U.S. patent application number 11/801402 was filed with the patent office on 2008-10-02 for compositions, kits, and methods for identification, assessment, prevention, and therapy of endometriosis.
This patent application is currently assigned to Praecis Pharmaceuticals Incorporated. Invention is credited to Dennis Benjamin, Jeffrey Messer, Eric Sigel, James Vath.
Application Number | 20080241852 11/801402 |
Document ID | / |
Family ID | 34084519 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241852 |
Kind Code |
A1 |
Messer; Jeffrey ; et
al. |
October 2, 2008 |
Compositions, kits, and methods for identification, assessment,
prevention, and therapy of endometriosis
Abstract
The invention relates to newly discovered marker polypeptides
associated with endometriosis. Compositions, kits, and methods for
detecting, characterizing, preventing, and treating endometriosis
are provided.
Inventors: |
Messer; Jeffrey; (Townsend,
MA) ; Benjamin; Dennis; (Redmond, WA) ; Vath;
James; (Lynnfield, MA) ; Sigel; Eric;
(Brookline, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
Praecis Pharmaceuticals
Incorporated
Waltham
MA
|
Family ID: |
34084519 |
Appl. No.: |
11/801402 |
Filed: |
May 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10887775 |
Jul 9, 2004 |
7268117 |
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11801402 |
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60486379 |
Jul 11, 2003 |
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60533430 |
Dec 29, 2003 |
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60575269 |
May 28, 2004 |
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Current U.S.
Class: |
435/7.1 ;
435/4 |
Current CPC
Class: |
G01N 2800/364 20130101;
G01N 33/689 20130101; C07K 14/472 20130101; G01N 33/57442 20130101;
C07K 14/57581 20130101; G01N 2500/00 20130101; C07K 14/75
20130101 |
Class at
Publication: |
435/7.1 ;
435/4 |
International
Class: |
G01N 33/566 20060101
G01N033/566; C12Q 1/00 20060101 C12Q001/00 |
Claims
1. A method of assessing whether a patient is afflicted with
endometriosis, the method comprising comparing: a) the abundance of
one or more marker polypeptides selected from the group consisting
of: iC3b (SEQ ID NO:33), or a fragment thereof; SEETKENEGFTVTAEG
(SEQ ID NO:34), or a fragment thereof; EETKENEGFTVTAEG (SEQ ID
NO:35), or a fragment thereof;
SDKPDMAEMEKFDKSKLKKTETQEKNLPSKETIEQEKQAGES (thymosin beta 1) (SEQ
ID NO:38), or a fragment thereof;
SDKPDMAEIEKFDKPKLKKTETQEKNPLPSKETIEQEKQAGES (thymosin beta 3) (SEQ
ID NO:39), or a fragment thereof; thymosin beta 4 (SEQ ID NO:40),
or a fragment thereof; SDKSDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES
(thymosin beta 6) (SEQ ID NO:41), or a fragment thereof;
TQEKNPLPSKETIEQEKQAGES (SEQ ID NO:42), or a fragment thereof; and
pyr-EKNPLPSKETIEQEKQAGES (SEQ ID NO:43), or a fragment thereof, in
a patient sample, wherein the fragment thereof consists of 7-15
consecutive amino acid residues of the marker polypeptide, and b)
the normal abundance of the one or more of marker polypeptides in a
sample from a control subject not afflicted with endometriosis,
wherein a significantly lower abundance of one or more of said
marker polypeptides in the patient sample as compared to the normal
abundance of one or more of said marker polypeptides indicates that
the patient is afflicted with endometriosis.
2. A method of assessing whether a patient is afflicted with
endometriosis, the method comprising comparing: a) the abundance of
each of a plurality of marker polypeptides independently selected
from the group consisting of: iC3b (SEQ ID NO:33), or a fragment
thereof; SEETKENEGFTVTAEG (SEQ ID NO:34), or a fragment thereof;
EETKENEGFTVTAEG (SEQ ID NO:35), or a fragment thereof;
SDKPDMAEMEKFDKSKLKKTETQEKNLPSKETIEQEKQAGES (thymosin beta 1) (SEQ
ID NO:38), or a fragment thereof;
SDKPDMAEIEKFDKPKLKKTETQEKNPLPSKETIEQEKQAGES (thymosin beta 3) (SEQ
ID NO:39), or a fragment thereof; thymosin beta 4 (SEQ ID NO:40),
or a fragment thereof; SDKSDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES
(thymosin beta 6) (SEQ ID NO:41), or a fragment thereof;
TQEKNPLPSKETIEQEKQAGES (SEQ ID NO:42), or a fragment thereof; and
pyr-EKNPLPSKETIEQEKQAGES (SEQ ID NO:43), or a fragment thereof, in
a patient sample, wherein the fragment thereof consists of 7-15
consecutive amino acid residues of the marker polypeptide, and b)
the normal abundance of each of the plurality of marker
polypeptides in a sample obtained from a control subject not
afflicted with endometriosis, wherein a significantly lower
abundance of one or more of said marker polypeptides in the patient
sample as compared to the normal abundance of one or more of said
marker polypeptides indicates that the patient is afflicted with
endometriosis.
3. The method of claim 1 or 2, wherein the sample comprises a fluid
selected from the group consisting of blood fluids, a blood
fraction, lymph, ascitic fluids, gynecological fluids, urine,
peritoneal fluid, cerebrospinal fluid, and fluids collected by
vaginal rinsing.
4. The method of claim 1 or 2, wherein the sample is blood serum or
blood plasma.
5. The method of claim 1 or 2, wherein the abundance of said marker
polypeptides in the samples is assessed by a method comprising the
step of detecting the presence in the samples of said marker
polypeptides or a fragment thereof.
6. The method of claim 5, wherein the presence of said marker
polypeptide or fragment thereof is detected using a reagent which
specifically binds with said marker polypeptide or fragment
thereof, wherein said reagent is selected from the group consisting
of an antibody, an antibody derivative, and an antibody
fragment.
7. The method of claim 2, wherein the plurality comprises at least
three of the marker polypeptides.
8. The method of claim 7, wherein the plurality comprises at least
five of the marker polypeptides.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 10/887,775, filed on Jul. 9, 2004,
which claims priority from U.S. provisional patent application Ser.
No. 60/575,269, filed on May 28, 2004, U.S. provisional patent
application Ser. No. 60/486,379, filed on Jul. 11, 2003, and from
U.S. provisional patent application Ser. No. 60/533,430, filed on
Dec. 29, 2003. The entire contents of each of the foregoing
applications are expressly incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Endometriosis is a condition afflicting women of
child-bearing age which is characterized by the growth of
endometrial tissue in areas outside the uterus. These extrauterine
endometrial growths are a leading cause of pelvic pain and can also
cause infertility. Endometrial growths can occur in a variety of
locations, including the lining of the pelvic cavity and the outer
surface of the uterus, and can also occur outside the abdomen, for
example, in the lung.
[0003] As is the case with the uterine lining, extrauterine
endometrial growths typically respond to the varying levels of
estrogen associated with the menstrual cycle. Thus, endometrial
growths proceed through a cycle of proliferation and breakdown.
Unlike the uterine lining, however, the body is unable to shed the
extrauterine endometrial growths, and breakdown of this tissue
results in internal bleeding, inflammation of the surrounding area
and formation of scar tissue. A number of complications can also
arise, including rupture of growths, which can spread the growths
to new regions of the body, and the formation of adhesions.
[0004] The most common symptoms of endometriosis include constant
pelvic pain, infertility, low sacral backache, and heavy or
irregular bleeding. The degree of pain does not correspond to the
size or extent of endometrial growths, and significant pain can
result even from microscopic growths. Endometrial implants can
destroy ovarian and tubal tissue. Several disorders of menstrual
cyclicity and ovulation have been suggested as a basis for the
infertility caused by mild endometriosis. More subtle problems in
folliculogenesis in endometriosis patients have been reported,
including lower serum estradiol levels, smaller follicle size
during follicular growth, and lower oocyte fertilization rates and
pregnancy rates in assisted reproduction. Problems with ovum pickup
by the fallopian tube and embryo implantation in the endometrium
have also been suggested.
[0005] Currently, a definitive diagnosis of endometriosis can be
made only upon laparoscopic examination of the abdomen. This is a
surgical procedure performed under local anesthesia and can
indicate the extent and location of extrauterine endometrial
growths. Laparoscopic examination is essential because symptoms of
endometriosis are similar to the symptoms of other conditions,
including ovarian cancer. Prevention of endometriosis is not
currently possible; however, treatment options are available based
on the patient's desire for future fertility, symptoms, the stage
of disease, and to some extent, age. Possible treatment options
include analgesic treatments, such as nonsteroidal
anti-inflammatory agents and prostaglandin synthetase-inhibiting
drugs, and hormonal therapy, which may be given as a means for
interrupting the cycles of stimulation and bleeding of
endometriotic tissue. Common hormonal therapies include oral
contraceptive pills; progestational agents, which cause
decidualization in the endometriotic tissue; danazol, a weak
androgen that is the isoxazole derivative of 17.alpha.-ethinyl
testosterone (ethisterone); and gonadotropin-releasing hormone
(GNRH) agonists, which are analogues of the 10-amino-acid
polypeptide hormone GnRH and act via the suppression of
gonadotropin secretion, resulting in elimination of ovarian
steroidogenesis and suppression of endometrial implants. Lastly,
surgical treatment, including laparoscopic resection, ablation of
minimal or mild endometriosis, presacral neurectomy and uterosacral
ligament ablation, may be performed to excise or destroy all
endometriotic tissue, remove all adhesions, and restore pelvic
anatomy to the best possible condition.
[0006] Despite the treatments available for endometriosis, it would
be beneficial to provide specific non-invasive methods and reagents
for the diagnosis, staging, prognosis, monitoring, and treatment of
endometriosis and endometriosis-related diseases, or to indicate a,
predisposition to such for preventative measures.
SUMMARY OF THE INVENTION
[0007] The invention relates to markers (hereinafter "markers",
"marker polypeptides" or "markers of the invention"), which are
listed in Tables 1-3. The invention provides nucleic acids and
polypeptides that encode or correspond to the markers (hereinafter
"marker nucleic acids" and "marker polypeptides," respectively).
Tables 1-3 provide the sequence identifiers of the sequences of
such marker peptides listed in the accompanying Sequence Listing.
The invention further provides antibodies, antibody derivatives and
antibody fragments which bind specifically with the marker
polypeptides and/or fragments of the marker polypeptides.
[0008] Table 1 lists all of the markers of the invention, whose
over- or under-abundance may be correlated with the diagnosis and
prognosis of endometriosis. In particular, Table 1 provides the
name of the gene corresponding to the marker ("Gene Name"), the
amino acid sequence ("Sequence") and the sequence listing
identifier of the amino acid sequence of the polypeptide marker
("SEQ ID NO (AAs)"). Table 2 lists markers whose over-abundance may
be correlated with endometriosis as compared to normal samples from
control subjects that do not have endometriosis. Table 3 lists
markers whose under-abundance may be correlated with endometriosis
as compared to normal samples from control subjects that do not
have endometriosis.
[0009] The invention also relates to various methods, reagents and
kits for diagnosing, staging, prognosticating, monitoring and
treating endometriosis. "Endometriosis" as used herein includes a
disorder in which abnormal growth of tissue, histologically
resembling the endometrium, is present in locations other than the
uterine lining (see Ainbinder et al., Current Obstetric &
Gynecologic Diagnosis & Treatment, 9.sup.th ed., Lange Medical
Books/McGraw-Hill, 2003). As used herein, an "endometriosis-related
disease" (also referred to herein as a "disorder" or "condition")
may include a disease, disorder, or condition, whose onset was
related to endometriosis. Such diseases, disorders and conditions
include infertility, and abdominal and/or pelvic pain.
[0010] In one embodiment, the invention provides a diagnostic
method of assessing whether a patient has endometriosis or has
higher than normal risk for developing endometriosis, comprising
the steps of comparing the abundance of a marker of the invention
in a patient sample and the normal abundance of the marker in a
control, e.g., a sample from a subject that does not have
endometriosis. A difference in the abundance of the marker in the
patient sample, as compared to the normal abundance, is an
indication that the patient is afflicted with endometriosis or has
higher than normal risk for developing endometriosis.
[0011] The methods of the present invention can be of use in
identifying patients having an enhanced risk of developing
endometriosis (e.g., patients having a familial history of
endometriosis and patients identified as having altered abundance
of a marker of the invention). The methods of the present invention
may further be of particular use in evaluating the specific stage
of endometriosis, as well as in assessing the progression of the
disease. The methods of the present invention are also useful in
predicting the clinical outcome for a patient with endometriosis,
or for a patient who has undergone therapy to eradicate
endometriosis. The methods of the present invention are also useful
in assessing the efficacy of treatment of a patient diagnosed with
endometriosis (e.g., the efficacy of hormonal suppression or
surgical ablation of endometrial implants).
[0012] The markers of the invention set forth in Table 1 may be
used in the methods of the invention. It will be appreciated that
in the methods of the invention, over-abundance of the markers set
forth in Table 2 in the patient sample may be correlated with
endometriosis as compared to normal samples from control subjects.
Likewise, in the methods of the invention, under-abundance of the
markers set forth in Table 3 in the patient sample may be
correlated with endometriosis as compared to normal samples from
control subjects.
[0013] In a preferred diagnostic method of assessing whether a
patient is afflicted with endometriosis (e.g., new detection
("screening"), detection of recurrence), the method comprises
comparing: [0014] a) the abundance of a marker listed in Table 1 in
a sample from the patient, and [0015] b) the normal abundance of
the marker. A different abundance of the marker in the patient
sample, as compared to the level in the control subject, i.e.,
increased or decreased as specified in Tables 2-3, is an indication
that the patient is afflicted with endometriosis.
