invention provides diagnostic and prognostic assays for detecting CPP
nucleic acids and
proteins, as further described. Also provided are diagnostic and prognostic
assays for detecting
interactions between CPPs and CPP target molecules, particularly natural
agonists and antagonists.
The present invention provides methods for identifying polypeptides that are
differentially
expressed between two or more samples. "Differential expression" refers to
differences in the
quantity or quality of a polypeptide between samples. Such differences could
result at any stage of
protein expression from transcription through post-translational modification.
For example, using
protein array methods, two samples are bound to affinity spots on different
sets of adsorbents (e.g.,
chips) and recognition maps are compared to identify polypeptides that are
differentially retained by
the two sets of adsorbents. Differential retention includes quantitative
retention as well as qualitative
differences in the polypeptide. For example, differences in post translational
modification of a
protein can result in differences in recognition maps detectable as
differences in binding
characteristics (e.g., glycosylated proteins bind differently to lectin
adsorbents) or differences in
mass (e.g., post-translational cleavage products). In certain embodiments, an
adsorbent can have an
array of affinity spots selected for a combination of markers diagnostic for a
disease or syndrome.
Differences in polypeptide levels between samples (e.g., differentially
expressed CPPs in
plasma samples) can be identified by exposing the samples to a variety of
conditions for analysis by
desorption spectrometry (e.g., mass spectrometry). Unknown proteins can be
identified by detecting
physicochemical characteristics (e.g., molecular mass), and this information
can be used to search
databases for proteins having similar profiles.
Preferred methods of detecting a CPP utilize mass spectrometry techniques.
Such methods
provide information about the size and character of the particular CPP isoform
that is present in a
sample, e.g., a biological sample submitted for diagnosis or prognosis. Mass
spectrometry techniques
are detailed in the section titled "Detection of CPPs by mass spectrometry".
Example 1 outlines a
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preferred detection scheme, wherein a biological sample is separated by
chromatography before
characterization by mass spectrometry. The invention provides a method of
detecting a CPP in a
biological sample comprising the steps of fractionating a biological sample
(e.g., plasma, serum,
lymph, cerebrospinal fluid, cell lysate of a particular tissue) by at least
one chromatographic step;
subjecting a fraction to mass spectrometry; and comparing the characteristics
of polypeptide species
observed in mass spectrometry with known characteristics of CPP polypeptides
(e.g., CPP 6-7, as
disclosed in Figure 1).
The isolated nucleic acid molecules of the invention can be used, for example,
to detect CPP
mRNA (e.g., in a biological sample) or a genetic alteration in a CPP-encoding
gene, and to modulate
a CPP activity, as described further below. The CPP can be used to treat
disorders characterized by
insufficient production of a CPP or by excessive production of a CPP target
molecule. In addition,
the CPPs can be used to screen for naturally occurring CPP target molecules,
to screen for drugs or
compounds which modulate, preferably activate CPP activity, as well as to
treat disorders
characterized by insufficient production of CPP or production of CPP forms
which have decreased
or aberrant activity compared to wild type CPP. Moreover, the anti- CPP
antibodies of the invention
can be used to detect and isolate CPP, regulate the bioavailability of CPP,
and modulate CPP
activity.
Accordingly one embodiment of the present invention involves a method of use
(e.g., a
diagnostic assay, prognostic assay, or a prophylactic/therapeutic method of
treatment) wherein a
molecule of the present invention (e.g., a CPP, CPP nucleic acid, or CPP
inhibitor or activator) is
used, for example, to diagnose, prognose and/or treat a disorder in which any
of the aforementioned
CPP activities is indicated. In another embodiment, the present invention
involves a method of use
(e.g., a diagnostic assay, prognostic assay, or a prophylactic/therapeutic
method of treatment)
wherein a molecule of the present invention is used, for example, for the
diagnosis, prognosis, and/or
treatment of subjects, preferably a human subject, in which any of the
aforementioned activities is
pathologically perturbed. In a preferred embodiment, the methods of use
involve administering to a
subject, preferably a human subject, a molecule of the present invention for
the diagnosis, prognosis,
and/or therapeutic treatment. In another embodiment, the methods of use
involve administering to a
human subject a molecule of the present invention.
For example, the invention encompasses a method of determining whether a CPP
is
expressed within a biological sample comprising: a) contacting said biological
sample with: i) a
polynucleotide that hybridizes under stringent conditions to a CPP nucleic
acid; or ii) a detectable
polypeptide (e.g. antibody) that selectively binds to a CPP; and b) detecting
the presence or absence
of hybridization between said polynucleotide and an RNA species within said
sample, or the presence
or absence of binding of said detectable polypeptide to a polypeptide within
said sample. Detection
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of said hybridization or of said binding indicates that said CPP is expressed
within said sample.
Preferably, the polynucleotide is a primer, and wherein said hybridization is
detected by detecting the
presence of an amplification product comprising said primer sequence, or the
detectable polypeptide
is an antibody.
In certain embodiments, detection involves the use of a probe/primer in a
polymerase chain
reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, the
disclosures of which are
incorporated herein by reference in their entireties), such as anchor PCR or
RACE PCR, or,
alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegren et al.
(1988) Science 241:1077-
1080; and Nakazawa et al. (1994) PNAS 91:360-364, the disclosures of which are
incorporated
herein by reference in their entireties), the latter of which can be
particularly useful for detecting
point mutations in the CPP-encoding-gene (see Abravaya et al. (1995) Nucleic
Acids Res. 23:675-
682, the disclosure of which is incorporated herein by reference in its
entirety).
Also envisioned is a method of determining whether a mammal, preferably
hu'rnan, has an
elevated or reduced level of expression of a CPP, comprising: a) providing a
biological sample from
said mammal; and b) comparing the amount of a CPP or of a CPP RNA species
encoding a CPP
within said biological sample with a.level detected in or expected from a
control sample. An
increased amount of said CPP or said CPP RNA species within said biological
sample compared to
said level detected in or expected from said control sample indicates that
said mammal has an
elevated level of CPP expression, and a decreased amount of said CPP or said
CPP RNA species
within said biological sample compared to said level detected in or expected
from said control sample
indicates that said mammal has a reduced level of expression of a CPP.
The present invention also pertains to the field of predictive medicine in
which diagnostic
assays, prognostic assays, and monitoring clinical trials are used for
prognostic purposes to thereby
treat an individual prophylactically. Accordingly, one aspect of the present
invention relates to
diagnostic assays for determining CPP and/or nucleic acid expression as well
as CPP activity, in the
context of a biological sample (e.g., blood, plasma, cells, tissue) to thereby
determine whether an
individual is afflicted with a disease or disorder, or is at risk of
developing a disorder, associated
with aberrant CPP expression or activity. The invention also provides for
prognostic (or predictive)
assays for determining whether an individual is at risk of developing a
disorder associated with a
CPP, nucleic acid expression or activity. For example, mutations in a CPP-
encoding gene can be
assayed in a biological sample. Such assays can be used for prognostic or
predictive purpose to
thereby prophylactically .treat an individual prior to the onset of a disorder
characterized by or
associated with CPP expression or activity.
The term "biological sample" is intended to include tissues, cells and
biological fluids
isolated from an individual, as well as tissues, cells and fluids present
within an individual. That is,
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the detection methods of the invention can be used to detect a CPP mRNA,
protein, or genomic DNA
in a biological sample in vitro as well as in vivo. Preferred biological
samples are biological fluids
such as lymph, cerebrospinal fluid, blood, and especially blood plasma. For
example, in vitro
techniques for detection of a CPP mRNA include Northern hybridizations and in
situ hybridizations.
In vitro techniques for detection of a CPP include mass spectrometry, Enzyme
Linked Immuno
Sorbent Assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro
techniques for detection of a CPP-encoding genomic DNA include Southern
hybridizations.
Furthermore, in vivo techniques for detection of a CPP include introducing
into an individual a
labeled anti- CPP antibody.
In preferred embodiments, the subject methods can be characterized by
generally comprising
detecting, in a tissue sample of the individual (e.g. a human patient), the
presence or absence of a
genetic lesion characterized by at least one of (i) a mutation of a gene
encoding one of the subject
CPP or (ii) the mis-expression of a CPP-encoding gene. To illustrate, such
genetic lesions can be
detected by ascertaining the existence of at least one of (i) a deletion of
one or more nucleotides from
the CPP-encoding gene, (ii) an addition of one or more nucleotides to the
gene, (iii) a substitution of
one or more nucleotides of the gene, (iv) a gross chromosomal rearrangement or
amplification of the
gene, (v) a gross alteration in the level of a messenger RNA transcript of the
gene, (vi) aberrant
modification of the gene, such as of the methylation pattern of the genomic
DNA, (vii) the presence
of a non-wild type splicing pattern of a messenger RNA transcript of the gene,
and (viii) reduced
level of expression, indicating lesion in regulatory element or reduced
stability of a CPP-encoding
transcript.
In yet another exemplary embodiment, aberrant methylation patterns of a CPP
nucleic acid
can be detected by digesting genomic DNA from a patient sample with one or
more restriction
endonucleases that are sensitive to rnethylation and for which recognition
sites exist in the CPP-
encoding gene (including in the flanking and intronic sequences). See, for
example, Buiting et al.
(1994) Human Mol Genet 3:893-895. Digested DNA is separated by gel
electrophoresis, and
hybridized with probes derived from, for example, genomic or cDNA sequences.
The methylation
status of the CPP-encoding gene can be determined by comparison of the
restriction pattern
generated from the sample DNA with that for a standard of known methylation.
In. yet another embodiment, a diagnostic assay is provided which detects the
ability of a CPP
to bind to a cell surface or extracellular protein. For instance, it will be
desirable to detect CPP
mutants which, while expressed at appreciable levels in the cell, are
defective at binding a CPP target
protein (having either diminished or enhanced binding affinity for the
target). Such mutants may
axise, for example, from mutations, e.g., point mutants, which may be
impractical to detect by the
diagnostic DNA sequencing techniques or by the immunoassays described above.
The present
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invention accordingly further contemplates diagnostic screening assays which
generally comprise
cloning one or more CPP-encoding gene from the sample tissue, and expressing
the cloned genes
under conditions which permit detection of an interaction between that
recombinant gene product and
a target protein. As will be apparent from the description of the various
assays set forth herein under
the Section entitled "Drug screening assays", a wide variety of techniques can
be used to determine
the ability of a CPP to bind to other components. These techniques can be used
to detect mutations in
a CPP-encoding gene which give rise to mutant proteins with a higher or lower
binding affinity for a
CPP target protein relative to the wild-type CPP. Conversely, by switching
which of the CPP target
protein and CPP is the "bait" and which is derived from the patient sample,
the subject assay can
also be used to detect CPP target protein mutants which have a higher or lower
binding affinity for a
CPP relative to a wild type form of that CPP target protein.