[0016] The invention additionally provides a diagnostic method for
assessing the aggressiveness of endometriosis, the method
comprising comparing: [0017] a) the abundance of a marker listed in
Table 1 in a sample from the patient, and [0018] b) the normal
abundance of the marker. A different abundance in the patient
sample, as compared to the normal level, i.e., increased or
decreased as specified in Tables 2-3, is an indication that the
patient has an aggressive form of endometriosis or is likely to
develop endometriosis.
[0019] The invention also provides methods for assessing the
efficacy of a therapy for inhibiting endometriosis in a patient.
Such methods comprise comparing: [0020] a) the abundance of a
marker of the invention in a first sample obtained from the patient
prior to providing at least a portion of the therapy to the
patient, and [0021] b) the abundance of the marker in a second
sample obtained from the patient following provision of the portion
of the therapy. An altered abundance of the marker in the second
sample relative to that in the first sample, i.e., increased or
decreased as specified in Tables 2-3, is an indication that the
therapy is efficacious for inhibiting endometriosis in the
patient.
[0022] It will be appreciated that in the methods of the present
invention, the "therapy" may be any therapy for treating
endometriosis including, but not limited to, analgesic treatments,
hormone therapy, surgical removal of endometrial implants, gene
therapy and biologic therapy such as the administering of
antibodies. Thus, the methods of the invention may be used to
evaluate a patient before, during and after therapy, for example,
to evaluate the reduction in endometriosis due to the therapy.
[0023] In a preferred embodiment, the methods are directed to
therapy using a chemical or biologic agent. These methods comprise
comparing: [0024] a) the abundance of a marker of the invention in
a first sample obtained from the patient and maintained in the
presence of the chemical or biologic agent, and [0025] b) the
abundance of the marker in a second sample obtained from the
patient and maintained in the absence of the agent. An altered
abundance of the marker in the second sample relative to that in
the first sample, i.e., increased or decreased as specified in the
above-described methods and in Tables 2-3, is an indication that
the agent is efficacious for inhibiting endometriosis, in the
patient. In one embodiment, the first and second samples can be
portions of a single sample obtained from the patient or portions
of pooled samples obtained from the patient.
[0026] The invention additionally provides a monitoring method for
assessing the progression of endometriosis in a patient, the method
comprising: [0027] a) detecting in a sample from the patient at a
first time point, the abundance of a marker of the invention;
[0028] b) repeating step a) at a subsequent time point in time; and
[0029] c) comparing the abundance detected in steps a) and b), and
therefrom monitoring the progression of endometriosis in the
patient. A different abundance of the marker in the sample at the
subsequent time point from that of the sample at the first time
point, i.e., increased or decreased as specified in the
above-described methods and in Tables 2 and 3, is an indication
that the endometriosis has progressed or regressed in the
patient.
[0030] The invention moreover provides a test method for selecting
a composition for inhibiting endometriosis in a patient. This
method comprises the steps of: [0031] a) obtaining a sample from
the patient; [0032] b) separately maintaining aliquots of the
sample in the presence of a plurality of test compositions; [0033]
c) comparing the abundance of a marker of the invention in each of
the aliquots; and [0034] d) selecting one of the test compositions
which significantly alters the abundance of the marker in the
aliquot containing that test composition, relative to the abundance
of the marker in the presence of the other test compositions.
[0035] In addition, the invention further provides a method of
inhibiting endometriosis in a patient. This method comprises the
steps of: [0036] a) obtaining a sample from the patient; [0037] b)
separately maintaining aliquots of the sample in the presence of a
plurality of compositions; [0038] c) comparing the abundance of a
marker of the invention in each of the aliquots; and [0039] d)
administering to the patient at least one of the compositions which
significantly alters the abundance of the marker in the aliquot
containing that composition, relative to the abundance of the
marker in the presence of the other compositions.
[0040] In the aforementioned methods, the samples or patient
samples comprise cells, tissues and/or fluids obtained from the
patient. The cells may be found in a cervical smear collected, for
example, by a cervical brush. In another embodiment, the sample is
a body fluid. Such fluids include, for example, blood fluids,
serum, plasma, a blood fraction, lymph, ascitic fluids,
gynecological fluids, urine, peritoneal fluid, cerebrospinal fluid,
and fluids collected by vaginal rinsing. In a further embodiment,
the patient sample is in vivo.
[0041] According to the invention, the abundance of a marker of the
invention in a sample can be assessed, for example, by detecting
the presence in the sample of: [0042] the marker polypeptide (e.g.,
a polypeptide having one of the sequences of SEQ ID NO (AAs)) or a
fragment of the polypeptide (e.g. by using a reagent, such as an
antibody, an antibody derivative, an antibody fragment or
single-chain antibody, which binds specifically with the protein or
protein fragment, or via chromatography with a suitable detector)
[0043] a metabolite which is produced directly (i.e., catalyzed) or
indirectly by the corresponding marker polypeptide; or [0044] a
metabolite of the marker polypeptide.
[0045] According to the invention, any of the aforementioned
methods may be performed using a plurality (e.g. 2, 3, 5, or 10 or
more) of markers of the invention, optionally in combination with
endometriosis markers known in the art. In such methods, the
abundance within the sample of each of a plurality of markers, at
least one of which is a marker of the invention, is compared with
the normal abundance of each of the plurality of markers. A
significantly altered (i.e., increased or decreased as specified in
the above-described methods using a single marker) abundance in the
sample of one or more markers of the invention, or some combination
thereof, relative to that marker's corresponding normal or control
level, is an indication that the patient is afflicted with
endometriosis.
[0046] In a further aspect, the invention provides an antibody, an
antibody derivative, or an antibody fragment, which binds
specifically with a marker polypeptide (e.g., a polypeptide having
the sequence of any of the SEQ ID NO (AAs)) or a fragment of the
polypeptide. The invention also provides methods for making such an
antibody, antibody derivative, and antibody fragment. Such methods
may comprise immunizing a mammal with a polypeptide comprising the
entirety, or a fragment, preferably a segment of 7 or more, more
preferably 10 or more, amino acids, of a marker polypeptide (e.g.,
a polypeptide having the sequence of any of the SEQ ID NO (AAs)),
wherein the polypeptide may be obtained from a cell or by chemical
synthesis. The methods of the invention also encompass producing
monoclonal and single-chain antibodies, which would further
comprise isolating splenocytes from the immunized mammal, fusing
the isolated splenocytes with an immortalized cell line to form
hybridomas, and screening individual hybridomas for those that
produce an antibody that binds specifically with a marker
polypeptide or a fragment of the polypeptide.
[0047] In another aspect, the invention relates to various
diagnostic and test kits. In one embodiment, the invention provides
a kit for assessing whether a patient is afflicted with
endometriosis. The kit comprises a reagent for assessing the
abundance of a marker or a plurality of markers of the invention.
In another embodiment, the invention provides a kit for assessing
the suitability of a chemical or biologic agent for inhibiting
endometriosis in a patient. Such a kit comprises a reagent for
assessing the abundance of a marker of the invention, and may also
comprise one or more additional such agents. Such kits comprise an
antibody, an antibody derivative, or an antibody fragment, which
binds specifically with a marker polypeptide, or a fragment of the
polypeptide. Such kits may also comprise a plurality of antibodies,
antibody derivatives, or antibody fragments wherein the plurality
of such antibody agents binds specifically with a marker
polypeptide, or a fragment of the marker polypeptide. In another
embodiment, the kit includes one or more synthetic standards and/or
internal standards.
[0048] It will be appreciated that the methods and kits of the
present invention may also include known endometriosis markers. It
will further be appreciated that the methods and kits may be used
to identify conditions related to endometriosis, such as
infertility or abdominal and/or pelvic pain of unknown
ideology.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIGS. 1-6 depict scatter plots showing the concentrations
(given as counts) of a given marker in the serum of women diagnosed
with endometriosis, the serum of healthy women, and, in certain
cases, the serum of women diagnosed with PCOS or PID.
[0050] FIGS. 1 and 2 demonstrate that certain markers of the
invention, including fibrinopeptide B derivative (SEQ ID NO:28),
full length fibrinopeptide A (SEQ ID NO:3) and fibrinopeptide A
fragment (SEQ ID NO:7), are present in higher concentrations in the
serum of patients diagnosed with endometriosis as compared to
healthy patients that do not have endometriosis.
[0051] FIG. 3 demonstrates that a marker of the invention, the
fibrinopeptide A derivative (SEQ ID NO:4), is present at higher
concentrations in the serum of patients diagnosed with
endometriosis as compared to serum of healthy patients that do not
have endometriosis or patients diagnosed with polycystic ovarian
syndrome (PCOS) and pelvic inflammatory disease (PID).
[0052] FIG. 4 demonstrates that certain markers of the invention,
thymosin fragments (SEQ ID NOS:42 and 43), are present at lower
concentrations in the serum of patients diagnosed with
endometriosis, PCOS and PID as compared to serum of healthy
patients that do not have endometriosis.
[0053] FIG. 5 demonstrates that a marker of the invention,
phosphoserine fibrinopeptide A (SEQ ID NO:22), is present at lower
concentrations in the serum of healthy patients as compared to
serum of patients diagnosed with endometriosis, PCOS or PID.
[0054] FIG. 6 demonstrates that a marker of the invention, the
internal fibrinogen alpha fragment (SEQ ID NO:29), is present at
lower concentrations in the serum of patients diagnosed with
endometriosis as compared to healthy patients that do not have
endometriosis, PCOS or PID.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The invention relates to newly discovered markers, whose
expression is correlated with the disease state of endometriosis.
In particular, the present invention relates to the discovery that
patient samples, e.g., serum samples, from women who have
endometriosis have higher levels of polypeptides derived from
fibrinogen proteolysis than women who do not have endometriosis, as
shown either through an absence of signs and symptoms of
endometriosis or by another diagnostic method, such as laparoscopy.
It has now been found that, compared to women who do not have
endometriosis, patient samples, e.g., serum samples, from women who
have endometriosis have higher levels of certain markers of the
invention and lower levels of certain other markers of the
invention. Thus, it has been discovered that for certain markers of
the invention an altered, e.g., significantly higher than normal or
significantly lower than normal abundance of these markers, or
combination of these markers, correlates with the presence of
endometriosis in a patient. Methods are provided for detecting the
presence or absence of endometriosis in a sample, the stage and
progression of endometriosis, predicting the likely clinical
outcome of a patient diagnosed with endometriosis, and other
characteristics of endometriosis that are relevant to prevention,
diagnosis, characterization, and therapy of endometriosis in a
patient. Methods of treating endometriosis are also provided.
[0056] Table 1 lists all of the markers of the invention, whose
over- or under-abundance correlates with endometriosis as compared
to normal samples from patients that do not have endometriosis.
Table 2 lists markers whose over-expression correlates with
endometriosis as compared to normal samples from control subjects.
Table 3 lists markers whose under-expression correlates with
endometriosis as compared to normal samples from control
subjects.
[0057] In one embodiment, a marker of the invention is fibrinogen,
such as the fibrinogen a (SEQ ID NO:1) or .beta.-chain (SEQ ID
NO:2) or a fragment thereof, such as fibrinopeptide A (SEQ ID
NO:3), fibrinopeptide B (SEQ ID NO:27) or a fragment thereof (SEQ
ID NO:28). The fragment of fibrinopeptide A can be, for example, a
fragment derived from N-terminal truncation of fibrinopeptide A
(SEQ ID NOS:5-21). Furthermore, the markers of the invention
include fibrinopeptide A and fragments thereof, wherein the serine
residue has been converted into dehydroalanine (SEQ ID NO:4) or in
which the serine residue is phosphorylated (SEQ ID NOS:22 to 26).
In another embodiment, the markers of the invention include
fibrinopeptide B (SEQ ID NO:27) and derivatives and fragments
thereof, including des-arginine-L-pyroglutamic acid fibrinopeptide
B (SEQ ID NO:28). In a further aspect, the markers of the invention
include an internal fragment of the fibrinogen a chain, such as a
polypeptide having the sequence set forth in (SEQ ID NO:29).
[0058] The markers of the invention also include polypeptides
derived from Complement component 3 (Complement C3) (SEQ ID NO:30).
Complement C3 is converted by the enzyme C3 convertase to two
protein products, C3a (SEQ ID NO:31) and C3b (SEQ ID NO:32). C3b is
in turn converted by thrombin to the product polypeptides iC3b (SEQ
ID NO:33) and C3f (SEQ ID NO:36). In one embodiment, the markers of
the invention include polypeptides which are N-terminal fragments
of iC3b, such as polypeptides having the sequence set forth in SEQ
ID NOS:34 and 35. The markers of the invention may also include
full length C3f (SEQ ID NO:36) and fragments thereof, including
polypeptides having the sequence set forth as (SEQ ID NO:37). In
yet another embodiment, the markers of the invention include
polypeptides derived from thymosin beta 1 (SEQ ID NO:38), thymosin
beta 3 (SEQ ID NO:39), thymosin beta 4 (SEQ ID NO:40), or thymosin
beta 6 (SEQ ID NO:41), and fragments thereof, including
polypeptides having the sequence set forth as SEQ ID NOS:42 and 43.
The invention also provides isolated nucleic acid molecules which
encode the amino acid sequences set forth herein.
TABLE-US-00001 TABLE 1 SEQ ID NO Gene Name Exemplary Sequence (AAs)
Fibrinogen (alpha chain) GenBank Accession No. P02671 1 Fibrinogen
(beta chain) GenBank Accession No. P02675 2 Fibrinopeptide A
H-ADSGEGDFLAEGGGVR-OH 3 Fibrinopeptide A
H-AD(dehydroA)GEGDFLAEGGGVR-OH 4 (wherein serine residue has been
converted into dehydroalanine) a fragment derived from
H-DSGEGDFLAEGGGVR-QH; 5 an N-terminal H-DSGEGDFLAEGGGV-OH; 6
truncation of H-SGEGDFLAEGGGVR-OH; 7 fibrinopeptide A
H-SGEGDFLAEGGGV-OH; 8 H-GEGDFLAEGGGVR-OH; 9 H-GEGDFLAEGGGV-OH; 10
H-EGDFLAEGGGVR-OH; 11 H-EGDFLAEGGGV-OH; 12 H-GDFLAEGGGVR-OH; 13
H-GDFLAEGGGV-OH; 14 H-DFLAEGGGVR-OH; 15 H-DFLAEGGGV-OH; 16
H-FLAEGGGVR-OH; 17 H-FLAEGGGV-OH; 18 H-LAEGGGV-OH; 19 H-AEGGGV-OH;
20 H-EGGGV-OH. 21 Phosphoserine- H-ADS*GEGDFLAEGGGVR-OH 22
containing (S* = phosphoserine) fibrinopeptide A Phosphoserine-
H-DS*GEGDFLAEGGGVR-OH; 23 containing H-DS*GEGDFLAEGGGV-OH; 24
fibrinopeptide A H-S*GEGDFLAEGGGVR.about.OH; 25 fragments derived
H-S*GEGDFLAEGGGV-OH 26 from an N-terminal (S* = phosphoserine)
truncation thereof fibrinopeptide B QGVNDNEEGFFSAR 27
des-arginine-L- pyr-EGVNDNEEGFFSA-OH 28 pyroglutamic acid
fibrinopeptide B internal fragment H-DEAGSEADHEGTHST-OH 29 of the
fibrinogen alpha chain Complement C3 GenBank Accession No. P01024
30 C3a SVQLTEKRM DKVGKYPKEL RKCCEDGMRE 31 NPMRFSCQRR TRFISLGEAC
KKVFLDCCNY ITELRRQHAR ASHLGLAR C3b See Sequence Listing 32 iC3b See
Sequence Listing 33 N-terminal fragments H-SEETKENEGFTVTAEG-OH; 34
of iC3b H-EETKENEGFTVTAEG-OH 35 C3f H-SSKITHRIHWESASLLR-OH 36 C3f
fragment H-HWESASLL-OH 37 Thymosin beta 1 SDKPDMAEME KFDKSKLKKT
ETQEKNPLPS 38 KETIEQEKQA GES Thymosin beta 3 SDKPDMAEIE KFDKPKLKKT
ETQEKNPLPS 39 KETIEQEKQA GES Thymosin beta 4 GenBank Accession No.
P01253 40 Thymosin beta 6 SDKSDMAEIE KFDKSKLKKT ETQEKNPLPS 41
KETIEQEKQA GES Fragments of thymosin H-TQEKNPLPSKETIEQEKQAGES-OH;
42 Pyr-EKNPLPSKETIEQEKQAGES-OH 43
TABLE-US-00002 TABLE 2 SEQ ID NO Gene Name Exemplary Sequence (AAs)
Fibrinogen (alpha chain) GenBank Accession No. P02671 1 Fibrinogen
(beta chain) GenBank Accession No. P02675 2 Fibrinopeptide A
H-ADSGEGDFLAEGGGVR-OH 3 Fibrinopeptide A (wherein
H-AD(dehydroA)GEGDFLAEGGGVR-OH 4 serine residue has been converted
into dehydroalanine) a fragment derived from H-DSGEGDFLAEGGGVR-OH;
5 an N-terminal truncation H-DSGEGDFLAEGGGV-OH; 6 of fibrinopeptide
A H-SGEGDFLAEGGGVR-OH; 7 H-SGEGDFLAEGGGV-OH; 8 H-GEGDFLAEGGGVR-OH;
9 H-GEGDFLAEGGGV-OH; 10 H-EGDFLAEGGGVR-OH; 11 H-EGDFLAEGGGV-OH; 12
H-GDFLAEGGGVR-OH; 13 H-GDFLAEGGGV-OH; 14 H-DFLAEGGGVR-OH; 15
H-DFLAEGGGV-OH; 16 H-FLAEGGGVR-OH; 17 H-FLAEGGGV-OH; 18
H-LAEGGGV-OH; 19 H-AEGGGV-OH; 20 H-EGGGV-OH 21
Phosphoserine-containing H-ADS*GEGDFLAEGGGVR-OH 22 fibrinopeptide A
(S* = phosphoserine) Phosphoserine-containing
H-DS*GEGDFLAEGGGVR-OH; 23 fibrinopeptide A fragments
H-DS*GEGDFLAEGGGV-OH; 24 derived from an N-terminal
H-S*GEGDFLAEGGGVR-OH; 25 truncation thereof H-S*GEGDFLAEGGGV-OH 26
(S* = phosphoserine) fibrinopeptide B QGVNDNEEGFFSAR 27
des-arginine-L- Pyr-EGVNDNEEGFFSA-OH 28 pyroglutamic acid
fibrinopeptide B internal fragment of the H-DEAGSEADHEGTHST-OH 29
fibrinogen alpha chain Complement C3 GenBank Accession No. P01024
30 C3a SVQLTEKRM DKVGKYPKEL RKCCEDGMRE 31 NPMRFSCQRR TRFISLGEAC
KKVFLDCCNY ITELRRQHAR ASHLGLAR C3b See Sequence Listing 32 C3f
H-SSKITHRIHWESASLLR-OH 36 C3f fragment H-HWESASLL-OH 37
TABLE-US-00003 TABLE 3 SEQ ID NO Gene Name Exemplary Sequence (AAs)
iC3b See Sequence Listing 33 N-terminal fragments of iC3b
H-SEETKENEGFTVTAEG-OH; 34 H-EETKENEGFTVTAEG-OH 35 Thymosin beta 1
SDKPDMAEME KFDKSKLKKT ETQEKNPLPS 38 KETIEQEKQA GES Thymosin beta 3
SDKPDMAEIE KFDKPKLKKT ETQEKNPLPS 39 KETIEQEKQA GES Thymosin beta 4
GenBank Accession No. P01253 40 Thymosin beta 6 SDKSDMAEIE
KFDKSKLKKT ETQEKNPLPS 41 KETIEQEKQA GES Fragments of
H-TQEKNPLPSKETIEQEKQAGES-OH; 42 thymosin
Pyr-EKNPLPSKETIEQEKQAGES-OH 43
[0059] Thus, in one embodiment, a marker polypeptide of the
invention is fibrinogen or a fragment thereof, such as
fibrinopeptide A or a fragment thereof. The fragment of
fibrinopeptide A can be, for example, a fragment derived from an
N-terminal truncation of fibrinopeptide A. For example, the marker
polypeptides of the invention can include one or more polypeptides
having the sequences ADSGEGDFLAEGGGVR (fibrinopeptide A) (SEQ ID
NO:3); DSGEGDFLAEGGGVR (SEQ ID NO:5); DSGEGDFLAEGGGV (SEQ ID NO:6);
SGEGDFLAEGGGVR (SEQ ID NO:7); SGEGDFLAEGGGV (SEQ ID NO:8);
GEGDFLAEGGGVR (SEQ ID NO:9); GEGDFLAEGGGV (SEQ ID NO:10);
EGDFLAEGGGVR (SEQ ID NO:11); EGDFLAEGGGV (SEQ ID NO:12);
GDFLAEGGGVR (SEQ ID NO:13); GDFLAEGGGV (SEQ ID NO:14); DFLAEGGGVR
(SEQ ID NO:15); DFLAEGGGV (SEQ ID NO:16); FLAEGGGVR (SEQ ID NO:17);
FLAEGGGV (SEQ ID NO:18); LAEGGGV (SEQ ID NO:19); AEGGGV (SEQ ID
NO:20); and EGGGV (SEQ ID NO:21). Fibrinopeptide A and fragments
thereof wherein the serine residue has been phosphorylated or
converted into dehydroalanine are also included. Such peptides
include AD(dehydroA)GEGDFLAEGGGVR (SEQ ID NO:4); ADS*GEGDFLAEGGGVR
(phosphorylated fibrinopeptide A) (SEQ ID NO:22); DS*GEGDFLAEGGGVR
(SEQ ID NO:23); DS*GEGDFLAEGGGV (SEQ ID NO:24); S*GEGDFLAEGGGVR
(SEQ ID NO:25); and S*GEGDFLAEGGGV (SEQ ID NO:26), where "dehydroA"
represents dehydroalanine and "S*" represents phosphoserine.
[0060] The marker polypeptides of the invention also include the
fibrinogen .beta.-chain or a fragment thereof, such as
fibrinopeptide B or a fragment thereof. In another embodiment, the
marker polypeptides of the invention include fibrinopeptide B and
derivatives and fragments thereof, including
des-arginine-L-pyroglutamic acid fibrinopeptide B,
pyr-EGVNDNEEGFFSA (SEQ ID NO:28). The N-terminal Gln has been
cyclized to form a pyroglutamate. It will be appreciated that pyr-E
in the sequences of the invention represents pyroglutamate.
[0061] In a further aspect, the marker polypeptides of the
invention include an internal fragment of the fibrinogen
.alpha.-chain, such as a peptide having the sequence
DEAGSEADHEGTHST (SEQ ID NO:29).
[0062] The marker polypeptides of the invention also include
peptides derived from Complement component 3 (Complement C3).
Complement C3 is converted by the enzyme C3 convertase to two
protein products, C3a and C3b. C3b is in turn converted by thrombin
to the product peptides iC3b and C3f. In one embodiment, the
biomarkers of the invention include peptides which are N-terminal
fragments of iC3b, such as peptides having the sequence
SEETKENEGFTVTAEG (SEQ ID NO:34) or EETKENEGFTVTAEG (SEQ ID NO:35).
The biomarkers of the invention also include full length C3f,
having the amino acid sequence SSKITHRIHWESASLLR (SEQ ID NO:36),
and fragments thereof, including peptides having the sequence
HWESASLL (SEQ ID NO:37).
[0063] In yet another embodiment, the marker polypeptides of the
invention include peptides derived from thymosin beta 1, thymosin
beta 3, thymosin beta 4, or thymosin beta 6, and fragments thereof,
including peptides having the sequence TQEKNPLPSKETIEQEKQAGES (SEQ
ID NO:42) or the sequence pyr-EKNPLPSKETIEQEKQAGES (SEQ ID NO:43).
It will be appreciated that pyr-E in the sequences of the invention
represents pyroglutamate.
[0064] Unless otherwise indicated, the marker polypeptides of the
invention have free N- and C-termini. This is shown in Tables 1 to
3 by the N-terminal "H-", which indicates a free amino group at the
N-terminus, and the C-terminal "--OH", which indicates a free
carboxyl group at the C-terminus.
[0065] In another embodiment, the present invention provides
isolated polypeptides which are fragments of fibrinogen, fibrinogen
.alpha.-chain, fibrinopeptide A, fibrinogen .beta.-chain,
fibrinopeptide B, C3a, C3b, iC3b, C3f, thymosin beta 1, thymosin
beta 3, thymosin beta 4, thymosin beta 6 or analogues thereof. The
isolated polypeptides of the invention include polypeptides having
an amino acid sequence selected from the group consisting of
ADSGEGDFLAEGGGVR (fibrinopeptide A) (SEQ ID NO:3); DSGEGDFLAEGGGVR
(SEQ ID NO:5); DSGEGDFLAEGGGV (SEQ ID NO:6); SGEGDFLAEGGGVR (SEQ ID
NO:7); SGEGDFLAEGGGV (SEQ ID NO:8); GEGDFLAEGGGVR (SEQ ID NO:9);
GEGDFLAEGGGV (SEQ ID NO: 10); EGDFLAEGGGVR (SEQ ID NO:11);
EGDFLAEGGGV (SEQ ID NO:12); GDFLAEGGGVR (SEQ ID NO:13); GDFLAEGGGV
(SEQ ID NO:14); DFLAEGGGVR (SEQ ID NO:15); DFLAEGGGV (SEQ ID
NO:16); FLAEGGGVR (SEQ ID NO:17); FLAEGGGV (SEQ ID NO:18); LAEGGGV
(SEQ ID NO:19); AEGGGV (SEQ ID NO:20); EGGGV (SEQ ID NO:21);
D(dehydroA)GEGDFLAEGGGVR (SEQ ID NO:4); ADS*GEGDFLAEGGGVR
(phosphorylated fibrinopeptide A) (SEQ ID NO:22); DS*GEGDFLAEGGGVR
(SEQ ID NO:23); DS*GEGDFLAEGGGV (SEQ ID NO:24); S*GEGDFLAEGGGVR
(SEQ ID NO:25); S*GEGDFLAEGGGV (SEQ ID NO:26); pyr-EGVNDNEEGFFSA
(SEQ ID NO:28); DEAGSEADHEGTHST (SEQ ID NO:29); SEETKENEGFTVTAEG
(SEQ ID NO:34); EETKENEGFTVTAEG (SEQ ID NO:35); SSKITHRIHWESASLLR
(SEQ ID NO:36); HWESASLL (SEQ ID NO:37); TQEKNPLPSKETIEQEKQAGES
(SEQ ID NO:42) and pyr-EKNPLPSKETIEQEKQAGES (SEQ ID NO:43).