In an exemplary embodiment, a target protein can be provided as an immobilized
protein (a
"target"), such as by use of GST fusion proteins and glutathione treated
microtiter plates as
described herein.
In another embodiment, the methods further involve obtaining a control
biological sample
from a control subject, contacting the control sample with a compound or agent
capable of detecting
a CPP, mRNA, or genomic DNA, such that the presence of a CPP, mRNA or genomic
DNA is
detected in the biological sample, and comparing the presence of a CPP, mRNA
or genomic DNA in
the control sample with the presence of a CPP, mRNA or genomic DNA in the test
sample. The
invention also encompasses kits for detecting the presence of a CPP, mRNA or
genomic DNA in a.
biological sample. For example, the kit can comprise: a labeled compound or
agent capable of
detecting a CPP, mRNA or genomic DNA in a biological sample; means for
determining the amount
of a CPP in the sample; and means for comparing the amount of CPP in the
sample with a standard.
The compound or agent can be packaged in a suitable container. The kit can
further comprise
instructions for using the kit to detect CPP or nucleic acid.
Drug screening assays
The invention provides a method (also referred to herein as a "screening
assay") for
identifying candidate modulators (e.g., small molecules, peptides, antibodies,
peptidomimetics or
other drugs) which bind to CPPs, have an inhibitory or activating effect on,
for example, CPP
expression or preferably CPP biological activity, or have an inhibitory or
activating effect on, for
example, the activity of a CPP target molecule. In some embodiments small
molecules can be
generated using combinatorial chemistry or can be obtained from a natural
products library. Assays
may be cell based or non-cell based assays. Drug screening assays may be
binding assays or more
preferentially functional assays; as further described.
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When the invention is used for drug development, e.g., to determine the
ability of a CPP
polypeptide or candidate modulator to induce an anti-cardiovascular disorder
response, the body
fluid analyzed for the level of at least one CPP is preferably from a non-
human mammal. The non-
human mammal is preferably one in which the induction of an anti-
cardiovascular disorder response
by endogenous andlor exogenous agents is predictive of the induction of such a
response in a human.
Rodents (mice, rats, etc.) and primates are particularly suitable for use in
this aspect of the
invention.
Agents that are found, using screening assays as further described herein, to
modulate CPP
activity by at least 5%, more preferably by at least 10%, still more
preferably by at least 30%, still
more preferably by at least 50%, still more preferably by at least 70%, even
more preferably by at
least 90 %, may be selected for further testing as a prophylactic and/or
therapeutic anti-
cardiovascular disease agent.
Agents that are found to increase CPP activity may be used, for example, to
reduce the
symptoms of a cardiovascular disorder alone or in combination with other
appropriate agents or
treatments. Agents found to inhibit CPP activity may be used, for example, to
modulate treatment
regimens for cardiovascular disorders.
In another aspect, agents that are found, using screening assays as further
described herein,
to modulate CPP expression by at least 5%, more preferably by at least 10%,
still more preferably
by at least 30%, still more preferably by at least 50%, still more preferably
by at least 70%, even
more preferably by at least 90 %, may be selected for further testing as a
prophylactic and/or
therapeutic anti-cardiovascular disease agent.
Protein array methods are useful for screening and drug discovery. For
example, one
member of a receptor/ ligand pair is docked to an adsorbent, and its ability
to bind the binding
partner is determined in the presence of the test substance. Because of the
rapidity with which
adsorption can be tested, combinatorial libraries of test substances can be
easily screened for their
ability to modulate the interaction. In preferred screening methods, CPPs are
docked to the
adsorbent. Binding partners are preferably labeled, thus enabling detection of
the interaction.
Alternatively, in certain embodiments, a test substance is docked to the
adsorbent. The
polypeptides of the invention are exposed to the test substance and screened
for binding. Preferred
test substances include substances correlated with a disease or disorder, such
as a protein, lipid, or
endocrine factor differentially present in disease (preferably, a
cardiovascular disease).
In other embodiments, an assay is a cell-based assay in which a cell which
expresses a CPP
or biologically active portion thereof is contacted with a test compound and
the ability of the test
compound to inhibit, activate, or increase CPP activity determined.
Determining the ability of the test
compound to inhibit, activate, or increase CPP activity can be accomplished by
monitoring the
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bioactivity of the CPP or biologically active portion thereof. The cell, for
example, can be of
mammalian origin, insect origin, bacterial origin or a yeast cell.
In one embodiment, the invention provides assays for screening candidate or
test compounds
which are target molecules of a CPP or biologically active portion thereof. In
another embodiment,
the invention provides assays for screening candidate or test compounds which
bind to or modulate
the activity of a CPP or biologically active portion thereof. The test
compounds of the present
invention can be obtained using any of the numerous approaches in
combinatorial library methods
known in the art, including: biological libraries; spatially addressable
parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution; the ' one-
bead one-compound'
library method; and synthetic library methods using affinity chromatography
selection. The
biological library approach is used with peptide libraries, while the other
four approaches are
applicable to peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam, I~. S.
(1997) Anticancer Drug Des. 12:145, the disclosure of which is incorporated
herein by reference in
its entirety).
Examples of methods for the synthesis of molecular libraries can be found in
the art, for
example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et
al. (1994) Proc.
Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al.
(1993) Science 261:1303; Garrell et al. (1994) Angew. Chem. Int. Ed. Engl.
33:2059 and 2061; and
in Gallop et al. (1994) J. Med. Chem. 37:1233, the disclosures of which are
incorporated herein by
reference in their entireties.
Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques
13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993)
Nature 364:555-
556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No.
'409), plasmids (Cull
et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and
Smith (1990) Science
249:386-390); (Devin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc.
Natl. Acad. Sci.
87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).
Determining the ability of the test compound to inhibit or increase CPP
activity can also be
accomplished, for example, by coupling the CPP or biologically active portion
thereof with a label
group such that binding of the CPP or biologically active portion thereof to
its cognate target
molecule can be determined by detecting the labeled CPP or biologically active
portion thereof in a
complex. For example, the extent of complex formation may be measured by
immunoprecipitating
the complex or by performing gel electrophoresis.
It is also within the scope of this invention to determine the ability of a
compound to interact
with its cognate target molecule without the labeling of any of the
interactants. For example, a
microphysiometer can be used to detect the interaction of a compound with its
cognate target
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WO 2004/061096 PCT/EP2004/000027
molecule without the labeling of either the compound or the target molecule.
McConnell, H. M. et al.
(1992) Science 257:1906-1912, the disclosure of which is incorporated by
reference in its entirety.
A microphysiometer such as a cytosensor is an analytical instrument that
measures the rate at which
a cell acidifies its environment using a Light-Addressable Potentiometric
Sensor (LAPS). Changes in
this acidification rate can be used as an indicator of the interaction between
compound and receptor.
In a preferred embodiment, the assay comprises: contacting a cell which
expresses a CPP or
biologically active portion thereof with a target molecule to form an assay
mixture, contacting the
assay mixture with a test compound, and determining the ability of the test
compound to inhibit or
increase the activity of the CPP or biologically active portion thereof.
Determining the ability of the
test compound to inhibit or increase the activity of the CPP or biologically
active portion thereof .
comprises: determining the ability of the test compound to inhibit or increase
a biological activity of
the CPP expressing cell (e.g., interaction with a CPP target molecule, as
discussed above)
In another preferred embodiment, the assay comprises contacting a cell which
is responsive
to a CPP or biologically active portion thereof with a CPP or biologically
active portion thereof, to
form an assay mixture, contacting the assay mixture with a test compound, and
determining the
ability of the test compound to modulate the activity of the CPP or
biologically active portion
thereof. Determining the ability of the test compound to modulate the activity
of the CPP or
biologically active portion thereof comprises determining the ability of the
test compound to
modulate a biological activity of the CPP-responsive cell.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a CPP target molecule (i.e. a molecule with which CPPs interact)
with a test compound
and determining the ability of the test compound to modulate (e.g. stimulate
or inhibit) the activity of
the CPP target molecule. Determining the ability of the test compound to
modulate the activity of a
CPP target molecule can be accomplished, for example, by assessing the
activity of a target
molecule, or by assessing the ability of the CPP to bind to or interact with
the CPP target molecule.
Determining the ability of the CPP to bind to or interact with a CPP target
molecule, for
example, can be accomplished by one of the methods described above for
directly or indirectly
determining binding. In a preferred embodiment, the assay includes contacting
the CPP or
biologically active portion thereof with a known compound which binds said CPP
(e.g., a CPP
antibody or target molecule) to form an assay mixture, contacting the CPP with
a test compound
before or after said known compound, and determining the ability of the test
compound to interact
with the CPP. Determining the ability of the test compound to interact with a
CPP comprises
determining the ability of the test compound to preferentially bind to the CPP
or biologically active
portion thereof as compared to the known compound. Determining the ability of
the CPP to bind to
a CPP target molecule can also be accomplished using a technology such as real-
time Biornolecular
CA 02512629 2005-07-05
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Interaction Analysis (BIA). Sjolander, S. and LTrbaniczky, C. (1991) Anal.
Chem. 63:233-2345 and
Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705, the disclosures of
which are incorporated
herein by reference in their entireties. As used herein, "BIA" is a technology
for studying biospecific
interactions in real time, without labeling any of the interactants (e.g.,
BIAcore). Changes in the
optical phenomenon of surface plasmon resonance (SPR) can be used as an
indication of real time
reactions between biological molecules.
In another embodiment, the assay is a cell-free assay in which a CPP or
biologically active
portion thereof is contacted with a test compound and the ability of the test
compound to modulate
(e.g., stimulate or inhibit) the activity of the CPP or biologically active
portion thereof is determined.
In a preferred embodiment, determining the ability of the CPP to modulate or
interact with a CPP
target molecule can be accomplished by determining the activity of the target
molecule. For example,
the activity of the target molecule can be determined by contacting the target
molecule with the CPP
or a fragment thereof and measuring induction of a cellular second messenger
of the target (e.g.,
cAMP, STAT3, Akt, intracellular Ca2+, diacylglycerol, IP3, etc.), detecting
catalytic/enzymatic
activity of the target for an appropriate substrate, detecting the induction
of a reporter gene
'.;(comprising a target-responsive regulatory element operatively linked to a
nucleic acid encoding a
detectable maxker, e.g., luciferase), or detecting a target-regulated cellular
response, for example,
signal transduction or protein:protein interactions.
The cell-free assays of the present invention are amenable to use of both
soluble and/or
membrane-bound forms of isolated proteins (e.g. CPPs or biologically active
portions thereof or
molecules to which CPPs targets bind). In the case of cell-free assays in
which a membrane-bound
form an isolated protein is used it may be desirable to utilize a solubilizing
agent such that the
membrane-bound form of the isolated protein is maintained in solution.
Examples of such
solubilizing agents include non-ionic detergents such as n-octylglucoside, n-
dodecylglucoside, n-
dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide,
Triton TM X-100,
Triton TM X-114, Thesit TM, Isotridecypoly(ethylene glycol ether)n,3-[(3-
cholamidopropyl)dimethylanuninio]- 1-propane sulfonate (CHAPS), 3-[(3-
cholamidopropyl)dimethylarnminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-
dodecyl=N,N-
dimethyl-3-ammonio-1-propane sulfonate.
In more than one embodiment of the above assay methods of the present
invention, it may be
desirable to immobilize either a CPP or its target molecule to facilitate
separation of cornplexed from
uncomplexed forms of one or both of the proteins, as well as to accommodate
automation of the
assay. Binding of a test compound to a CPP, or interaction of a CPP with a
target molecule in the
presence and absence of a candidate compound, can be accomplished in any
vessel suitable for
containing the reactants and by any immobilization protocol described herein.
Alternatively, the
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complexes can be dissociated from the matrix, and the level of CPP binding or
activity determined
using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening
assays of the invention. For example, either a CPP or a CPP target molecule
can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated CPP or target
molecules can be
prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known
in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the
wells of streptavidin-
coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive
with CPP or target
molecules but which do not interfere with binding of the CPP to its target
molecule can be
derivatized to the wells of the plate, and unbound target or CPP trapped in
the wells by antibody
conjugation. Methods for detecting such complexes, in addition to those
described above for the
GST-immobilized complexes, include immunodetection of complexes using
antibodies reactive with
the CPP or target molecule, as well as enzyme-linked assays which rely on
detecting an enzymatic
activity associated with the CPP or target molecule.
In another embodiment, modulators of CPP expression are identified in a method
wherein a
cell is contacted with a candidate compound and the expression of CPP mRNA or
protein in the cell
is determined. The level of expression of CPP mRNA or protein in the presence
of the candidate
compound is compared to the level of expression of CPP mRNA or protein in the
absence of the
candidate compound. The candidate compound can then be identified as a
modulator of CPP
expression based on this comparison. For example, when expression of CPP mRNA
or protein is
greater (statistically significantly greater) in the presence of the candidate
compound than in its
absence, the candidate compound is identified as a stimulator of CPP mRNA or
protein expression.
Alternatively, when expression of CPP mRNA or protein is less (statistically
significantly less) in the
presence of the candidate compound than in its absence, the candidate compound
is identified as an
inhibitor of CPP mRNA or protein expression. The level of CPP rnRNA or protein
expression in the
cells can be determined by methods described herein for detecting CPP mRNA or
protein.
In yet another aspect of the invention, the CPP can be used as "bait proteins"
in a two-hybrid
assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.
(1993) Cell 72:223
232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)
Biotechniques
14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent W094/10300,
the disclosures
of which are incorporated herein by reference in their entireties), to
identify other proteins, which
bind to or interact with CPPs ("CPP-binding proteins" or "CPP-by") and are
involved in CPP
activity. Such CPP-binding proteins are also likely to be involved in the
propagation of signals by the
CPP or CPP targets as, for example, downstream elements of a CPP-mediated
signaling pathway.
Alternatively, such CPP-binding proteins are likely to be CPP inhibitors.
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The two-hybrid system is based on the modular nature of most transcription
factors, which
consist of separable DNA-binding and activation domains. Briefly, the assay
utilizes two difFerent
DNA constructs. In one construct, the gene that codes for a CPP or a fragment
thereof is fused to a
gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-4). In the other
construct, a DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein
("prey" or "sample") is fused to a gene that codes for the activation domain
of the known
transcription factor. If the "bait" and the "prey" proteins are able to
interact, in vivo, forming a CPP
-dependent complex, the DNA-binding and activation domains of the
transcription factor are brought
into close proximity. This proximity allows transcription of a reporter gene
(e.g., LacZ) which is
operably linked to a transcriptional regulatory site responsive to the
transcription factor. Expression
of the reporter gene can be detected and cell colonies containing the
functional transcription factor
can be isolated and used to obtain the cloned gene which encodes the protein
which interacts with the
CPP.
This invention further pertains to novel agents identified by the above-
described screening
assays and to processes for producing such agents by use of these assays.
Accordingly, in one
embodiment, the present invention includes a compound or agent obtainable by a
method comprising
the steps of any one of the aforementioned screening assays (e.g., cell-based
assays or cell-free
assays).
Accordingly, it is within the scope of this invention to further use an agent
identified as
described herein in an appropriate animal model. For example, an agent
identified as described herein
(e.g., a CPP modulating agent, or a CPP -binding partner) can be used in an
animal model to
determine the efficacy, toxicity, or side effects of treatment with such an
agent. Alternatively, an
agent identified as described herein can be used in an animal model to
determine the mechanism of
action of such an agent. Furthermore, this invention pertains to uses of novel
agents identified by the,
above-described screening assays for treatments as described herein.
Preferably, such agents activate
or enhance CPP biological activity.
The present invention also pertains to uses of novel agents identified by the
above-described
screening assays for diagnoses, prognoses, prevention, and treatments as
described herein.
Accordingly, it is within the scope of the present invention to use such
agents in the design,
formulation, synthesis, manufacture, and/or production of a drug or
pharmaceutical composition for
use in diagnosis, prognosis, or treatment, as described herein. For example,
in one embodiment, the
present invention includes a method of synthesizing or producing a drug or
pharmaceutical
composition by reference to the structure and/or properties of a compound
obtainable by one of the
above-described screening assays. For example, a drug or pharmaceutical
composition can be
synthesized based on the structure and/or properties of a compound obtained by
a method in which a
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cell which expresses a CPP target molecule is contacted with a test compound
and the ability of the ,
test compound to bind to, or modulate the activity of, the CPP target molecule
is determined. In
another exemplary embodiment, the present invention includes a method of
synthesizing or producing
a drug or pharmaceutical composition based on the structure andlor properties
of a compound
obtainable by a method in which a CPP or biologically active portion thereof
is contacted with a test
compound and the ability of the test compound to bind to, or modulate (e.g.,
stimulate or inhibit) the
activity of, the CPP or biologically active portion thereof is determined.
Animal based drug sc~ee~ihg
It is also advantageous to carry out drug screening assays ih vivo. In vivo
screening assays
are carried out in nonhuman animals to discover effective CPP modulators that
may play a role in
cardiovascular disease. Animal-based model systems of cardiovascular disease
include, but are not
limited to, non-recombinant animals and transgenic animals.
Non-recombinant animal models for cardiovascular disease may include, for
example,
genetic models. Such genetic cardiovascular disease models include apoB or
apoR deficient pigs
(Rapacz, et al., 1986, Science 234:1573-1577) and Watanabe heritable
hyperlipidemic (V~HI~L)
rabbits (Kita et al., 1987, Proc. Natl. Acad. Sci U.S.A. 84: 5928-5931). Non-
recombinant, non-
genetic animal models of atherosclerosis may include, for example, pig,
rabbit, or rat models in
which the animal has been exposed to either chemical.wounding through dietary
supplementation of
LDL, or mechanical wounding through balloon catheter angioplasty, for example.
As indicated in the prior art (Ferns, G. A. A. et al. (1991) Science, 253:1129-
1132) the rat
carotid artery injury model of restenosis can be a useful indication of
potential therapeutic action. An
example of this method is described in US Patent 6500859, the disclosure of
which is incorporated
herein by reference. Briefly, the protocol approved by the National Institute
on Aging Animal Care
and use Committee used 6 month Wistar rats from the GRC colony anesthetized
with 20 mg/kg body
weight pentobarbital, 2 rng/kg body weight ketamine, and 4 mg/kg body weight
xylazine
intraperitoneally. The left external carotid artery was cannulated with 2-
French Fogarty
embolectomy catheter, inflated with saline and passed three times up and down
the common carotid
artery to produce a distending, deendothelializing injury. The animals were
treated with an
appropriate dosage of the test substance or with vehicle alone (e.g., based on
body weight per day in
an appropriate solution such as 1:2:2:165 DMSO:Cremophor EL:Dehydrated
ethanol:phosphate
buffered saline) by intraperitoneal injection beginning 2 hours after injury.
Test substance or vehicle
alone was administered once daily, as an intraperitoneal injection, for the
next 4 days. After 11 days
the animals (8 treated and 10 vehicle-treated) were anesthetized as above and
the carotid artery was
isolated and fixed in 10% buffered formalin and embedded in paraffin. Cross
sections of the carotids
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were mounted on microscope slides and stained with hematoxylin and eosin
stain. The image of the
carotid artery was projected onto a digitizing board and the cross sectional
areas of the intima and
the media were measured. Reduction of the neointimal area (thickening)
indicates that the test
substance is an effective antirestenosis agent.
Interfering with the recirculation of bile acids from the lumen of the
intestinal tract is found
to reduce the levels of serum. cholesterol in a causal relationship.
Epidemiological data has
accumulated which indicates such reduction Leads to an improvement in the
disease state of
atherosclerosis (Stedronsky, Biochimica et Biophysica Acta, 1210, 255-287
(1994)). Inhibition of
cholesteryl ester transfer protein (CETP) has been shown to effectively modify
plasma HDL/LDL
. ratios, and is expected to check the progress and/or formation of certain
cardiovascular diseases.
Inhibition of CETP should lead to elevation of plasma HDL cholesterol and
lowering of plasma LDL_
cholesterol, thereby providing a therapeutically beneficial plasma lipid
profile (MeCarthy, Medicinal
Res. Revs., 13, 139-59 (1993)). An in vivo assay for compounds that inhibit
rat ileal uptake of'4C-
w Taurocholate into bile (CETP inhibition) is disclosed in US Patent 6489366
and Une, et al.
Biochimica et Biophysica Acta, 833, 196-202 (1985), disclosures of which are
incorporated herein
by reference.