[0066] The invention also provides isolated nucleic acid molecules
which encode an amino acid sequence set forth herein.
[0067] In another embodiment, the invention relates to antibodies
that bind to and/or can detect a polypeptide of the invention. The
antibodies can be polyclonal antibodies or monoclonal antibodies,
humanized or chimeric antibodies or fragments thereof.
DEFINITIONS
[0068] As used herein, each of the following terms has the meaning
associated with it in this section.
[0069] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0070] The "abundance" of a polypeptide marker in a sample is the
amount of the marker in the sample, as determined by quantitative
analysis. The abundance can be expressed as the absolute amount of
the marker polypeptide within the sample, or as a relative amount,
such as amount of the polypeptide per unit mass of the sample or
the concentration of the polypeptide within the sample. The
abundance of the polypeptide in the sample can depend upon a number
of factors, such as the level of expression of the polypeptide
within the tissue sampled, the extent of transport of the
polypeptide from its source to the tissue sampled and the extent of
metabolism or processing of a parent protein to produce the
polypeptide.
[0071] An "analogue" of a reference polypeptide is a polypeptide
having substantial identity to the reference polypeptide. For
example, an analogue can exhibit 70%, 75%, 80%, 85%, 90% or 95%
identity to the reference polypeptide. The analogue can also be a
truncated polypeptide resulting from C- and/or N-terminal
truncation of the reference polypeptide. An analogue can also have
one or more amino acid substitutions compared to the reference
polypeptide, such as the substitution of one or more residues with
another naturally occurring L-configuration residue, or a
non-natural amino acid residue, such as a D-configuration residue
or a D- or L-configuration residue bearing a side chain which is
different from the side chains of the twenty naturally-occurring
L-amino acids. In one embodiment, the analogue results from
conservative substitution of one or more residues in the reference
polypeptide. The term "conservative substitution" is known in the
art and relates to the substitution of an amino acid residue with a
residue bearing a side chain of similar properties. In a preferred
embodiment, an analogue of a reference polypeptide is
immunologically cross-reactive with the reference polypeptide, that
is, antibodies raised to the analogue are able to bind to and/or
detect the reference polypeptide.
[0072] A "marker", as this term is used herein, is a polypeptide
whose altered abundance in a tissue, cell or bodily fluid compared
to its abundance in normal or healthy tissue, cell or bodily fluid
is associated with a disease state, such as endometriosis. A
"marker nucleic acid" is a nucleic acid (e.g., mRNA, cDNA) which
encodes a marker of the invention. Such marker nucleic acids
include DNA (e.g., cDNA) encoding the entire or a partial sequence
of any of the SEQ ID NO (AA) or the complement of such a sequence.
The marker nucleic acids also include RNA corresponding to the
entire or a partial sequence of any marker nucleic acid sequence or
the complement of such a sequence, wherein all thymidine residues
are replaced with uridine residues.
[0073] The term "probe" refers to any molecule which is capable of
selectively binding to a specifically intended target molecule, for
example, a polynucleotide, a polynucleotide transcript or a
polypeptide. Probes can be either synthesized by one skilled in the
art, or derived from appropriate biological preparations. For
purposes of detection of the target molecule, probes may be
specifically designed to be labeled, as described herein. Examples
of molecules that can be utilized as probes include, but are not
limited to, RNA, DNA, proteins, antibodies, and organic
molecules.
[0074] As used herein, a "patient sample" or "bodily fluid" can be
any bodily fluid in which differences in the amount of at least one
marker of the invention is indicative of the presence of
endometriosis. Suitable bodily fluids include blood, a blood
fraction, urine, saliva, tears, and cerebrospinal fluid. In a
preferred embodiment, the bodily fluid is blood or a fraction
thereof, such as serum or plasma. More preferably, the bodily fluid
is serum. An "endometrial-associated fluid" is a fluid which, when
in the body of a patient, contacts or passes through the
endometrium or into which cells, nucleic acids or proteins are shed
from endometrial cells. Exemplary endometrial-associated body
fluids include peritoneal fluid, which is a fluid obtained from the
peritoneal cavity, the resulting fluids obtained from a PAP smear
procedure, gynecological fluids, and fluids collected by vaginal
rinsing.
[0075] The term "normal abundance" of a marker is the abundance of
the marker in a particular sample, such as blood serum, which is
indicative of the absence of endometriosis. For example, the normal
abundance of a marker is the abundance found in a sample from a
control subject not afflicted with endometriosis. Preferably, the
normal abundance is an average value or a range of values found by
analysis of samples derived from a plurality of control subjects
not afflicted with endometriosis.
[0076] The terms "significantly greater abundance" and
"overabundance" of a marker refers to an amount of the marker in a
test sample that is greater than the normal abundance of the marker
by an amount equal to at least the standard error of the assay
employed to determine abundance, and is preferably at least twice,
the normal abundance of the marker.
[0077] The terms "significantly lower abundance" and
"under-abundance" of a marker refer to an amount of the marker in a
test sample that is that is less than the normal abundance of the
marker by an amount equal to at least the standard error of the
assay employed to determine abundance, and is preferably 50% or
less of the normal abundance of the marker.
[0078] The term "altered abundance" refers to a significantly
greater abundance or a significantly lower abundance when compared
to the normal abundance.
[0079] When comparing the abundance of a marker in a subject to the
normal abundance, the comparison is made using like samples. For
example, the abundance of a given marker in a serum sample derived
from a subject is compared to the normal abundance of the marker in
serum. Thus, a marker which is over-abundant or under-abundant in
the serum of a subject has an altered abundance relative to the
normal abundance in serum.
[0080] As used herein, a "patient," "subject" or "female subject"
can be any female mammal, and is preferably a human female. More
preferably, the subject is a human female of child-bearing age,
i.e., a post-pubescent human female who has not yet entered
menopause. The woman may be selected by a physician for evaluation
by the method of the invention on the basis of the presence of one
or more symptoms of endometriosis, such as abdominal or pelvic
pain, irregular menstruation, or infertility.
[0081] As used herein, the term "promoter/regulatory sequence"
means a nucleic acid sequence which is required for expression of a
gene product operably linked to the promoter/regulatory sequence.
In some instances, this sequence may be the core promoter sequence
and in other instances, this sequence may also include an enhancer
sequence and other regulatory elements which are required for
expression of the gene product. The promoter/regulatory sequence
may, for example, be one which expresses the gene product in a
tissue-specific manner.
[0082] A "constitutive" promoter is a nucleotide sequence which,
when operably linked with a polynucleotide which encodes or
specifies a gene product, causes the gene product to be produced in
a living human cell under most or all physiological conditions of
the cell.
[0083] An "inducible" promoter is a nucleotide sequence which, when
operably linked with a polynucleotide which encodes or specifies a
gene product, causes the gene product to be produced in a living
human cell substantially only when an inducer which corresponds to
the promoter is present in the cell.
[0084] A chemical entity, such as a protein, polypeptide or
antibody, is "isolated" if a composition comprising the entity is
substantially free of other macromolecules, such as other proteins.
A chemical entity is "purified" in a composition in which the
entity is present in substantially greater relative concentration
than it exists in its natural state, for example in a body fluid of
a subject. Preferably the chemical entity comprises at least 80%,
more preferably at least 90%, and even more preferably at least 95%
by weight of the macromolecular species present in the composition.
Most preferably, the chemical entity is purified to homogeneity,
i.e., other macromolecular species are not significantly detectable
using standard techniques, such as polyacrylamide gel
electrophoresis and high performance liquid chromatography.
[0085] A "transcribed polynucleotide" or "nucleotide transcript" is
a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such
RNA or cDNA) which is complementary to or homologous with all or a
portion of a mature mRNA made by transcription of a marker of the
invention and normal post-transcriptional processing (e.g.
splicing), if any, of the RNA transcript, and reverse transcription
of the RNA transcript.
[0086] "Homologous" as used herein, refers to amino acid sequence
similarity between regions of two different polypeptide sequences.
When an amino acid residue position in both regions is occupied by
the same amino acid residue, then the regions are homologous at
that position. A first region is homologous to a second region if
at least one amino acid residue position of each region is occupied
by the same residue. Homology between two regions is expressed in
terms of the proportion of amino acid residue positions of the two
regions that are occupied by the same amino acid residue.
Preferably, the first region comprises a first portion and the
second region comprises a second portion, whereby, at least about
50%, and preferably at least about 75%, at least about 90%, or at
least about 95% of the amino acid residue positions of each of the
portions are occupied by the same amino acid residue.
[0087] A molecule is "fixed" or "affixed" to a substrate if it is
covalently or non-covalently associated with the substrate such the
substrate can be rinsed with a fluid (e.g. standard saline citrate,
pH 7.4) without a substantial fraction of the molecule dissociating
from the substrate.
[0088] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in an organism found in nature.
[0089] The term "synthetic standard" refers to a synthetic or
recombinant polypeptide which is structurally equivalent to one of
the marker polypeptides of the invention. The term "internal
standard" refers to a compound which is chemically and structurally
similar to a marker polypeptide, but which differs from the marker
polypeptide in mass.
[0090] Endometriosis is "inhibited" if at least one symptom of the
disease is alleviated, terminated, slowed, or prevented. As used
herein, endometriosis is also "inhibited" if recurrence or
progression of the disease is reduced, slowed, delayed, or
prevented.
[0091] A kit is any manufacture (e.g. a package or container)
comprising at least one reagent, e.g. a probe, an antibody, a
synthetic standard or an internal standard, for specifically
determining the abundance of a marker of the invention. The kit may
be promoted, distributed, or sold as a unit for performing the
methods of the present invention.
[0092] "Polypeptides of the invention" encompass marker
polypeptides, such as the polypeptides set forth in Table 1 and
their fragments; variant marker polypeptides, such as polypeptides
which are homologous to the polypeptides set forth in Table 1, for
example, polypeptides which can be used as internal standards, and
their fragments; polypeptides and polypeptides comprising an at
least 7, 10 or 15 amino acid segment of a marker or variant marker
polypeptide; and fusion proteins comprising a marker or variant
marker polypeptide, or an at least 7, 10 or 15 amino acid segment
of a marker or variant marker polypeptide.
[0093] Unless otherwise specified herein, the terms "antibody" and
"antibodies" broadly encompass naturally-occurring forms of
antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies
such as single-chain antibodies, chimeric and humanized antibodies
and multi-specific antibodies, as well as fragments and derivatives
of all of the foregoing, which fragments and derivatives have at
least an antigenic binding site. Antibody derivatives may comprise
a protein or chemical moiety conjugated to an antibody.
DESCRIPTION
[0094] The present invention is based, in part, on newly identified
markers which are over- or under-abundant in samples, such as blood
serum, from patients who have endometriosis as compared to
unaffected control samples, e.g., serum samples from control
subjects that do not have endometriosis. The altered abundance of
one or more of these markers in a patient sample is herein
correlated with the endometriosis. An altered abundance of some of
these markers is also correlated with the stage and clinical
outcome of the patient. The invention provides compositions, kits,
and methods for assessing the stage of the endometriosis, as well
as for treating patients afflicted with endometriosis.
[0095] The compositions, kits, and methods of the invention have
the following uses, among others: [0096] 1) assessing whether a
patient is afflicted with endometriosis; [0097] 2) assessing the
stage of endometriosis in a human patient; [0098] 3) predicting the
clinical outcome of a patient diagnosed with endometriosis; [0099]
4) assessing the nature of endometriosis in a patient; [0100] 5)
making antibodies, antibody fragments or antibody derivatives that
are useful for assessing whether a patient is afflicted with
endometriosis; [0101] 6) assessing the efficacy of one or more test
compounds for inhibiting endometriosis in a patient; [0102] 7)
assessing the efficacy of a therapy for inhibiting endometriosis in
a patient; [0103] 8) monitoring the progression of endometriosis in
a patient; and [0104] 9) selecting a composition or therapy for
inhibiting endometriosis in a patient.
[0105] The invention thus includes a method of assessing whether a
patient is afflicted with endometriosis. This method comprises
comparing the abundance of a marker of the invention (listed in
Table 1) in a patient sample and the normal abundance of the
marker. An altered abundance of the marker in the patient sample as
compared to the normal abundance is an indication that the patient
is afflicted with endometriosis.
[0106] The invention further includes polypeptides comprising the
entirety, or a segment of any of the sequences of SEQ ID NO (AAs)
and homologues thereof. Gene delivery vehicles, host cells and
compositions (all described herein) containing nucleic acids
encoding the polypeptides of the invention are also included.
[0107] As described herein, endometriosis in a patient is
associated with an altered abundance of one or more markers of the
invention in a sample or tissue obtained from the patient.
[0108] Any marker or combination of markers of the invention, as
well as any known markers in combination with the markers of the
invention, may be used in the compositions, kits, and methods of
the present invention. In general, it is preferable to use markers
for which the difference between the abundance of the marker in a
sample from a patient having endometriosis and the normal abundance
of the same marker is as great as possible. Although this
difference can be as small as the limit of detection of the method
for assessing abundance of the marker, it is preferred that the
difference be at least greater than the standard error of the
assessment method, and preferably a difference of at least 2-, 3-,
4-, 5-, 6-, 7-, 8-, 9-, or 10-fold or greater than the normal
abundance of the same marker.
[0109] It will be appreciated that a variety of patient samples may
be used in the methods of the present invention. In these
embodiments, the abundance of the marker can be assessed by
assessing the amount (e.g. absolute amount or concentration) of the
marker in the patient sample, e.g., a bodily fluid, such as a serum
sample. The bodily fluid can be obtained from the subject using any
available method, which may be selected on the basis of the amount
of fluid required. In certain cases, a collected blood sample may
be used in a variety of tests and only a portion or aliquot of the
sample drawn will be required for use in the methods described
herein. The amount of the marker(s) of the invention can be
determined in whole blood or in a fraction of the blood.