Briefly, male Wistar rats (200-300 g) are anesthetized with inactin (100
mg/kg). Bile ducts
are cannulated with a l flinch length of PE10 tubing. The small intestine is
to be exposed and laid out
on a gauze pad. A canulae (1/8" luer lock, tapered female adapter) is inserted
at 12 crn from the
junction of the small intestine and the cecum. A slit is cut at 4 cm from this
same junction (utilizing a
8 cm length of ileum). Twenty milliliters of warm Dulbecco's phosphate
buffered saline, pH 6.5
(PBS) is to be used to flush out the intestine segment. The distal opening is
cannulated with a 20 cm
length of silicone tubing (0.02" LD.×0.037" O.D.). The~proximal cannulae
is hooked up to a
peristaltic pump and the intestine is washed for 20 min with warm PBS at 0.25
ml/min. Temperature
of the gut segment is to be monitored continuously. At the start of the
experiment, 2.0 m1 of control
sample (14C-taurocholate at 0.05 mCi/mL with 5 mM non-radiolabeled
taurocholate) is loaded into
the gut segment with a 3 ml syringe and bile sample collection is begun.
Control sample is infused at
a rate of 0.25 ml/min for 21 min. Bile samples fractions are to be collected
every 3 minute for the
first 27 minutes of the procedure. After the 21 min of sample infusion, the
ileal loop is to be washed
out with 20 ml of warm PBS (using a 30 ml syringe), and then the loop is to be
washed out for 21
min with warm PBS at 0.25 ml/min. A second perfusion is initiated as described
above but this with
test compound being administered as well (2I min administration followed by 21
min of wash out)
and bile sampled every 3 min for the first 27 min. If necessary, a third
perfusion is performed as
above that typically contains the control sample.
In addition, measurement of hepatic cholesterol concentration is a useful
assay for
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
determining the effectiveness of a test substance against cardiovascular
disorders. In this assay, liver
tissue is weighed and homogenized in chloroform:methanol (2:1). After
homogenization and
centrifugation the supernatant is separated and dried under nitrogen. The
residue is to be dissolved in
isopropanol and the cholesterol content measured enzymatically, using a
combination of cholesterol
oxidase and peroxidase, as described by Allain, C. A. et al., Clin. Chem., 20,
470 (1974) (herein
incorporated by reference).
Similarly, serum cholesterol may be determined as follows. Total serum
cholesterol is
measured enzymatically using a commercial kit from Wako Fine Chemicals
(Richmond, Va.);
Cholesterol C 11, Catalog No. 276-64909. HDL cholesterol may be assayed using
this same kit after
precipitation of VLDL and LDL with Sigma Chemical Co. HILL Cholesterol
reagent, Catalog No.
352-3 (dextran sulfate method). Total serum triglycerides (blanked) (TGI) is
also assayed
enzymatically with Sigma Chemical Co. GPO-Trinder, Catalog No. 337-B. VLD,L
and LDL
(VLDL+LDL) cholesterol concentrations are calculated as the difference between
total and fiDL
w cholesterol. A reduction in VLDL+LDL cholesterol in the test substance-
treated sample relative to
control is indicative of an effective anti-cardiovascular disorder agent.
A dog model for evaluating lipid lowering drugs may also be utilized, for
example, as
described in US Patent 6489366.
Briefly, male beagle dogs, obtained from a vendor such as Marshall farms and
weighing 6-
12 kg are fed once a day for two hours and given water ad libitum. Dogs may be
randomly assigned
to a dosing groups consisting of 6 to 12 dogs each, such as: vehicle, i.g.; 1
mg/kg, i.g.; 2 mg/kg, i.g.;
4 mg/kg, i.g.; 2 mg/kg, p.o. (powder in capsule). Intra-gastric dosing of a
therapeutic material
dissolved in aqueous solution (for example, 0.2% Tween 80 solution
[polyoxyethylene mono-oleate,
Sigma Chemical Co., St. Louis, Mo.]) may be done using a gavage tube. Prior to
initiating dosing,
blood samples may be drawn from the cephalic vein in the morning before
feeding in order to
evaluate serum cholesterol (total and HDL) and triglycerides. For several
consecutive days animals
are dosed in the morning, prior to feeding. Animals are to be allowed 2 hours
to eat before any
remaining food is removed. Feces are to be collected over a 2 day period at
the end of the study and
may be analyzed for bile acid or lipid content. Blood samples are also to be
taken, at the end of the
treatment period, for comparison with pre-study serum lipid levels.
Statistical significance will be
determined using the standard student's T-test with p<0.05.
Serum lipid measurement is measured similarly. Blood is collected from the
cephalic vein of
fasted dogs in serum separator tubes (Vacutainer SST, Becton Dickinson and
Co., Franklin Lakes,
N.J.). The blood is centrifuged at 2000 rpm for 20 minutes and the serum
decanted. Total cholesterol
may be measured in a 96 well format using a Wako enzymatic diagnostic kit
(Cholesterol CII)
(Wako Chemicals, Richmond, Va.), utilizing the cholesterol oxidase reaction to
produce hydrogen
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peroxide which is measured colorimetrically. A standard curve from 0.5 to 10
ug cholesterol is to be
prepared in the first 2 columns of the plate. The serum samples (20-40 ul,
depending on the expected
lipid concentration) or known serum control samples are added to separate
wells in duplicate. Water
is added to bring the volume to 100 ul in each well. A 100 ul aliquot of color
reagent is added to each
well and the plates will be read at 500 nm after a 15 minute incubation at 37
degrees centigrade.
HDL cholesterol may be assayed using Sigma kit No. 352-3 (Sigma Chemical Co.,
St.
Louis, Mo.) which utilizes dextran sulfate and Mg ions to selectively
precipitate LDL and VLDL. A
volume of 150 ul of each serum sample is to be added to individual microfuge
tubes, followed by 15
ul of HDL cholesterol reagent (Sigma 352-3). Samples are to be mixed and
centrifuged at 5000 rpm
for 5 minutes. A 50 ul aliquot of the supernatant is to be then mixed with 200
ul of saline and
assayed using the same procedure as for total cholesterol measurement.
Triglycerides are measured using Sigma kit No. 337 in a 96 well plate format.
This
procedure will measure glycerol, following its release by reaction of
triglycerides with lipoprotein
lipase. Standard solutions of glycerol (Sigma 339-11) ranging from 1 to 24 ug
are to be used to
generate the standard curve. Serum samples (20-40 ul, depending on the
expected lipid
concentration) are added to wells in duplicate. Water is added to bring the
volume to 100 ul in each
well and 100 ul of color reagent is also added to each well. After mixing and
a 15 minute incubation,
the plates will be read at 540 ~nm and the triglyceride values calculated from
the standard curve. A
replicate plate is also to be run using a blank enzyme reagent to correct for
any endogenous glycerol
in the serum samples.
Test compounds may be evaluated for their effect on serum glucose and serum
insulin in
db/db mice (C578BL/KsJ-db/db Jcl) as described in US 6462046, disclosure of
which is
incorporated herein. The compounds are dissolved in a vehicle (e.g.,
consisting of 2% Tween80 in
distilled water) and administered orally. Dosage is determined by body weight.
All aspects of the
work including experimentation and disposal of the animals is performed in
general accordance with
the International Guiding Principles for Biomedical Research Involving Animals
(CIOMS
Publication No. ISBN 92 90360194, 1985). Glucose-HA Assay kits (Wako, Japan)
are used for
determination of serum glucose and ELISA Mouse Insulin Assay kits (SPI bio,
France) are utilized
for determination of insulin. An appropriate positive control is troglitazone
(Helios Pharmaceutical,
Louisville, Ky.).
The animals are divided into twenty groups of four animals each. The animals
weigh 52 +/-
5 gms at age 8-10 weeks. During the experiment the animals are provided free
access to laboratory
chow (Fwusow Industry Co., Taiwan) and water. Prior to any treatment a blood
sample
(pretreatment blood) is taken from each animal. Four groups of animals, the
vehicle groups, receive
only doses of the vehicle. Each of the vehicle groups receive 100, 30, 10 or 1
ml/kg body weight of
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the vehicle orally. A triglitazone solution (10 ml/kg body weight in tween
80/water) is administered
orally to the four positive control groups in doses of 100, 30, 10 and 1 ml/kg
body weight
respectively. The test compound is similarly administered orally as a solution
to four groups of
animals with each group receiving a different dose of the compound. The
vehicle, positive control
and test compound solutions are administered to the groups immediately, 24
hours and 48 hours after
drawing the pretreatment blood. Blood is withdrawn (post treatment blood) 1.5
hours after
administration of the last dose. The serum glucose are determined
enzymatically (Mutaratose-GOD)
and the insulin levels by ELISA (mouse insulin assay kit). The mean SEM of
each group is
calculated and the percent inhibition of serum glucose and insulin obtained by
comparison between
pretreatment blood and post treatment blood. The percentage of reduction of
the serum glucose and
insulin levels in the post treatment blood relative to the pretreatment blood
is determined and the
Unpaired students t test applied for the comparison between the control and
test solution groups and
the vehicle group. A significant difference is considered at P<0.05.
Troglitazone, as an effective anti-
cardiovascular disorder agent, results in a reduced glucose level at 10 mg/kg
body weight (25 +/-
2%).
US Patent 6121319, disclosure of which is incorporated herein, describes an
assay for the
progression of atherosclerosis in hypercholesterolemic rabbits. The rabbits
are sacrificed and aortas
obtained. The aortas are stained with sudan-4 and the extent of staining
analyzed. The percent aortic
surface area covered by lesions in test substance treated and untreated lipid-
fed rabbits is graphed.
The aortas of the rabbits treated with an effective anti-atherosclerotic agent
have less staining,
indicating decreased atherosclerosis. In addition, sections of the aortas are
immunostained for
VCAM-1 expression or macrophage accumulation using antibodies for VCAM-1 or
Ram-11
antigen. Reduced VCAM-1 expression and macrophage accumulation compared to
control treated
samples are indicative of an effective agent.
Reduction in LDL cholesterol may also be determined in a primate model. For
example,
Cynomolgus monkeys are made hypercholesterolemic prior to test compound dosing
by feeding a
high fat cholesterol diet. The monkeys are then dosed orally with the test
compound or control
vehicle for two weeks. A reduction in the percentage serum LDL cholesterol in
the monkeys over this
time period is indicative of an effective anti-atherosclerotic agent.