Preferably, the amount of marker is determined for a cell-free
fraction of the blood, such as the plasma or the serum. It is
particularly preferred to determine the amount of marker(s) in the
serum. In embodiments in which only a fraction of the blood is used
in the analysis, the method also includes the steps of separating
the desired blood fraction from the whole blood acquired from the
subject. This separation of blood fractions can be achieved using
methods which are well-known in the art.
[0110] The bodily fluid may be further processed, as is known in
the art, prior to the measurement of the marker. For example, the
fluid can be processed to remove a particular protein, such as
serum albumin, a set of proteins, or cells or cell components which
are present in the fluid and which may interfere with the analysis.
The processing can include steps such as precipitation,
chromatography, centrifugation, ultrafiltration and dialysis.
[0111] The cell sample can, of course, be subjected to a variety of
well-known post-collection preparative and storage techniques
(e.g., nucleic acid and/or protein extraction, fixation, storage,
freezing, ultrafiltration, concentration, evaporation,
centrifugation, etc.) prior to assessing the amount of the marker
in the sample.
[0112] In one embodiment the abundance of the marker(s) of the
invention in the sample is determined using chromatography, such as
liquid chromatography or gas chromatography, with a suitable
detection system. In one embodiment, the chromatographic step
separates the bodily fluid into fractions, and at least one of the
fractions comprises the marker polypeptide. The fraction comprising
the marker can be identified using, for example, mass spectrometry,
and the amount of marker present can be determined using methods
which are well known in the art, for example, by comparing the ion
current generated in the mass spectrometer for the marker to that
generated by an internal standard of known concentration.
[0113] In another embodiment, the abundance of a marker is assessed
using an antibody (e.g. a radio-labeled, chromophore-labeled,
fluorophore-labeled, or enzyme-labeled antibody), an antibody
derivative (e.g. an antibody conjugated with a substrate or with
the protein or ligand of a protein-ligand pair {e.g.
biotin-streptavidin}), or an antibody fragment (e.g. a single-chain
antibody, an isolated antibody hypervariable domain, etc.) which
binds specifically with a marker protein or fragment thereof,
including a marker protein which has undergone all or a portion of
its normal post-translational modification.
[0114] Because the compositions, kits, and methods of the invention
rely on detection of a difference in the abundance of one or more
markers of the invention, it is preferable that the abundance of
the marker is significantly greater than the minimum detection
limit of the method used to assess abundance.
[0115] It is understood that by routine screening of additional
patient samples using one or more of the markers of the invention,
it will be realized that certain of the markers are associated with
endometriosis of various stages based on a weighted point system.
The number, size, and location of endometrial implants, plaques,
endometriomas, and/or adhesions are considered. According to the
American Society for Reproductive Medicine revised classification
of endometriosis, Stage I (minimal): 1-5; stage 11 (mild): 6-15;
stage III (moderate): 16-40; stage 1V (severe): >40 (Revised
ASRM classification. Fertil Steri 1997; 67:819.) In addition, as a
greater number of patient samples are assessed for expression of
the markers of the invention and the outcomes of the individual
patients from whom the samples were obtained are correlated, it
will also be confirmed that altered abundance of certain of the
markers of the invention is strongly correlated with endometriosis
and other endometriosis-related diseases or conditions. The
compositions, kits, and methods of the invention are thus useful
for characterizing the stage, and nature of endometriosis in
patients.
[0116] When the compositions, kits, and methods of the invention
are used for characterizing the stage and nature of endometriosis
in a patient, it is preferred that the marker or panel of markers
of the invention is selected such that a positive result is
obtained in at least about 20%, and preferably at least about 40%,
60%, or 80%, and more preferably in substantially all patients
afflicted with endometriosis of the corresponding stage and
nature.
[0117] When a plurality of markers of the invention are used in the
compositions, kits, and methods of the invention, the abundance of
each marker in a patient sample can be compared with the normal
abundance of each of the plurality of markers in normal samples of
the same type, either in a single reaction mixture (i.e. using
reagents, such as different fluorescent probes, for each marker) or
in individual reaction mixtures corresponding to one or more of the
markers. In one embodiment, a significantly increased abundance of
more than one of the plurality of markers in the sample, relative
to the corresponding normal levels, is an indication that the
patient is afflicted with endometriosis. In another embodiment, a
significantly lowered abundance of more than one of the plurality
of markers in the sample, relative to the corresponding normal
levels, is an indication that the patient is afflicted with
endometriosis. In another embodiment, a significantly lowered
abundance of a first marker in the sample and a significantly
increased abundance of a second marker, relative to the
corresponding normal levels, is an indication that the patient is
afflicted with endometriosis. When a plurality of markers is used,
it is preferred that 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, or
50 or more individual markers be used. In general, it is preferable
to use the fewest markers required to provide a test with the
desired characteristics.
[0118] In one preferred embodiment, a marker polypeptide derived
from each of two or more parent proteins can be monitored. For
example, one set of marker polypeptides which can be monitored
includes H-ADSGEGDFLAEGGGVR-OH (SEQ ID NO:22); Pyr-EGVNDNEEGFFSA-OH
(SEQ ID NO:28); H-HWESASLL-OH (SEQ ID NO:37); H-EETKENEGFTVTAEG-OH
(SEQ ID NO:35); and H-TQEKNPLPSKETIEQEKQAGES-OH (SEQ ID NO:42).
[0119] Markers associated with the presence of endometriosis have
been described, including those disclosed in U.S. Pat. Nos.
5,843,673; 5,618,689 and 6,525,187 and published PCT applications
WO 00/43789; WO 99/55902; WO 00/63675; WO 96/20404; WO 99/63116; WO
01/62959; WO 95/13821; and WO 00/47739, the contents of all of
which are incorporated herein by reference. These markers are not,
of course, included among the markers of the invention, although
they may be used together with one or more markers of the invention
in a panel of markers, for example.
[0120] It is recognized that the compositions, kits, and methods of
the invention will be of particular utility to patients having an
enhanced risk of developing endometriosis and their medical
advisors. Patients recognized as having an enhanced risk of
developing endometriosis include, for example, patients having a
familial history of endometriosis, patients identified as having
altered abundance of a marker of the invention, and patients with a
history of pelvic pain, abdominal pain, irregular menstruation
and/or infertility.
[0121] The normal abundance of a marker in a particular type of
biological sample (i.e., in samples from patients that do not have
endometriosis) can be assessed in a variety of ways. In one
embodiment, as further information becomes available as a result of
routine performance of the methods described herein,
population-average values for normal abundance of the markers of
the invention may be used. In other embodiments, the normal
abundance of a marker may be determined by assessing abundance of
the marker in a patient sample obtained from a
non-endometriosis-afflicted patient, from a patient sample obtained
from a patient before the suspected onset of endometriosis in the
patient, from archived patient samples, and the like.
[0122] The invention includes compositions, kits, and methods for
assessing the presence of endometriosis in a sample (e.g. an
archived tissue sample or a sample obtained from a patient). These
compositions, kits, and methods are substantially the same as those
described above, except that, where necessary, the compositions,
kits, and methods are adapted for use with samples other than
patient samples. For example, when the sample to be used is a
parafinized, archived human tissue sample, it can be necessary to
adjust the ratio of compounds in the compositions of the invention,
in the kits of the invention, or the methods used to assess levels
of marker expression in the sample. Such methods are well known in
the art and within the skill of the ordinary artisan.
[0123] The invention includes a kit for assessing the presence of
endometriosis (e.g. in a sample such as a patient sample). The kit
comprises a plurality of reagents, each of which is capable of
binding specifically with a marker polypeptide. Suitable reagents
for binding with a marker polypeptide include antibodies, antibody
derivatives, antibody fragments, and the like.
[0124] The kit of the invention may optionally comprise additional
components useful for performing the methods of the invention. By
way of example, the kit may comprise fluids (e.g. SSC buffer)
suitable for binding an antibody with a polypeptide with which it
specifically binds, one or more sample compartments, instructional
material, such as a instruction manual, which describes performance
of a method of the invention, and a positive and negative
control.
[0125] The invention also includes a method of making an isolated
hybridoma which produces an antibody useful for assessing whether a
patient is afflicted with endometriosis. In this method, a protein
or polypeptide comprising the entirety or a segment of a marker
polypeptide is synthesized or isolated (e.g. by purification from a
cell in which it is expressed or by transcription and translation
of a nucleic acid encoding the protein or polypeptide in vivo or in
vitro using known methods). A vertebrate, preferably a mammal such
as a mouse, rat, rabbit, or sheep, is immunized using the protein
or polypeptide. The vertebrate may optionally (and preferably) be
immunized at least one additional time with the protein or
polypeptide, so that the vertebrate exhibits a robust immune
response to the protein or polypeptide. Splenocytes are isolated
from the immunized vertebrate and fused with an immortalized cell
line to form hybridomas, using any of a variety of methods well
known in the art. Hybridomas formed in this manner are then
screened using standard methods to identify one or more hybridomas
which produce an antibody which specifically binds with the marker
protein or a fragment thereof. The invention also includes
hybridomas made by this method and antibodies made using such
hybridomas.
[0126] The invention also includes a method of assessing the
efficacy of a test compound for inhibiting endometriosis. As
described above, differences in the abundance of the markers of the
invention correlate with endometriosis. Although it is recognized
that changes in the levels of certain of the markers of the
invention likely result from endometriosis, it is likewise
recognized that changes in the abundance of other of the markers of
the invention may be directly or indirectly associated with the
induction, maintenance, and/or promotion of endometriosis. Thus,
compounds which inhibit endometriosis in a patient will cause the
abundance of one or more of the markers of the invention to change
to a level nearer the normal abundance level for that marker.
[0127] This method thus comprises comparing abundance of a marker
in a first patient sample and maintained in the presence of the
test compound and expression of the marker in a second patient
sample and maintained in the absence of the test compound. A
significantly altered abundance of a marker of the invention in the
presence of the test compound is an indication that the test
compound inhibits endometriosis. The patient samples may, for
example, be aliquots of a single sample of normal endometrial cells
obtained from a patient, pooled samples of normal endometrial cells
obtained from a patient, cells of a normal endometrial cell line,
aliquots of a single sample of endometrial cells obtained from a
patient, pooled samples of endometrial cells obtained from a
patient, cells of an endometrial cell line, a serum sample, or the
like. In one embodiment, the samples include serum from the patient
or endometrial cells obtained from a patient and a plurality of
compounds known to be effective for inhibiting endometriosis are
tested in order to identify the compound which is likely to best
inhibit the endometriosis in the patient.
[0128] This method may likewise be used to assess the efficacy of a
therapy for inhibiting endometriosis in a patient. In this method,
the abundance of one or more markers of the invention in a pair of
samples (one subjected to the therapy, the other not subjected to
the therapy) is assessed. As with the method of assessing the
efficacy of test compounds, if the therapy induces a significantly
altered abundance (i.e., causes the abundance to approach normal
values) of a marker of the invention then the therapy is
efficacious for inhibiting endometriosis. As above, if samples from
a selected patient are used in this method, then alternative
therapies can be assessed in vitro in order to select a therapy
most likely to be efficacious for inhibiting endometriosis in the
patient.
[0129] Various aspects of the invention are described in further
detail in the following subsections.
I. Isolated Nucleic Acid Molecules
[0130] One aspect of the invention pertains to isolated nucleic
acid molecules, including nucleic acids which encode a marker
polypeptide or a portion (fragment) thereof. Isolated nucleic acids
of the invention also include nucleic acid molecules sufficient for
use as hybridization probes to identify these nucleic acid
molecules and fragments of these nucleic acid molecules, e.g.,
those suitable for use as PCR primers for the amplification or
mutation of the nucleic acid molecules. As used herein, the term
"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.
[0131] 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 molecule. Preferably, an
"isolated" nucleic acid molecule is free of sequences (preferably
protein-encoding 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 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.
[0132] A nucleic acid molecule of the present invention can be
isolated using standard molecular biology techniques and the
sequence information in the database records described herein.
Using all or a portion of such nucleic acid sequences, nucleic acid
molecules of the invention can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd
ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0133] A nucleic acid molecule of the invention can be amplified
using cDNA, mRNA, or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, nucleotides corresponding to all or a portion of a
nucleic acid molecule of the invention can be prepared by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
[0134] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
has a nucleotide sequence complementary to the nucleotide sequence
of a marker nucleic acid or to the nucleotide sequence of a nucleic
acid encoding a marker polypeptide. A nucleic acid molecule which
is complementary to a given nucleotide sequence is one which is
sufficiently complementary to the given nucleotide sequence that it
can hybridize to the given nucleotide sequence thereby forming a
stable duplex.
[0135] Moreover, a nucleic acid molecule of the invention can
comprise only a portion of a nucleic acid sequence, wherein the
full length nucleic acid sequence comprises a marker nucleic acid
or which encodes a marker polypeptide. Such nucleic acids can be
used, for example, as a probe or primer. The probe/primer typically
is used as one or more substantially purified oligonucleotides. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 7,
preferably about 15, more preferably about 25, 50, 75, 100, 125,
150, 175, 200, 250, 300, 350, or 400 or more consecutive
nucleotides of a nucleic acid of the invention.
[0136] Probes based on the sequence of a nucleic acid molecule of
the invention can be used to detect transcripts or genomic
sequences corresponding to one or more markers of the invention.