Phar-rnaceutical Compositions
When polypeptides of the present invention are expressed in soluble form, for
example as ~a
secreted product of transformed yeast or mammalian cells, they can be purified
according to standard
procedures of the art, including steps of ammonium sulfate precipitation, ion
exchange
78
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
chromatography, gel filtration, electrophoresis, affinity chromatography,
according to, e.g., "Enzyme
Purification and Related Techniques," Methods in Enzymology, 22:233-577
(1977), and Scopes, R.,
Protein Purification: Principles and Practice (Springer-Verlag, New York,
1982) provide guidance
in such purifications. Likewise, when polypeptides of the invention are
expressed in insoluble form,
for example as aggregates or inclusion bodies, they can be purified by
appropriate techniques,
including separating the inclusion bodies from disrupted host cells by
centrifugation, solublizing the
inclusion bodies with chaotropic and reducing agents, diluting the solubilized
mixture, and lowering
the concentration of chaotropic agent and reducing agent so that the
polypeptide takes on a
biologically active conformation. The latter procedures are disclosed in the
following references,
which are incorporated by reference: Winkler et al, Biochemistry, 25: 4041-
4045 (1986); Winkler et
al, Biotechnology, 3: 992-998 (1985); Koths et al, U.S. patent 4,569,790; and
European patent
applications 86306917.5 and 86306353.3.
Compounds capable of modulating and preferably increasing a CPP biological
activity,
preferably small molecules but also including peptides, CPP nucleic acid
molecules, CPP, and anti-
CPP antibodies of the invention can be incorporated into pharmaceutical
compositions suitable for
administration. Such compositions typically comprise a pharmaceutically
acceptable carrier. As used
herein the language "pharmaceutically acceptable carrier" is intended to
include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying
agents, and the like, compatible with pharmaceutical administration. The use
of such media and
agents for pharmaceutically active substances is well known in the art. Except
insofar as any
conventional media or agent is incompatible with the active compound, use
thereof in the
compositions is contemplated. Supplementary active compounds can also be
incorporated into the
compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with its
intended route of administration. Examples of routes of administration include
parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal,
and rectal administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous
application can include the following components: a sterile diluent such as
water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or
other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants
such as ascorbic acid or
sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid;
buffers such as acetates,
citrates or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose.
pH can be adjusted with acids ox bases, such as hydrochloric acid or sodium
hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made
of glass or plastic.
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Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions
(where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of
sterile injectable solutions or dispersion. For intravenous administration,
suitable carriers include
physiological saline, bacteriostatic water, Cremophor EL~ (BASF, Parsippany,
N.J.) or phosphate
buffered saline (PBS). In all cases, the composition must be sterile and
should be fluid to the extent
that easy syringability exists. It must be stable under the conditions of
manufacture and storage and
must be preserved against the contaminating action of microorganisms such as
bacteria and fungi:
The carrier can be a solvent or dispersion medium containing, for example,
water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene glycol, and
the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for example, by the
use of a coating such as
lecithin, by the maintenance of the required particle size in the case of
dispersion and by the use of
surfactants. Prevention of the action microorganisms can be achieved by
various antibacterial and
antifungal agents, for example, parabens, c111orobutanol, phenol, ascorbic
acid, thimerosal, and the
like. In many cases, it will be preferable to include isotonic agents; for
example, sugars, polyalcohols'
such as manitol, sorbitol, sodium chloride in the composition. Prolonged
absorption of the injectable
compositions can be brought about by including in the composition an agent
which delays
absorption, for example, aluminum monostearate and gelatin.
Where the active compound is a protein, e.g., an anti-CPP antibody, sterile
injectable
solutions can be prepared by incorporating the active compound in the required
amount in an
appropriate solvent with one or a combination of ingredients enumerated above,
as required,
followed by filtered sterilization. Generally, dispersions are prepared by
incorporating the active
compound into a sterile vehicle which contains a basic dispersion medium and
other required
ingredients from those enumerated above. In the case of sterile powders for
the preparation of sterile
injectable solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which
. yields a powder of the active ingredient plus any additional desired
ingredient from a previously
sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible Garner. They
can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the active compound can be incorporated with excipients and
used in the form of
tablets, troches, or capsules. For administration by inhalation, the compounds
are delivered in the
form of an aerosol spray from pressured container or dispenser which contains
a suitable propellant, ,
e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration
can also be by
transmucosal or transdermal means. For transmucosal or transdermal
administration, penetrants
appropriate to the barner to be permeated are used in the formulation. Such
penetrants are generally
known in the art, and include, for example, for transmucosal administration,
detergents, bile salts,
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
and fusidic acid derivatives. Transmucosal administration can be accomplished
through the use of
nasal sprays or suppositories. For transdermal administration, the active
compounds are formulated
into ointments, salves, gels, or creams as generally lcr~own in the art. Most
preferably; active
compound is delivered to a subject by intravenous injection.
In one embodiment, the active compounds are prepared with carriers that will
protect the
compound against rapid elimination from the body, such as a controlled release
formulation,
including implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers
can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen,
polyoxthoesters, and polylactic acid. Methods for preparation of such
formulations will be apparent
to those skilled in the art. The materials can also be obtained commercially
from Alza Corporation
and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected
cells with monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable
carriers. These can be prepared according to methods known to those skilled in
the art, for example,
as described in U.S. Pat. No. 4,522,81 l, the disclosure of which is
incorporated herein by refexence
in its entirety.
In a further embodiment; the active compound may be coated on a microchip drug
delivery .
device. Such devices are useful for controlled delivery of proteinaceous
compositions into the
bloodstream, cerebrospinal fluid, lymph, or tissue of an individual without
subjecting such
compositions to digestion or subjecting the individual to injection. Methods
of using microchip drug
delivery devices are described in US Patents 6,123,861 and 5,797,898 and US
Patent application
20020119176A1, disclosures of which are hereby incorporated in their
entireties.
It is especially advantageous to formulate oral or preferably parenteral
compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form as used
herein refers to physically discrete units suited as unitary dosages for the
subject to be treated; each
unit containing a predetermined quantity of active compound calculated to
produce the desired
therapeutic effect in association with the required pharmaceutical carrier.
The specification for the
dosage unit forms of the invention are dictated by and directly dependent on
the unique
characteristics of the active compound and the particular therapeutic effect
to be achieved, and the
limitations inherent in the art of compounding such an active compound for the
treatment of
individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., for
determining the LD50
(the dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of
the population). The dose~ratio between toxic and therapeutic effects is the
therapeutic index and it
can be expressed as the ratio LD50/ED50. Compounds which exhibit large
therapeutic indices are
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CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
preferred. While compounds that exhibit toxic side effects may be used, care
should be taken to
design a delivery system that targets such compounds to the site of affected
tissue in order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
The data obtained from the cell culture assays and animal studies can be used
in formulating
a range of dosage for use in humans. The dosage of such compounds lies
preferably within a range
of circulating concentrations that include the ED50 with little or no
toxicity. The dosage may vary
within this range depending upon the dosage form employed and the route of
administration utilized.
For any compound used in the method of the invention, the therapeutically
effective dose can be
estimated initially from cell culture assays. A dose may be formulated in
animal models to achieve a
circulating used to more accurately determine useful doses in humans. Levels
in plasma may be
measured, for example, by high performance liquid chromatography.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together
with instructions for administration.
Therapeutic Uses of CPPs
The CPPs, CPP modulators, and anti-CPP antibodies of the invention can be used
in the
treatment or prevention of CPP-related disorders. Unlike manufactured small
molecule therapeutics
that may cause an immune reaction or prove to be toxic to the host, the
peptides of the invention are
normally secreted and are thought not to be toxic to a subject to which they
are administered. Thus,
in one aspect the invention relates to pharmaceutical compositions containing
CPP as active
ingredients, preferably containing a pharmaceutically acceptable carrier or
diluent. The earner or
diluent is preferably adapted for oral, intravenous, intramuscular or
subcutaneous administration.
CPPs can also be prepared as solutions for inhalation. Pharmaceutical
compositions may comprise,
consist or consist essentially any of the CPPs described herein. Optionally, a
CPP can be
administered as a propeptide or prepropeptide.
A number of agents axe useful for the treatment and prevention of
cardiovascular disorders.
Such agents may be used advantageously in combination with a CPP.
For example, cell cycle inhibitors and proto-oncogenes (Simari and Nabel,
Semin. Intervent.
Cardiol. 1:77-83 (1996)); NO (nitric oxide) donor drugs; pro-apoptotic agents
such as bcl-x
(Pollinan et al., Nature Med. 2:222-227 (1998)); herpes virus thymidine kinase
(tk) gene and
systemic ganciclovir (Ohno et al., Science 265:781-784 (1994); Guzman et al.,
Proc. Natl. Acad.
Sci. USA 91:10732-10736 (1994); Chang et al., Mol. Med. 1:172-181 (1995); and
Simari et al.,
Circulation 92:1-501 (1995)) have been exploited to treat atherosclerosis,
restenosis and neointimal
smooth muscle proliferation. Disclosures of the above references are hereby
incorporated in their
entireties.
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WO 2004/061096 PCT/EP2004/000027
Anti-thrombotic agents useful in combination with the peptides of the
invention include, for
example, inhibitors of the ITb/ITIa integrin; tissue factor inhibitors; and
anti-thrombin agents. An
antiarrhythmic agent, such as a local anesthetic (class I agent), sympathetic
antagonist (class II
agent), antifibrillatory agent (class III agent) calcium channel agent (class
IV agent) or anion
antagonist (class V agent) as described in Vukmir, Am. J. Emer. Med. 13:459-
470 (1995); Grant,
PACE 20:432-444 (1997); Assmann L, Curr. Med. Res. Opin. 13:325-343 (I995);
and Lipka et aL,
' Am. Heart J. 130:632-640 (1995), disclosures of which are hereby
incorporated by reference in their
entireties, may also be used. Examples of class I agents include:
procainamide; quinidine or
disopyramide; lidocaine; phenytoin; tocainide or mexiletine; encainide;
flecainide; lorcainide;
propafenone (III) or moricizine. Sympathetic antagonists include: propranolol,
esmolol, metoprolol,
atenelal, or acebutolol. Examples of antifibrillatory agents are bretylium,
amiodarone, sotalol (II) or
N-acetylprocainamide. Class IV agents include verapamil, diltiazem, and
bepridil, and anion
antagonists such as alinidine.
Congestive heart failure therapeutic agents include TNF inhibitors such as
Embxel.TM.
(Immunex Corp.; Seattle, Wash.), TBC11251, or an ACE (angiotensin converting
enzyme) inhibitor,
such as Natrecor (nesiritide; Scios, Inc.). Angiogenic agents, for example,
recombinant VEGF
isoforms, such as rhVEGF developed by Genentech; a nucleic acid molecule
encoding the I21 amino
acid isoform of VEGF (BioByPass.TM.; GenVec/Parke Davis); or a nucleic acid
encoding VEGF-2
(Vascular Genetics, Inc.); FIBLAST.TM., a recombinant form of FGF-2 being
developed by Scios,
Tnc. (Mountain View, Cali~) and Wyeth Ayerst Laboratories (Radnor, Pa.),
GENERX.TM., ox an
adenoviral gene therapy vector encoding FGF-4 developed by Collateral
Therapeutics (San Diego,
Calif.) and Schering AG (see Miller and Abrams, Gen. Engin. News 18:1 (1998),
disclosure of
which is hereby incorporated by reference in its entirety), are also useful in
combination with the
CPP-related compositions of the invention. Finally, calcium antagonists, such
as amlodipine
(Marche et al., Int. J. Cardiol. 62(Suppl.):S17-S22 (1997); Schachter, Int. J.
Cardiol.
62(Suppl.):S85-S90 (1997)); nicardipine; nifedipine; propanolol; isosorbide
dinitrate; diltiazem; and
isradipine (Nayler (Ed.) Calcium Antagonists pages 157-260 London: Academic
Press (1988);
Schachter, Int. J. Cardiol. 62(SuppL):S9-SI5 (I997)) are also advantageous
therapeutic agents for
cardiovascular disorders.
Having generally described this invention, a further understanding can be
obtained by
reference to certain specific examples which are provided herein for purposes
of illustration only,
and are not intended to be limiting unless otherwise specified.
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EXAMPLES
Example 1: Characterization of CPP levels in experimental and control
populations
Subjects enrolled in the Duke Databank for Cardiovascular Disease were
selected on the
basis of coronary artery disease (CAD). A total of 241 CAD patients and
control individuals were
further matched for gender, age, and ethnicity and individuals with plasma
abnormalities were
excluded. A set of 53 CAD patients and a set of 53 control individuals were
established. Six liters
of plasma were pooled from each set. An aliquot of plasma was retained from
each individual, thus
allowing a positive result in the pooled sample to be confirmed for each
member of the population.
Such confirmation is valuable to erase possible confounding effects of an
individual with an aberrant
level of a specific.. polypeptide that is not related to a cardiovascular
disorder. Two and a half liters
of pooled plasma from each population was subjected to separation by multiple
chromatography
steps according to the Microprot.TM process as follows:
Step l: HSAlIgG depletion
125 ml frozen plasma were defrost and filtered on 0.45 pm sterile filter in a
sterile hood.
Filtrate was injected on two inline columns of respectively 300 ml of HSA
ligand Sepharose
fast Flow column (Amersham, Upsala, Sweden), Scm ID, 15 cm length; and 100 ml
Protein G
Sepharose fast Flow column (Amersham, Upsala, Sweden), 5 cm ID, 5 cm length.
Columns were equilibrated and washed with 50 mM P04 buffer, pH 7.1, O.15M
NaCI. Flow
rate was 5 ml/min.
Non-retained fraction (350 ml) was frozen until second step. Twenty runs were
performed.
Step 2: Gel Filtration /Reverse Phase Capture step
Sample from step 1 was defrosted and filtered on 0.45 ~n sterile filter in a
sterile hood.
Filtrate was injected on two in line gel filtration columns: 2 X 9.5 litres
Superdex 75
(Amersham, UK) column, 14 cm ID, 62 cm length. Column was equilibrated with
50mM P04 buffer
pH 7.4, 0.1 M NaCI, 8M urea. Hydrophobic impurities were retained on a reverse
phase precolumn:
150 ml PLRPS (Polymer Labs, UK). Precolumn was switched for sample injection.
Gel filtration
was performed at a flow rate of 40 ml/min.
Low molecular weight proteins (<20 kDa) were oriented to in line reverse phase
capture
column: 50 ml PLRPS 100 angstroms (Polymer labs, UK). The three-way valve
controlling injection
on PLRl'S column was switched at a cut-off of 33 mAU (280 nm) to send gel
filtration eluate into
reverse phase capture column. This cut-off value was established by first
using SDS-PAGE to
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WO 2004/061096 PCT/EP2004/000027
provide an estimated range of OD values and by subsequently evaluating three
cut-off values (high,
median and low values of OD range). The final cut-off value was chosen to
maximize the low
molecular weight protein obtained, with a low molecular protein proportion of
at least 85%. Low
molecular weight proteins and peptides were eluted from reverse phase capture
PLRPS column by
one column volume gradient of 0.1% TFA, 80% CH3CN in water.
Eluate fractions (50 ml) were frozen until next step. Twenty runs were
performed. At the end of this
step, all reverse phase eluates were defrosted, pooled (1 liter) and shared in
7 polypropylene
containers (143 ml). Containers were kept at -20°C until use for next
step.
to Step 3: Cation Exchange
Sample from step 2 (147 ml) was defrosted and mixed with an equal volume of
cation
exchange buffer A (Gly/HCl buffer 50 mM, pH 2.7, urea 8M).
Sample was injected on a 100 xnl Source 15S column (Amersham, Upsala, Sweden),
35 mm
ID, 100 mm length. Column was equilibrated and washed with buffer A. Flow rate
was 10 ml/min.
15 Proteins and peptides were eluted with step gradient from 100% buffer A
until 100 % buffer
B (buffer A containing 1M NaCI):
3 column volumes 7.5% B (75 mM NaCI)
3 column volumes 10% B (100 mM NaCI)
3 column volumes 17.5% B (175 mM NaCI)
20 2 column volumes 22.5% B (225 mM NaCI)
2 column volumes 27.5% B (275 mM NaCI)
2 column volumes 100% B (1 M NaCI)
45 to 60 fractions were collected based on peak. Seven runs were conducted.
After 7 runs
were achieved, fractions were pooled intra and inter run in order to obtain 18
fractions. Fractions
25 were kept at -20°C until use for next step. .
Step 4: RedicctionlAlkylation and Reverse Phase HPLC F~actionatioh 1
After adjusting the pH to 8.5 with concentrated Tris-HCI, each of the 18
cation exchange
fractions was reduced with dithioerythritol (DTE, 30 mM, 3 hours at
37°C) and alkylated with
30 iodoacetamid (120 mM, 1 hour 25°C in the dark). The latter reaction
was stopped with the addition
of DTE (30 mM) followed by acidification (TFA, 0.1%). The fractions were then
injected on an
Uptispher C8, 5 microm, 300 angstroms column (Interchim, France), 21 mm ID,
150 mm length.
Injection was performed with a 10 ml/min flow rate.
C8 column was equilibrated and washed with 0.1% TFA in water (solution A).
Proteins and
35 peptides were eluted with a biphasic gradient from 100% A until 100% B
(0.1% TFA, 80% CH3CN
CA 02512629 2005-07-05
WO 2004/061096 PCT/EP2004/000027
in water) in 60 min. Flow rate was 20 ml/min. Thirty fractions of 40 ml were
collected.
Based on the measured optical density (OD) at 280 nm of each fraction, which
reflects the
protein concentration in that fraction, aliquots of similar protein content
were created for each
fraction.
All aliquots were frozen and kept for further use except one per fraction
which was dried
with a Speed Vac (Savant, Fischer, Geneva) after addition of 500 microl 10%
glycerol in water in
each fraction, in order to prevent excess drying. Dried fractions were kept at
-20°C until use for next
step.
l0 Step 5: Reverse Phase HPLC Fractionation 2
Dried samples from step 4 were resuspended in 1 ml of solution A (0.03% TFA in
water)
and injected on a Vydac LCMS C4 column, 5 micrometers, 300 angstroms (Vydac,
USA), 4.6 mm
ID, 150 mm length. Flow rate was 0.8 ml/min.
C4 column was equilibrated and washed with solution A and proteins and
peptides were
15 eluted with a biphasic gradient adapted to elution position of the sample
in Reverse Phase HPLC
Fractionation 1. Intact mass data were acquired using Electrospray Ion Trap
Mass spectrometry.
Sixteen different gradients were used with a CH3CN concentration range minus
and plus S%
CH3 CN of RP 1 fraction corresponding solvent concentration. For proteins
eluted in RP 1 with a
solvent concentration equal to or greater than 30% CH3CN, the starting elution
conditions for the
20 RP2 gradient was set, in CH3CN percentage, at the RP1 elution concentration
minus 30%. Twenty-
four eluted fractions were collected in a deep well plate, adopting optimized
different collection
configurations designed for optimal SpeedVac concentration and further robotic
treatment.
Step 6: Mass detection
25 About 13,000 fractions were collected following reverse phase HPLC
fractionation 2 into
96-well deep well plates (DWP). A small proportion (2.5%) of the volume was
diverted to online
analysis using LC-ESI-MS (Broker Esquire). Aliquots of undigested proteins
were mixed with
MALDI matrices, and spotted on MALDI plates together with mass calibration
standards and
sensitivity standards. Automated spotting devices (Broker MALDI sample prep.
Robots) were used.
30 Two different MALDI matrices were employed: sinapic acid (SA), also known
as sinapinic acid,
trans-3,5-dimethoxy-4-hydroxycinnarnic acid, and alpha-cyano-4-hydroxycinnamic
acid (HCCA).
MALDI plates were subjected to mass detection using Broker Reflex III MALDI MS
apparati. The
96-well plates were stored at +4 C.
96-well plates (DWP) were recovered and subjected to two sequential
concentration steps.
35 Volumes were concentrated from 0.8 ml to about 50 microl per well by drying
with a SpeedVac, and
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WO 2004/061096 PCT/EP2004/000027
then xesolubilized to ca. 200 microl and reconcentrated to about 50 microl per
well, and stored at +4
C. Proteins were then digested by re-buffering, adding trypsin to the wells,
sealing and incubating
the plates at 37 C for 12 hours, followed by quenching (addition of formic
acid to bring the pH down
to 2.0). The concentration of txypsin to be added to the wells was adjusted
based on the OD at 280
nm recorded for each particular fraction. This ensured an optimal use of
trypsin and a complete
digestion of the most concentrated fractions. Automated spotting devices
(Broker MALDI sample
prep. Robots) were used to deposit a volume from each well, pre-mixed with a
HCCA matrix onto a
MALDI plate together with sensitivity and mass calibration standards. MALDI
plates ware
analyzed using a Broker Reflex III MALDI MS device. Contents from each well of
the 96 well
plates were analyzed with LC-ESI-MS-MS Bruleer Esquire ESI Ion-Trap MS
devices.