The probe comprises a label group attached thereto, e.g., a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as part of a diagnostic test kit
for identifying cells or tissues which mis-express the polypeptide,
such as by measuring levels of a nucleic acid molecule encoding the
polypeptide in a sample of cells from a subject, e.g., detecting
mRNA levels or determining whether a gene encoding the polypeptide
has been mutated or deleted.
[0137] It will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequence can exist within a population (e.g., the human
population). Such genetic polymorphisms can exist among individuals
within a population due to natural allelic variation. An allele is
one of a group of genes which occur alternatively at a given
genetic locus. In addition, it will be appreciated that DNA
polymorphisms that affect RNA expression levels can also exist that
may affect the overall expression level of that gene (e.g., by
affecting regulation or degradation).
[0138] As used herein, the phrase "allelic variant" refers to a
nucleotide sequence which occurs at a given locus or to a
polypeptide encoded by the nucleotide sequence.
[0139] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding a polypeptide corresponding to a marker of the invention.
Such natural allelic variations can typically result in 1-5%
variance in the nucleotide sequence of a given gene. Alternative
alleles can be identified by sequencing the gene of interest in a
number of different individuals. This can be readily carried out by
using hybridization probes to identify the same genetic locus in a
variety of individuals. Any and all such nucleotide variations and
resulting amino acid polymorphisms or variations that are the
result of natural allelic variation and that do not alter the
functional activity are intended to be within the scope of the
invention.
[0140] In another embodiment, an isolated nucleic acid molecule of
the invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150,
200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200,
1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000,
4500, or more nucleotides in length and hybridizes under stringent
conditions to a to a nucleic acid encoding a marker polypeptide. As
used herein, the term "hybridizes under stringent conditions" is
intended to describe conditions for hybridization and washing under
which nucleotide sequences at least 60% (65%, 70%, preferably 75%)
identical to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in sections 6.3.1-6.3.6 of Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989). A preferred,
non-limiting example of stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50-65.degree. C.
[0141] In addition to naturally-occurring allelic variants of a
nucleic acid molecule of the invention that can exist in the
population, the skilled artisan will further appreciate that
sequence changes can be introduced by mutation thereby leading to
changes in the amino acid sequence of the encoded polypeptide,
without altering the biological activity of the polypeptide encoded
thereby. For example, one can make nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues.
A "non-essential" amino acid residue is a residue that can be
altered from the wild-type sequence without altering the biological
activity, whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are not
conserved or only semi-conserved among homologs of various species
may be non-essential for activity and thus would be likely targets
for alteration. Alternatively, amino acid residues that are
conserved among the homologs of various species (e.g., murine and
human) may be essential for activity and thus would not be likely
targets for alteration.
[0142] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding a variant marker polypeptide that
contains changes in amino acid residues that are not essential for
activity. Such variant marker polypeptides differ in amino acid
sequence from the naturally-occurring marker polypeptides, yet
retain biological activity. In one embodiment, such a variant
marker polypeptide has an amino acid sequence that is at least
about 40% identical, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical
to the amino acid sequence of a marker polypeptide.
[0143] An isolated nucleic acid molecule encoding a variant marker
polypeptide can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of marker nucleic acids, such that one or more amino acid residue
substitutions, additions, or deletions are introduced into the
encoded polypeptide. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
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), non-polar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded polypeptide can be expressed recombinantly and the activity
of the polypeptide can be determined.
II. Isolated Polypeptides and Antibodies
[0144] One aspect of the invention pertains to isolated marker
polypeptides and biologically active portions thereof, as well as
polypeptide fragments suitable for use as immunogens to raise
antibodies directed against a marker polypeptide or a fragment
thereof. In one embodiment, the native marker polypeptide can be
isolated from cells or tissue sources by an appropriate
purification scheme using standard polypeptide purification
techniques. In another embodiment, a polypeptide comprising the
whole or a segment of the marker polypeptide is produced by
recombinant DNA techniques. Alternative to recombinant expression,
such a polypeptide can be synthesized chemically using standard
polypeptide synthesis techniques.
[0145] An "isolated" or "purified" polypeptide or biologically
active portion thereof is substantially free of cellular material
or other contaminating proteins from the cell or tissue source from
which the polypeptide is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of polypeptide in which the polypeptide is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. Thus, polypeptide that is substantially
free of cellular material includes preparations of polypeptide
having less than about 30%, 20%, 10%, or 5% (by dry weight) of
heterologous polypeptide (also referred to herein as a
"contaminating polypeptide"). When the polypeptide 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%, 10%, or 5% of the volume of
the polypeptide preparation. When the polypeptide is produced by
chemical synthesis, it is preferably substantially free of chemical
precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the polypeptide. Accordingly such preparations of the
polypeptide have less than about 30%, 20%, 10%, 5% (by dry weight)
of chemical precursors or compounds other than the polypeptide of
interest.
[0146] Preferred marker polypeptides are those having an amino acid
sequence of any of the SEQ ID NO (AAs). Other useful polypeptides
are substantially identical (e.g., at least about 40%, preferably
50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and
retain the functional activity of the corresponding
naturally-occurring marker polypeptide yet differ in amino acid
sequence due to natural allelic variation or mutagenesis.
[0147] To determine the percent identity 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). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions 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 identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In one embodiment the two sequences are the
same length.
[0148] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the BLASTN and BLASTX
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the BLASTN program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to a nucleic acid molecules of the invention. BLAST polypeptide
searches can be performed with the BLASTP program, score=50,
wordlength=3 to obtain amino acid sequences homologous to a
polypeptide molecules of the invention. To obtain gapped alignments
for comparison purposes, a newer version of the BLAST algorithm
called Gapped BLAST can be utilized as described in Altschul et al.
(1997) Nucleic Acids Res. 25:3389-3402, which is able to perform
gapped local alignments for the programs BLASTN, BLASTP and BLASTX.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules. When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., BLASTX and BLASTN) can
be used. See http://www.ncbi.nlm.nih.gov. Another preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
(1988) CABIOS 4:11-17. 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. Yet
another useful algorithm for identifying regions of local sequence
similarity and alignment is the FASTA algorithm as described in
Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448.
When using the FASTA algorithm for comparing nucleotide or amino
acid sequences, a PAM120 weight residue table can, for example, be
used with a k-tuple value of 2.
[0149] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0150] The invention also provides polypeptides which can be used
as internal standards for the marker polypeptides of the invention,
for example, in methods employing mass spectrometry. Such
polypeptides include polypeptides expected to behave in the
analysis substantially similar to the marker, but that have a mass
which is distinct from the marker. Such polypeptides include
isotopically-labelled versions of the marker polypeptides,
including versions of the marker polypeptides which include one or
more deuterium, tritium, .sup.15N, .sup.13C, .sup.14C, .sup.32P,
.sup.35S or a combination thereof. Other suitable polypeptides for
use as internal standards include those differing from the marker
polypeptides by a small structural element, for example, the
addition or deletion of a methylene (--CH.sub.2--) group, a methyl
group, or a halogen atom, such as a fluorine, chlorine, bromine or
iodine atom. Included are polypeptides which are highly homologous
to one of the marker polypeptides, for example, those differing by
the identity of one amino acid residue from the marker sequence.
Suitable amino acid substitutions include conservative
substitutions, including substitutions with homologous amino acid
residues, such as substitution of praline with homoproline,
substitutions among valine and leucine or isoleucine, and others as
can be determined by the skilled artisan.
[0151] In another embodiment, the invention relates to chimeric or
fusion proteins comprising a marker polypeptide of the invention or
a segment thereof. As used herein, a "chimeric protein" or "fusion
protein" comprises all or part (preferably a biologically active
part) of a marker polypeptide operably linked to a heterologous
polypeptide (i.e., a polypeptide other than the marker
polypeptide). Within the fusion protein, the term "operably linked"
is intended to indicate that the marker polypeptide or segment
thereof and the heterologous polypeptide are fused in-frame to each
other. The heterologous polypeptide can be fused to the
amino-terminus or the carboxyl-terminus of the marker polypeptide
or segment.
[0152] One useful fusion protein is a GST fusion protein in which a
marker polypeptide or segment is fused to the carboxyl terminus of
GST sequences. Such fusion proteins can facilitate the purification
of a recombinant polypeptide of the invention.
[0153] In another embodiment, the fusion protein contains a
heterologous signal sequence at its amino terminus. For example,
the native signal sequence of a marker polypeptide can be removed
and replaced with a signal sequence from another protein. For
example, the gp67 secretory sequence of the baculovirus envelope
protein can be used as a heterologous signal sequence (Ausubel et
al., ed., Current Protocols in Molecular Biology, John Wiley &
Sons, NY, 1992). Other examples of eukaryotic heterologous signal
sequences include the secretory sequences of melittin and human
placental alkaline phosphatase (Stratagene; La Jolla, Calif.). In
yet another example, useful prokaryotic heterologous signal
sequences include the phoA secretory signal (Sambrook et al.,
supra) and the protein A secretory signal (Pharmacia Biotech;
Piscataway, N.J.).
[0154] Chimeric and fusion proteins of the invention can be
produced by standard recombinant DNA techniques. In another
embodiment, the fusion gene can be synthesized by conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
re-amplified to generate a chimeric gene sequence (see, e.g.,
Ausubel et al., supra). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A nucleic acid encoding a polypeptide of the
invention can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to the polypeptide of the
invention.
[0155] A signal sequence can be used to facilitate secretion and
isolation of marker polypeptides. Signal sequences are typically
characterized by a core of hydrophobic amino acids which are
generally cleaved from the mature protein during secretion in one
or more cleavage events. Such signal polypeptides contain
processing sites that allow cleavage of the signal sequence from
the mature proteins as they pass through the secretory pathway.
Thus, the invention pertains to marker polypeptides, fusion
proteins or segments thereof having a signal sequence, as well as
to such proteins from which the signal sequence has been
proteolytically cleaved (i.e., the cleavage products). In one
embodiment, a nucleic acid sequence encoding a signal sequence can
be operably linked in an expression vector to a protein of
interest, such as a marker polypeptide or a segment thereof. The
signal sequence directs secretion of the protein, such as from a
eukaryotic host into which the expression vector is transformed,
and the signal sequence is subsequently or concurrently cleaved.
The protein can then be readily purified from the extracellular
medium by art recognized methods. Alternatively, the signal
sequence can be linked to the protein of interest using a sequence
which facilitates purification, such as with a GST domain.
[0156] Another aspect of the invention pertains to antibodies
directed against a polypeptide of the invention. In preferred
embodiments, the antibodies specifically bind a marker polypeptide
or a fragment thereof. The terms "antibody" and "antibodies", as
used interchangeably herein, refer to immunoglobulin molecules as
well as fragments and derivatives thereof that comprise an
immunologically active portion of an immunoglobulin molecule,
(i.e., such a portion contains an antigen binding site which
specifically binds an antigen, such as a marker polypeptide, e.g.,
an epitope of a marker protein). An antibody which specifically
binds to a polypeptide of the invention is an antibody which binds
the polypeptide, but does not substantially bind other molecules in
a sample, e.g., a biological sample, which naturally contains the
polypeptide. Examples of an immunologically active portion of an
immunoglobulin molecule include, but are not limited to,
single-chain antibodies (scAb), F(ab) and F(ab').sub.2
fragments.
[0157] An isolated polypeptide of the invention or a fragment
thereof can be used as an immunogen to generate antibodies. The
full-length polypeptide can be used or, alternatively, the
invention provides antigenic polypeptide fragments for use as
immunogens. The antigenic polypeptide of the invention comprises at
least 7 (preferably 8, 10, 15, 20, or 30 or more) amino acid
residues of the amino acid sequence of one of the proteins of the
invention, and encompasses at least one epitope of the protein such
that an antibody raised against the polypeptide forms a specific
immune complex with the protein. Preferred epitopes encompassed by
the antigenic polypeptide are regions that are located on the
surface of the protein, e.g., hydrophilic regions. Hydrophobicity
sequence analysis, hydrophilicity sequence analysis, or similar
analyses can be used to identify hydrophilic regions. In preferred
embodiments, an isolated marker polypeptide or fragment thereof is
used as an immunogen.
[0158] An immunogen typically is used to prepare antibodies by
immunizing a suitable (i.e. immunocompetent) subject such as a
rabbit, goat, mouse, or other mammal or vertebrate. An appropriate
immunogenic preparation can contain, for example,
recombinantly-expressed or chemically-synthesized polypeptide or
polypeptide. The preparation can further include an adjuvant, such
as Freund's complete or incomplete adjuvant, or a similar
immunostimulatory agent. Preferred immunogen compositions are those
that contain no other human proteins such as, for example,
immunogen compositions made using a non-human host cell for
recombinant expression of a polypeptide of the invention. In such a
manner, the resulting antibody compositions have reduced or no
binding of human proteins other than a polypeptide of the
invention.
[0159] The invention provides polyclonal and monoclonal antibodies.
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. Preferred
polyclonal and monoclonal antibody compositions are ones that have
been selected for antibodies directed against a polypeptide of the
invention. Particularly preferred polyclonal and monoclonal
antibody preparations are ones that contain only antibodies
directed against a marker polypeptide or fragment thereof.
[0160] Polyclonal antibodies can be prepared by immunizing a
suitable subject with a polypeptide of the invention as an
immunogen The antibody titer in the immunized subject can be
monitored over time by standard techniques, such as with an enzyme
linked immunosorbent assay (ELISA) using immobilized polypeptide.