Step 7: Detection aid Identification of Low Abundance Peptides in Human Plasma
Separated fractions are further subjected to mass spectrometry (both matrix-
assisted laser
desorption/ionization (MALDI) and MS-MS) for separation and detection.
Intact mass data, Peptide Mass Fingerprints and peptide sequence data were
integrated for
protein identification and characterization. Proteins were identified using
Mascot software (Matrix
Science Ltd., London, IJI~), and results from peptide identification were
checked by manual analysis
of the spectra.
Among the proteins identified by this process, Calgxanulin A (S 100 calcium-
binding protein
A8, of SwissProt accession number POS 109), was found to be expressed to a
greater extent in the
pooled sample from controls than in the pooled sample from CAD patients (e.g.,
peptides from the
protein were observed in twice as many control fractions compared with disease
fractions, and the
cumulated scores obtained during mass spectra identification of this protein
were 2.5-fold higher for
the control sample). Calgranulin A has been characterized as a pro-
inflammatory protein (Odink, et
al., Nature 330 (6143), 80-82 (1987) and numerous Iater references). It is
expressed by
extravasating myeloid cells during inflammatory responses, where it binds to a
glycosaminoglycan
structure on epithelial cells (Robinson, et al., JBC 277:3658-65 (2002)).
Interestingly, PCT
publication WO 00/61742 discloses the use of Calgranulin A for the treatment
of cardiac
insufficiency, e.g. caused by arteriosclerosis. Moreover, PCT publication WO
00/18970 discloses
the use of Calgranulin A as an inhibitor of vasculax membrane growth for
pxevention of myocardial
infarction and hypertension. It appears therefore that the protein separation
and identification
approach described herein is efficient at providing proteins which, when
detected at higher levels in
the control sample than in the disease sample, have a beneficiary effect for
the treatment of the
studied disease.
Conversely, the methods of protein separation and identification described in
this Example
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WO 2004/061096 PCT/EP2004/000027
have allowed the identification of the Matrix Gla Protein (of SwissProt
accession number P08493)
as overexpressed in the pooled sample from CAD patients by comparison with the
pooled sample
from controls (e.g., peptides from the protein were observed in alinost twice
as many disease
fractions compared with control fractions, and the cumulated scores obtained
during mass spectra
identification of this protein were 2-fold higher for the disease sample). MGP
is a vitamin K-
dependent protein which associates with the organic matrix of bone and
cartilage. Mori, et al.
demonstrated that MGP is capable of inhibiting vascular calcification (FEBS
Letters 433:19-22
(1998)). MGP levels are increased in atherosclerotic plaques as a likely
feedback response to vessel
calcification. PCT publications WO 01/02863 and WO 01/25427 describe MGP as a
biomarker for
atherosclerosis and cardiovascular disorders. It appears therefore that the
protein separation and
identification approach described herein is efficient at providing proteins
which have a recognized
use in the diagnosis of the studied disease.
Example 2: PuYificatioh and Characterisation of CPPs
Human plasma was fractionated by a series of multiple chromatography columns
as described in
Example 1. The fractions were analysed by mass spectrometry. A distinct
tryptic fragment was
found as described in Table l and SEQ ID NO:S.
The tryptic peptide listed in Table 1 (SEQ ID NO:S) was observed by MS-MS in
the plasma
of control individuals without Coronary Artery Disease. CPP peptides present
in the plasma of CAD
patients include CPPs 6 and 7 of SEQ ID NOs:3 and 4. The full-length
polypeptide sequences
correlated with the tryptic peptide are described as SEQ ID NOs: l and 2. The
listed peptides were
not found in the CAD sample. The Microprot.TM process is able to detect very
low abundance
proteins with a plasma concentration in the range of 50 pM. Thus, the absence
of the listed peptides
in CAD plasma indicates that the CPPs are present at vanishingly low levels in
CAD patients, if at
all.
The presence of a tryptic peptide indicates that a polypeptide with a
molecular mass below
20kD and comprising the amino acid sequence FTTLVQDLANAFQQEAQTSGK was present
in the
starting human plasma sample. Such polypeptides include those comprising the
amino acid sequence
selected from the group consisting of SEQ ID NOs:3-4.
Example 3: Chemical Synthesis of CPPs
In this example, a CPP of the invention is synthesized. Peptide fragment
intermediates are
first synthesized and then assembled into the desired polypeptide.
A CPP can initially be prepared in, e.g. 5 fragments, selected to have a Cys
residue at the N-
terminus of the fragment to be coupled. Fragment 1 is initially coupled to
fragment 2 to give a first
88
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product, then after preparative HPLC purification, the first product is
coupled to fragment 3 to give
a second product. After preparative HPLC purification, the second product is
coupled to fragment 4
to give a third product. Finally, after preparative HPLC purification, the
t~rd product is coupled to
fragment 5 to give the desired polypeptide, which is purified and refolded.
Thioeste~ formation
Fragments 2, 3, 4~ and 5 are synthesized on a thioester generating resin, as
described above.
For this purpose the following resin is prepared: ~S-acetylthioglycolic acid
pentafluorophenylester is
coupled to a Leu-PAM resin under conditions essentially as described by
Hackeng et al (1999). In
the first case, the resulting resin is used as a starting resin for peptide
chain elongation on a 0.2 mmol
scale after removal of the acetyl protecting group with a 30 min treatment
with 10%
mercaptoethanol, 10% piperidine in DMF. The N" of the N-terminal Cys residues
of fragments 2
through 5 are protected by coupling a Boc-thioproline (Boc-SPr, i.e. Boc-L-
thioproline) to the
terminus of the respective chains instead of a Cys having conventional N" or
Sa protection, e.g. Brik
et al, J. Org. Chem., 65: 3829-3835 (2000).
Peptide synthesis
Solid-phase synthesis is performed on a custom-modified 433A peptide
synthesizer from
Applied Biosystems, using in situ neutralization/2-(1H-benzotriazol-1-yl)-
1,1,1,3,3-
tetramethyluronium hexafluoro-phosphate (HBTU) activation protocols for
stepwise Boc chemistry
chain elongation, as described by Schnolzer et al, Int. J. Peptide Protein
Res., 40: 180-193 (1992).
Each synthetic cycle consists of N"-Boc -removal by a 1 to 2 min treatment
with neat TFA, a 1-miri
DMF flow wash, a 10-min coupling time with 2.0 mmol of preactivated Boc-amino
acid in the
presence of excess DIEA and a second DMF flow wash. Na-Boc-amino acids (2
mmol) are
preactivated for 3min with l.8mmol HBTU (O.SM in DMF) in the presence of
excess DIEA
(6mmol). After coupling of Gln residues, a dichloromethane flow wash is used
before and after
deprotection using TFA, to prevent possible high temperature (TFA/DMF)-
catalyzed pyrrolidone
carboxylic acid formation. Side-chain protected amino acids are Boc-Arg(p-
toluenesulfonyl)-OH,
Boc-Asn(Xanthyl)-OH, Boc-Asp(O-cyclohexyl)-OH, Boc-Cys(4-methylbenzyl)-OH, Boc-
Glu(0-
cyclohexyl)-OH, Boc-His(dinitrophenylbenzyl)-OH, Boc-Lys(2-Cl-Z)-OH, Boc-
Ser(benzyl)-OH,
Boc-Thr(benzyl)-OH, Boc-Trp(cyclohexylcarbonyl)-OH and Boc-Tyr(2-Br-Z)-OH
(Orpagen
Pharma, Heidelberg, Germany). Other amino acids are used without side chain
protection. C-
terminal Fragment 1 is synthesized on Boc-Leu-0-CHZ-Pam resin (0.71mmol/g of
loaded resin),
while for Fragments 2 through 5 machine-assisted synthesis is started on the
Boc-Xaa-S-CHz-CO-
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Leu-Pam resin. This resin is obtained by the coupling of S-acetylthioglycolic
acid
pentaffuorophenylester to a Leu-PAM resin under standard conditions. The
resulting resin is used as
a starting resin for peptide chain elongation on a 0.2 mmol scale after
removal of the acetyl
protecting group with a 30min treatment with 10% mercaptoethanol, 10%
piperidine in DMF.
After chain assembly is completed, the peptide fragments are deprotected and
cleaved from
the resin by treatment with anhydrous hydrogen fluoride for lhr at 0°C
with 5% p-cresol as a
scavenger. In all cases except Fragment 1, the imidazole side chain 2,4-
dinitrophenyl (DNP)
protecting groups remain on His residues because the DNP-removal procedure is
incompatible with
C-terminal thioester groups. However DNP is gradually removed by thiols during
the ligation
reaction, yielding unprotected His. After cleavage, peptide fragments are
precipitated with ice-cold
diethylether, dissolved in aqueous acetonitrile and lyophilized. The peptide
fragments are purified by
RP-HPLC with a C 18 column from Waters by using linear gradients of buffer B
(acetonitile/0.1
trifluoroacetic acid) in buffer A (Hz0/0.1 % trifluoroacetic acid) and UV
detection at 214nrn.
Samples are analyzed by electrospray mass spectrometry (ESMS) using an Esquire
instrument
(Brucker, Bremen , Germany), or like instrument.
Native chemical ligatiohs
As described more fully below, the ligation of unprotected fragments is
performed as
follows: the dry peptides are dissolved in equimolar amounts in 6M guanidine
hydrochloride
(GuHCI), 0.2M phosphate, pH 7.5 in order to get a final peptide concentration
of 1-8 mM at a pH
around 7, and 1 % benzylinercaptan, 1 % thiophenol is added. Usually, the
reaction is carried out
overnight and is monitored by HPLC and electrospray mass spectrometry. The
ligation product is
subsequently treated to remove protecting groups still present. Opening of the
N terminal thiazolidine
ring further required the addition of solid methoxamine to a O.SM final
concentration at pH3.5 and a
further incubation for 2h at 37°C. A 10-fold excess of Tris(2-
carboxyethyl)phosphine is added
before preparative HPLC purification. Fractions containing the polypeptide
chain are identified by
ESMS, pooled and lyophilized.
The ligation of fragments 4 and 5 is performed at pH7.0 in 6 M GuHCI. The
concentration
of each reactant is 8rnM, and 1 % benzylinercaptan and 1 % thiophenol were
added to create a
reducing environment and to facilitate the ligation reaction. An almost
quantitative ligation reaction
is observed after overnight stirring at 37°C. At this point in the
reaction, CH3-O NHz.HCI is added
to the solution to get a O.SM final concentration, and the pH adjusted to 3.5
in order to open the N-
terminal thiazolidine ring. After 2h incubation at 37°C, ESMS is used
to confirm the completion of
the reaction. The reaction mixture is subsequently treated with a 10-fold
excess of Tris(2-
carboxyethylphosphine) over the peptide fragment and after l5min, the ligation
product is purified
CA 02512629 2005-07-05
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using the preparative HPLC (e.g., C4, 20-60% CH3CN, 0.5 % per min),
lyophilized, and stored at -
20°C.
The same procedure is repeated for the remaining ligations with slight
modifications.
Polypeptide Folding
The full length peptide is refolded by air oxidation by dissolving the reduced
lyophilized protein
(about 0.1 mglmL) in 1M GuHCl, 100mM Tris, l OmM methionine, pH 8.6. After
gentle stirring
overnight, the protein solution is purified by RP-HPLC as described above.
to Example 4: Preparation of CPP antibody compositions
Substantially pure CPP or a portion thereof is obtained. The concentration of
protein in the
final preparation is adjusted, for example, .by concentration on an Amicon
filter device, to the level of
a few micrograms per ml. Monoclonal or polyclonal antibodies to the protein
are then prepared as
described in the sections titled "Monoclonal antibodies" and "Polyclonal
antibodies." '
15 Briefly, to produce an anti-CPP monoclonal antibody, a mouse is
repetitively inoculated with a few
micrograms of the CPP or a poxtion thereof 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 by
means of polyethylene glycol with mouse myeloma cells, and the excess unfused
cells destroyed by
growth of the system on selective media comprising aminopterin (HAT media).
The successfully
20 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, E., Meth. Enzymol. 70: 419 (1980), the disclosure of which is
incozporated herein by
reference in its entixety. Selected positive clones can be expanded and their
monoclonal antibody
25 , product harvested for use. Detailed procedures for monoclonal antibody
production are described in
Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York.
Section 21-2, the
disclosure of which is incorporated herein by reference in its entirety.
For polyclonal antibody production by immunization, polyclonal antiserum
containing
antibodies to heterogeneous epitopes in the CPP or a portion thereof are
prepared by immunizing a
30 mouse with the CPP or a portion thereof, which can be unmodified or
modified to enhance
immunogenicity. Any suitable nonhuman animal, preferably a non human mammal,
may be selected
including rat, rabbit, goat, or horse.
Antibody preparations prepared according to either the monoclonal or the
polyclonal
protocol are useful in quantitative immunoassays which determine
concentrations of CPP in
35 biological samples; or they are also used semi-quantitatively or
qualitatively to identify the presence
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of antigen in a biological sample. The antibodies may also be used in
therapeutic compositions for
killing cells expressing the protein or reducing the levels of the protein in
the body.
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SEQUENCE LISTING
<110> GeneProt, InC.
Bougueleret, Lydie
Cusin, Isabelle
<120> SECRETED POLYPEPTIDE SPECIES REDUCED IN CARDTOVASCULAR DISORDERS
<130> 5031-W001
<150> US 60/438,643
<151> 2003-01-07
<160> 5
<170> PatentTn version 3.1
<210> l
<211> 466
<212> PRT
<213> Homo Sapiens
<400> 1
Met Val Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu Met Ile
1 5 10 15
Pro Trp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala
20 25 30
Gln Tyr Arg Gln Gly Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro
35 40 45
Ser Leu Cys Thr His Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His
50 55 60
Gln Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe
65 70 75 80
Asn Gly Leu Lys Lys Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile
85 90 95
Gly Gly Trp Asn Phe Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr
200 105 110
Ala Asn Asn Arg Gln Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg
115 120 125
Lys Tyr~Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser
130 135 140
Gln Gly Ser Pro Ala Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln
145 150 155 160
Asp Leu Ala Asn Ala Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu
165 170 175
1/6
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Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp
180 185 190
Ala Gly Tyr Glu Val Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn
195 200 205
Leu Met Ala Tyr Asp Phe His Gly Ser Trp Glu Lys Val Thr Gly His
210 215 220
Asn Ser Pro Leu Tyr Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser
225 230 235 240
Leu Asn Val Asp Ala Ala Val Gln Gln Trp Leu Gln Lys Gly Thr Pro
245 250 255
Ala Ser Lys Leu Ile Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr
260 265 270
Leu Ala Ser Ser Sex Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser
275 280 285
Gly Thr Pro Gly Pro Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr
290 295 300
Glu Val Cys Ser Trp Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln
305 310 ~ 315 320
Lys Val Pro Tyr Ile Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp
325 330 335
Val Glu Ser Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu
340 345 350
Gly Gly Ala Met Val Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe
355 360 365
Ser Cys Asn Gln Gly Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu
370 375 380
Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val Pro
385 390 395 400
Lys Pro Gly Gln Pro Ser Glu Pro Glu His Gly Pro Ser Pro Gly GIn
405 410 415
Asp Thr Phe Cys Gln Gly Lys Ala Asp Gly Leu Tyr Pro Asn Pro Arg
420 425 430
Glu Arg Ser Ser Phe Tyr Ser Cys Ala Ala Gly Arg Leu Phe Gln Gln
435 440 445
Ser Cys Pro Thr Gly Leu Val Phe Ser Asn Ser Cys Lys Cys Cys Thr
450 455 460
Trp Asn
465
2/6
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<210> 2
<211> 444
<212> PRT
<213> Homo Sapiens
<400> 2
Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala Gln Tyr Arg Gln Gly Glu
1 5 10 15
Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro Ser Leu Cys Thr His Leu
20 25 30
Ile Tyr Ala Phe Ala Gly Met Thr Asn His Gln Leu Sex Thr Thr Glu
35 40 45
Trp ,Asn Asp Glu Thr Leu Tyr Gln Glu Phe Asn Gly Leu Lys Lys Met
50 55 60
:Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile Gly Gly Trp Asn Phe Gly
65 70 75 80
Thr Gln Lys Phe Thr Asp Met Val Ala Thr Ala Asn Asn Arg Gln Thr
85 90 95
Phe Val Asn Ser Ala Ile Arg Phe Leu Arg Lys Tyr Ser Phe Asp Gly
100 105 110
Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Ala Val
115 120 125
Asp Lys Glu Arg Phe Thr Thr Leu Val Gln Asp Leu Ala Asn Ala Phe
130 135 140
Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser Ala
145 150 155 160
Ala Val Pro Ala Gly Gln Thr Tyr Val Asp Ala Gly Tyr Glu Val Asp
165 170 175
Lys Ile Ala Gln Asn Leu Asp Phe Val Asn Leu Met Ala Tyr Asp Phe
180 . 185 190
His Gly Ser Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr Lys
195 200 205
Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser Leu Asn Val Asp Ala Ala
210 215 220
Val Gln Gln Trp Leu Gln Lys Gly Thr Pro Ala Ser Lys Leu Ile Leu
225 230 235 240
Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr Leu Ala Ser Ser Ser Asp
245 250 255
3/6
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Thr Arg Val Gly Ala Pro Ala Thr Gly Ser Gly Thr Pro Gly Pro Phe
260 265 270
Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp Lys
275 280 285
Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln Lys Val Pro Tyr Ile Phe
290 295 300
Arg Asp Asn Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys Thr
305 310 315 320
Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu Gly Gly Ala Met Val Trp
325 330 335
Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe Ser Cys Asn Gln Gly Arg
340 345 350
Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu Leu Ser Leu Pro Tyr Leu
355 360 365
Pro Ser Gly Thr Pro Glu Leu Glu Val Pro Lys Pro Gly Gln Pro Ser
370 375 380
Glu Pro Glu His Gly Pro Ser Pro Gly Gln Asp Thr Phe Cys Gln Gly
385 390 395 400
Lys Ala Asp Gly Leu Tyr Pro Asn Pro Arg Glu Arg Ser Ser Phe Tyr
405 410 415
Ser Cys Ala Ala Gly Arg Leu Phe Gln Gln Ser Cys Pro Thr Gly Leu
420 425 430
Val Phe Ser Asn Ser Cys Lys Cys Cys Thr Trp Asn
435 440
<210> 3
<211> 146
<212> PRT
<213> Homo Sapiens
<400> 3
Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile Gly Gly Trp Asn Phe
1 5 10 15
Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr Ala Asn Asn Arg Gln
20 25 30
Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg Lys Tyr Ser Phe Asp
35 40 45
Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Ala
50 55 60
4/6
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Slal Asp Lys Glu Arg Phe Thr Thr Leu VaI Gln Asp Leu Ala Asn AIa
65 70 75 80
Phe G1n Gln Glu Ala Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Sex
85 90 95
Ala Ala Val Pro A1a Gly Gln Thr Tyr Val Asp Ala Gly Tyr Glu Val
100 105 110
Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn Leu Met Ala Tyr Asp
115 120 125
Phe His Gly Ser Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr
130 135 140
Lys Arg
145
<210> 4
<211> 102
<212> PRT
<213> Homo Sapiens
<400> 4
Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Gln
1 5 10 15
Gly Ser Pro Ala Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln Asp
20 25 30
Leu Ala Asn Ala Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu Arg
35 40 45
Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp Ala
50 55 60
GIy Tyr GIu Val Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn Leu
65 70 75 80
Met Ala Tyr Asp Phe His Gly Ser Trp Glu Lys Val Thr Gly His Asn
85 90 95
Ser Pro Leu Tyr Lys Arg
100
<210> 5
<211> 21
<212> PRT
<213> Homo Sapiens
<400> 5
Phe Thr Thr Leu Val Gln Asp Leu Ala Asn Ala Phe Gln Gln GIu Ala
1 5 ZO 15
5/6
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Gln Thr Ser Gly Lys
6/6
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Table 1
Peptide Sequence (SEQ
ID CEX Salt RP1 % B Run #
N0:5)
FTTLVQDLANAFQQEAQTSGK9 175 mM 16 49.0 154234
15
FTTLVQDLANAFQQEAQTSGK9 175 mM 16 49.0 154234'13
FTTLVQDLANAFQQEAQTSGK9 175 mM 20 56.7 130966,07
FTTLVQDLANAFQQEAQTSGK9 175 mM 16 49.0 154234'16
FTTLVQDLANAFQQEAQTSGK9 175 mM 16 49.0 154234_14
FTTLVQDLANAFQQEAQTSGK9 175 mM 16 49.0 154234
12