At an appropriate time after immunization, e.g., when the specific
antibody titers are highest, antibody-producing cells can be
obtained from the subject and used to prepare monoclonal antibodies
(mAb) by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature
256:495-497, the human B cell hybridoma technique (see Kozbor et
al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see
Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., 1985) or trioma techniques. The technology for
producing hybridomas is well known (see generally Current Protocols
in Immunology, Coligan et al. ed., John Wiley & Sons, New York,
1994). Hybridoma cells producing a monoclonal antibody of the
invention are detected by screening the hybridoma culture
supernatants for antibodies that bind the polypeptide of interest,
e.g., using a standard ELISA assay.
[0161] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a polypeptide of
the invention can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody
phage display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfZ4P Phage Display
Kit, Catalog No. 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No.
WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No.
WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No.
WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No.
WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; Griffiths et al. (1993) EMBO J.
12:725-734.
[0162] The invention also provides recombinant antibodies that
specifically bind a polypeptide of the invention. In preferred
embodiments, the recombinant antibodies specifically binds a marker
polypeptide or fragment thereof. Recombinant antibodies include,
but are not limited to, chimeric and humanized monoclonal
antibodies, comprising both human and non-human portions,
single-chain antibodies and multi-specific antibodies. A chimeric
antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region
derived from a murine mAb and a human immunoglobulin constant
region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and
Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein
by reference in their entirety.) Single-chain antibodies have an
antigen binding site and consist of a single polypeptides. They can
be produced by techniques known in the art, for example using
methods described in Ladner et. al U.S. Pat. No. 4,946,778 (which
is incorporated herein by reference in its entirety); Bird et al.,
(1988) Science 242:423-426; Whitlow et al., (1991) Methods in
Enzymology 2:1-9; Whitlow et al., (1991) Methods in Enzymology
2:97-105; and Huston et al., (1991) Methods in Enzymology Molecular
Design and Modeling: Concepts and Applications 203:46-88.
Multi-specific antibodies are antibody molecules having at least
two antigen-binding sites that specifically bind different
antigens. Such molecules can be produced by techniques known in the
art, for example using methods described in Segal, U.S. Pat. No.
4,676,980 (the disclosure of which is incorporated herein by
reference in its entirety); Holliger et al., (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Whitlow et al., (1994) Protein Eng.
7:1017-1026 and U.S. Pat. No. 6,121,424.
[0163] The antibodies of the invention can be isolated after
production (e.g., from the blood or serum of the subject) or
synthesis and further purified by well-known techniques. For
example, IgG antibodies can be purified using protein A
chromatography. Antibodies specific for a polypeptide of the
invention can be selected or (e.g., partially purified) or purified
by, e.g., affinity chromatography. For example, a recombinantly
expressed and purified (or partially purified) polypeptide of the
invention is produced as described herein, and covalently or
non-covalently coupled to a solid support such as, for example, a
chromatography column. The column can then be used to affinity
purify antibodies specific for the polypeptides of the invention
from a sample containing antibodies directed against a large number
of different epitopes, thereby generating a substantially purified
antibody composition, i.e., one that is substantially free of
contaminating antibodies. By a substantially purified antibody
composition is meant, in this context, that the antibody sample
contains at most only 30% (by dry weight) of contaminating
antibodies directed against epitopes other than those of the
desired polypeptide of the invention, and preferably at most 20%,
yet more preferably at most 10%, and most preferably at most 5% (by
dry weight) of the sample is contaminating antibodies. A purified
antibody composition means that at least 99% of the antibodies in
the composition are directed against the desired polypeptide of the
invention.
[0164] An antibody directed against a polypeptide of the invention
can be used to isolate the polypeptide by standard techniques, such
as affinity chromatography or immunoprecipitation. Moreover, such
an antibody can be used to detect the marker polypeptide or
fragment thereof (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the level and pattern of expression of the
marker. The antibodies can also be used diagnostically to monitor
polypeptide levels in tissues or body fluids (e.g. in an
endometrial-associated body fluid) as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by the use of an
antibody derivative, which comprises an antibody of the invention
coupled to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0165] Accordingly, in one aspect, the invention provides
substantially purified antibodies, antibody fragments and
derivatives, all of which specifically bind to a polypeptide of the
invention and preferably, a marker polypeptide. In various
embodiments, the substantially purified antibodies of the
invention, or fragments or derivatives thereof, can be human,
non-human, chimeric and/or humanized antibodies. In another aspect,
the invention provides non-human antibodies, antibody fragments and
derivatives, all of which specifically bind to a polypeptide of the
invention and preferably, a marker polypeptide. Such non-human
antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or
rat antibodies. Alternatively, the non-human antibodies of the
invention can be chimeric and/or humanized antibodies. In addition,
the non-human antibodies of the invention can be polyclonal
antibodies or monoclonal antibodies. In still a further aspect, the
invention provides monoclonal antibodies, antibody fragments and
derivatives, all of which specifically bind to a polypeptide of the
invention and preferably, a marker polypeptide. The monoclonal
antibodies can be human, humanized, chimeric and/or non-human
antibodies.
[0166] The invention also provides a kit containing an antibody of
the invention conjugated to a detectable substance, and
instructions for use. Still another aspect of the invention is a
pharmaceutical composition comprising an antibody of the invention
and a pharmaceutically acceptable carrier. In one embodiment, the
pharmaceutical composition comprises an antibody of the invention,
a therapeutic moiety, and a pharmaceutically acceptable
carrier.
III. Recombinant Expression Vectors and Host Cells
[0167] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding a
marker polypeptide (or a portion of such a polypeptide). As used
herein, the term "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) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors, namely expression vectors, are capable
of directing the expression of genes to which they are operably
linked. In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids (vectors).
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.
[0168] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell. This means that the recombinant
expression vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for expression,
which is operably linked to the nucleic acid sequence to be
expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner which
allows for expression of the nucleotide sequence (e.g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel,
Methods in Enzymology: Gene Expression Technology vol. 185,
Academic Press, San Diego, Calif. (1991). Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cell and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of polypeptide
desired, and the like. The expression vectors of the invention can
be introduced into host cells to thereby produce proteins or
polypeptides, including fusion proteins or polypeptides, encoded by
nucleic acids as described herein.
[0169] The recombinant expression vectors of the invention can be
designed for expression of a marker polypeptide or a segment
thereof in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g.,
insect cells {using baculovirus expression vectors}, yeast cells or
mammalian cells). Suitable host cells are discussed further in
Goeddel, supra. Alternatively, the recombinant expression vector
can be transcribed and translated in vitro, for example using T7
promoter regulatory sequences and T7 polymerase.
[0170] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0171] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET
11d (Studier et al., p. 60-89, In Gene Expression Technology:
Methods in Enzymology vol. 185, Academic Press, San Diego, Calif.,
1991). Target gene expression from the pTrc vector relies on host
RNA polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
co-expressed viral RNA polymerase (T7 gn1). This viral polymerase
is supplied by host strains BL21(DE3) or HMS174(DE3) from a
resident prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0172] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, p. 119-128, In Gene Expression Technology: Methods in
Enzymology vol. 185, Academic Press, San Diego, Calif., 1990.
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al., 1992, Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be carried out by standard DNA synthesis
techniques.
[0173] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari et al., 1987, EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943),
pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San
Diego, Calif.).
[0174] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al., 1983, Mol. Cell. Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).
[0175] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987, Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO
J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook et al.,
supra.
[0176] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0177] A host cell can be any prokaryotic (e.g., E. coli) or
eukaryotic cell (e.g., insect cells, yeast or mammalian cells).
[0178] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (supra), and other
laboratory manuals.
[0179] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs, such as
G418, hygromycin and methotrexate. Cells stably transfected with
the introduced nucleic acid can be identified by drug selection
(e.g., cells that have incorporated the selectable marker will
survive, while the other cells die).
[0180] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce a marker
polypeptide or a segment thereof. Accordingly, the invention
further provides methods for producing a marker polypeptide or a
segment thereof using the host cells of the invention. In one
embodiment, the method comprises culturing the host cell of the
invention (into which a recombinant expression vector encoding a
marker polypeptide or a segment thereof has been introduced) in a
suitable medium such that the polypeptide is produced. In another
embodiment, the method further comprises isolating the marker
polypeptide or a segment thereof from the medium or the host
cell.
IV. Pharmaceutical Compositions
[0181] The invention also provides methods (also referred to herein
as "screening assays") for identifying modulators, i.e., candidate
or test compounds or agents (e.g., polypeptides, peptidomimetics,
peptoids, small molecules or other drugs) which have a modulatory
effect on the abundance of the marker.
[0182] The test compounds of the present invention may be obtained
from any available source, including systematic libraries of
natural and/or synthetic compounds. Test compounds may also be
obtained by any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
polypeptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem.
37:2678-85); 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 and peptoid library approaches are limited to polypeptide
libraries, while the other four approaches are applicable to
polypeptide, non-peptide oligomer or small molecule libraries of
compounds (Lam, 1997, Anticancer Drug Des. 12:145).
[0183] 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. USA. 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; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J Med. Chem.
37:1233.
[0184] 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 and/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids
(Cull et al., 1992, Proc Natl Acad Sci USA 89:1865-1869) or on
phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 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.).
[0185] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
polypeptide encoded by or corresponding to a marker or biologically
active portion thereof. In another embodiment, the invention
provides assays for screening candidate or test compounds which
bind to a polypeptide encoded by or corresponding to a marker or
biologically active portion thereof. Determining the ability of the
test compound to directly bind to a polypeptide can be
accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to the marker can be determined by detecting the labeled marker
compound in a complex. For example, compounds (e.g., marker
substrates) can be labeled with .sup.125I, .sup.35S, .sup.14C, or
.sup.3H, either directly or indirectly, and the radioisotope
detected by direct counting of radioemission or by scintillation
counting. Alternatively, assay components can be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to
product.
[0186] In another embodiment, the invention provides assays for
screening candidate or test compounds which modulate the expression
of a marker
[0187] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a marker polypeptide can be further confirmed in vivo, e.g., in
a whole animal model for cellular transformation and/or
tumorigenesis.
[0188] This invention further pertains to novel agents identified
by the above-described screening 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 marker modulating
agent) 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.
[0189] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
V. Predictive Medicine
[0190] The present invention pertains to the field of predictive
medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining the level of
expression of one or more marker polypeptides in order to determine
whether an individual is at risk of developing endometriosis. Such
assays can be used for prognostic or predictive purposes to thereby
prophylactically treat an individual prior to the onset of
endometriosis.
[0191] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs or other compounds
administered either to inhibit endometriosis or to treat or prevent
any other endometriosis-related disease, disorder or condition) on
the abundance of a marker of the invention in clinical trials.
These and other agents are described in further detail in the
following sections.
[0192] A. Diagnostic Assays
[0193] An exemplary method for detecting the presence, absence or
abundance of a marker polypeptide in a biological sample involves
obtaining a biological sample (e.g. an endometrial-associated body
fluid, blood, blood plasma or serum) from a test subject and
contacting the biological sample with a compound or an agent
capable of detecting the polypeptide. In vitro techniques for
detection of a marker polypeptide include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence.
[0194] A general principle of such diagnostic and prognostic assays
involves preparing a sample or reaction mixture that may contain a
marker, and a probe, under appropriate conditions and for a time
sufficient to allow the marker and probe to interact and bind, thus
forming a complex that can be removed and/or detected in the
reaction mixture. These assays can be conducted in a variety of
ways.
[0195] For example, one method to conduct such an assay comprises
anchoring the marker or probe onto a solid phase support, also
referred to as a substrate, and detecting target marker/probe
complexes anchored on the solid phase at the end of the reaction.
In one embodiment of such a method, a sample from a subject, which
is to be assayed for presence and/or concentration of marker, can
be anchored onto a carrier or solid phase support. In another
embodiment, the reverse situation is possible, in which the probe
can be anchored to a solid phase and a sample from a subject can be
allowed to react as an unanchored component of the assay.
[0196] There are many established methods for anchoring assay
components to a solid phase. These include, without limitation,
marker or probe molecules which are immobilized through conjugation
of biotin and streptavidin. Such biotinylated assay components can
be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques 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). In certain
embodiments, the surfaces with immobilized assay components can be
prepared in advance and stored.
[0197] Other suitable carriers or solid phase supports for such
assays include any material capable of binding the class of
molecule to which the marker or probe belongs. Well-known supports
or carriers include, but are not limited to, glass, polystyrene,
nylon, polypropylene, nylon, polyethylene, dextran, amylases,
natural and modified celluloses, polyacrylamides, gabbros, and
magnetite.
[0198] In order to conduct assays with the above mentioned
approaches, the non-immobilized component is added to the solid
phase upon which the second component is anchored. After the
reaction is complete, uncomplexed components may be removed (e.g.,
by washing) under conditions such that any complexes formed will
remain immobilized upon the solid phase. The detection of
marker/probe complexes anchored to the solid phase can be
accomplished in a number of methods outlined herein.
[0199] In a preferred embodiment, the probe, when it is the
unanchored assay component, can be labeled for the purpose of
detection and readout of the assay, either directly or indirectly,
with detectable labels discussed herein and which are well-known to
one skilled in the art.
[0200] It is also possible to directly detect marker/probe complex
formation without further manipulation or labeling of either
component (marker or probe), for example by utilizing the technique
of fluorescence energy transfer (see, for example, Lakowicz et al.,
U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.
4,868,103). A fluorophore label on the first, `donor` molecule is
selected such that, upon excitation with incident light of
appropriate wavelength, its emitted fluorescent energy will be
absorbed by a fluorescent label on a second `acceptor` molecule,
which in turn is able to fluoresce due to the absorbed energy.
Alternately, the `donor` polypeptide molecule may simply utilize
the natural fluorescent energy of tryptophan residues. Labels are
chosen that emit different wavelengths of light, such that the
`acceptor` molecule label may be differentiated from that of the
`donor`. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, spatial
relationships between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be
maximal. An FET binding event can be conveniently measured through
standard fluorometric detection means well known in the art (e.g.,
using a fluorimeter).
[0201] In another embodiment, determination of the ability of a
probe to recognize a marker can be accomplished without labeling
either assay component (probe or marker) by utilizing a technology
such as real-time Biomolecular Interaction Analysis (BIA) (see,
e.g., Sjolander, S, and Urbaniczky, C., 1991, Anal. Chem.
63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol.
5:699-705). As used herein, "BIA" or "surface plasmon resonance" is
a technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore). Changes
in the mass at the binding surface (indicative of a binding event)
result in alterations of the refractive index of light near the
surface (the optical phenomenon of surface plasmon resonance
(SPR)), resulting in a detectable signal which can be used as an
indication of real-time reactions between biological molecules.
[0202] Alternatively, in another embodiment, analogous diagnostic
and prognostic assays can be conducted with marker and probe as
solutes in a liquid phase. In such an assay, the complexed marker
and probe are separated from uncomplexed components by any of a
number of standard techniques, including but not limited to:
differential centrifugation, chromatography, electrophoresis and
immunoprecipitation. In differential centrifugation, marker/probe
complexes may be separated from uncomplexed assay components
through a series of centrifugal steps, due to the different
sedimentation equilibria of complexes based on their different
sizes and densities (see, for example, Rivas, G., and Minton, A.
P., 1993, Trends Biochem Sci. 18(8):284-7). Standard
chromatographic techniques may also be utilized to separate
complexed molecules from uncomplexed ones. For example, gel
filtration chromatography separates molecules based on size, and
through the utilization of an appropriate gel filtration resin in a
column format, for example, the relatively larger complex may be
separated from the relatively smaller uncomplexed components.
Similarly, the relatively different charge properties of the
marker/probe complex as compared to the uncomplexed components may
be exploited to differentiate the complex from uncomplexed
components, for example through the utilization of ion-exchange
chromatography resins. Such resins and chromatographic techniques
are well known to one skilled in the art (see, e.g., Heegaard, N.
H., 1998, J. Mol. Recognit. Winter 11(1-6):141-8; Hage, D. S., and
Tweed, S. A. J Chromatogr B Biomed Sci Appl 1997 Oct. 10;
699(1-2):499-525). Gel electrophoresis may also be employed to
separate complexed assay components from unbound components (see,
e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, 1987-1999). In this technique,
polypeptide or nucleic acid complexes are separated based on size
or charge, for example. In order to maintain the binding
interaction during the electrophoretic process, non-denaturing gel
matrix materials and conditions in the absence of reducing agent
are typically preferred. Appropriate conditions to the particular
assay and components thereof will be well known to one skilled in
the art.
[0203] Alternatively, the abundance can be provided as a relative
abundance. To determine a relative abundance of a marker, the
abundance of the marker is determined for 10 or more samples of
normal (non-endometriosis) samples, preferably 50 or more samples,
prior to the determination of the abundance for the sample in
question. The mean abundance of each of the markers assayed in the
larger number of samples is determined and this is used as a
baseline abundance level for the marker. The abundance of the
marker determined for the test sample (absolute level of
expression) is then divided by the mean abundance value obtained
for that marker. This provides a relative abundance.
[0204] Preferably, the samples used in the baseline determination
will be from blood, serum or plasma. The choice of the sample is
dependent on the use of the relative abundance and the ease of
obtaining and processing the sample. Using normal abundance as a
mean abundance score aids in validating whether the marker assayed
is endometriosis specific (versus normal). In addition, as more
data is accumulated, the mean expression value can be revised,
providing improved relative abundance values based on accumulated
data. Abundance data provide a means for grading the severity of
the endometriosis state.
[0205] In another embodiment of the present invention, a marker
polypeptide is detected. A preferred agent for detecting a marker
polypeptide of the invention is an antibody capable of binding to
the polypeptide or a fragment thereof, preferably an antibody with
a detectable label. Antibodies can be polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment or
derivative thereof (e.g., Fab or F(ab').sub.2) can be used. The
term "labeled", with regard to the probe or antibody, is intended
to encompass direct labeling of the probe or antibody by coupling
(i.e., physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin.
[0206] A variety of formats can be employed to determine whether a
sample contains a polypeptide that binds to a given antibody.
Examples of such formats include, but are not limited to, enzyme
immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis
and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan
can readily adapt known polypeptide/antibody detection methods for
use in determining the abundance of a marker of the present
invention within a given sample.
[0207] In one format, antibodies, or antibody fragments or
derivatives, can be used in methods such as Western blots or
immunofluorescence techniques to detect the expressed polypeptides.
In such uses, it is generally preferable to immobilize either the
antibody or polypeptides on a solid support. Suitable solid phase
supports or carriers include any support capable of binding an
antigen or an antibody. Well-known supports or carriers include
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite.
[0208] One skilled in the art will know many other suitable
carriers for binding antibody or antigen, and will be able to adapt
such support for use with the present invention. For example,
polypeptide isolated from endometrial cells can be run on a
polyacrylamide gel electrophoresis and immobilized onto a solid
phase support such as nitrocellulose. The support can then be
washed with suitable buffers followed by treatment with the
detectably labeled antibody. The solid phase support can then be
washed with the buffer a second time to remove unbound antibody.
The amount of bound label on the solid support can then be detected
by conventional means.
[0209] In one embodiment, the abundance of a marker or collection
of markers of the invention is determined using mass spectrometric
methods. For example, following pretreatment, if any, the sample
can be subjected to liquid chromatography/mass spectrometry
("LC/MS"), a method is which the components of the sample are
separated by the chromatographic step and then analyzed by mass
spectrometry. The method thus provides mass scan data as a function
of retention time. Because of the possibility that the analyte of
interest will co-elute with another sample component having the
same mass, the sample is preferably analyzed using liquid
chromatography/mass spectrometry/mass spectrometry ("LC/MS/MS"), a
method in which the molecular ions from the first mass
spectrometric step are fragmented. Such a method typically provides
one or more fragments which is unique to the analyte of interest
and can be monitored. The LC/MS/MS method thus provides a means of
monitoring a specific analyte without interference from other
sample components and is particularly useful when the sample
includes a complex mixture of components.
[0210] In one embodiment, the mass spectrometer is set to scan over
a small mass range which includes the mass of the analyte. In an
LC/MS/MS method, the analyte can be determined using Selective
Reaction Monitoring ("SRM"). In SRM, the parent mass of the analyte
is specified for further fragmentation, and a specific fragment ion
is monitored. In a Multiple Reaction Monitoring ("MRM") experiment
two or more parent-fragment pairs are monitored.
[0211] In a preferred embodiment, a known quantity of internal
standard is added to the sample prior analysis of the sample.
Preferably, an internal standard is added for each marker
polypeptide to be analyzed. The internal standard is preferably
added to the sample prior to any processing of the sample. The
internal standard is a compound which is closely related
structurally to the analyte of interest, but that has a different
mass than the analyte. For example, the internal standard should be
a compound which behaves essentially identically to the marker
during sample preparation, chromatographic separation and
ionization in the mass spectrometer. Thus, the internal standard is
preferably, a compound which has structural, physical and chemical
features which are very close to those of the marker polypeptide.
Suitable internal standards for the present marker polypeptides
include synthetic polypeptides, such as synthetic, isotopically
labeled polypeptides having the same structure, other than the
isotopic label, as the marker polypeptide to be analyzed. The
internal standard polypeptides can be isototopically labeled in a
suitable manner, as is known in the art, such as labeling with
.sup.2H (deuterium), .sup.3H (tritium), .sup.13C, .sup.33S,
.sup.32P, .sup.15N and .sup.17O. Preferably, the isotopic label is
not a radioisotope. Preferred isotopic labels include .sup.2H,
.sup.13C, .sup.15N and .sup.17O. The internal standard for a given
polypeptide marker can also be a polypeptide which differs
structurally from the marker polypeptide in a minor way, such as
the addition or subtraction of a methylene group, methyl group,
hydroxyl group or halogen atom. For example, the following pairs of
amino acid residues can be substituted for each other in preparing
an internal standard for a given marker polypeptide:
gycine/alanine; valine/leucine; valine/isoleucine;
cysteine/homocysteine; asparagine/glutamine; aspartic acid/glutamic
acid; serine/threonine; and phenylalanine/tyrosine. Similar
substitutions can be made using structurally related non-natural
amino acid residues, as is known in the art. Examples of suitable
internal standards for use in the present method include, but are
not limited to, polypeptides of the formula H-EETKENEGFTVTAEG-OH
(d.sub.8-Val) (SEQ ID NO:35) and Pyr-EGLNDNEEGFFSA-OH (SEQ ID
NO:44).
[0212] Quantitation of a marker polypeptide of the invention can be
accomplished using methods known in the art. For example, for a
given marker polypeptide, the synthetic standard can be used to
generate a calibration curve. To generate a calibration curve, the
synthetic standard is added to blank or surrogate matrix in varying
amounts, the samples are pretreated using the desired protocol and
then analyzed by LC/MS or LC/MS/MS. The area under the counts per
second vs time curve for the ion current (peak area) for the
fragment (SIM, SRM) or each of the multiple fragments (MRM) is
determined. A calibration curve is constructed in which the
concentration ratio of analyte to internal standard is plotted
versus peak area ratio. The peak area of the corresponding fragment
or fragments of the marker polypeptide is determined and the
concentration of the marker polypeptide is found using the
calibration curve.
[0213] The mass spectrometer to be used in the methods of the
invention can be, for example, an ion trap mass spectrometer or a
(triple)quadrupole mass spectrometer. Electrospray (atmospheric
pressure) ionization is preferably used, but other methods for
ionization can also be used, as is known in the art.
[0214] The invention also encompasses kits for detecting the
presence of a marker polypeptide in a biological sample. Such kits
can be used to determine if a subject is suffering from or is at
increased risk of developing endometriosis. For example, the kit
can comprise a labeled compound or agent capable of detecting one
or more marker polypeptides in a biological sample and means for
determining the amount of the polypeptide in the sample (e.g., an
antibody which binds the polypeptide or fragment thereof.
[0215] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a marker polypeptide; and, optionally, (2) a second,
different antibody which binds to either the polypeptide or the
first antibody and is conjugated to a detectable label.
[0216] Kits to be used in mass spectrometric methods can include
one or more synthetic standards and/or one or more internal
standards.
[0217] The kit can also comprise, e.g., a buffering agent, a
preservative, or a protein stabilizing agent. The kit can further
comprise components necessary for detecting the detectable label
(e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples which can be assayed
and compared to the test sample. Each component of the kit can be
enclosed within an individual container and all of the various
containers can be within a single package, along with instructions
for interpreting the results of the assays performed using the
kit.
[0218] C. Monitoring Clinical Trials
[0219] Monitoring the influence of agents (e.g., drug compounds) on
the level of expression of a marker of the invention can be applied
not only in basic drug screening, but also in clinical trials. For
example, the ability of an agent to affect marker abundance can be
monitored in clinical trials of subjects receiving treatment for
endometriosis. In a preferred embodiment, the present invention
provides a method for monitoring the effectiveness of treatment of
a subject with an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, polypeptide, nucleic acid, small molecule,
or other drug candidate) comprising the steps of (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) determining the abundance of one or more selected
markers of the invention in the pre-administration sample; (iii)
obtaining one or more post-administration samples from the subject;
(iv) determining the abundance of the marker(s) in the
post-administration samples; (v) comparing the abundance of the
marker(s) in the pre-administration sample with the abundance of
the marker(s) in the post-administration sample or samples; and
(vi) altering the administration of the agent to the subject
accordingly. For example, increased abundance of certain marker
polypeptide(s) during the course of treatment may indicate
ineffective dosage and the desirability of increasing the dosage.
Conversely, decreased expression of these marker polypeptide(s) may
indicate efficacious treatment and no need to change dosage.
[0220] D. Electronic Apparatus Readable Media and Arrays
[0221] Electronic apparatus readable media comprising a marker of
the present invention is also provided. As used herein, "electronic
apparatus readable media" refers to any suitable medium for
storing, holding or containing data or information that can be read
and accessed directly by an electronic apparatus. Such media can
include, but are not limited to: magnetic storage media, such as
floppy discs, hard disc storage medium, and magnetic tape; optical
storage media such as compact disc; electronic storage media such
as RAM, ROM, EPROM, EEPROM and the like; general hard disks and
hybrids of these categories such as magnetic/optical storage media.
The medium is adapted or configured for having recorded thereon a
marker of the present invention.
[0222] As used herein, the term "electronic apparatus" is intended
to include any suitable computing or processing apparatus or other
device configured or adapted for storing data or information.
Examples of electronic apparatus suitable for use with the present
invention include stand-alone computing apparatus; networks,
including a local area network (LAN), a wide area network (WAN)
Internet, Intranet, and Extranet; electronic appliances such as a
personal digital assistants (PDAs), cellular phone, pager and the
like; and local and distributed processing systems.
[0223] As used herein, "recorded" refers to a process for storing
or encoding information on the electronic apparatus readable
medium. Those skilled in the art can readily adopt any of the
presently known methods for recording information on known media to
generate manufactures comprising the markers of the present
invention.
[0224] A variety of software programs and formats can be used to
store the marker information of the present invention on the
electronic apparatus readable medium. For example, the marker
nucleic acid sequence can be represented in a word processing text
file, formatted in commercially-available software such as
WordPerfect and MicroSoft Word, or represented in the form of an
ASCII file, stored in a database application, such as DB2, Sybase,
Oracle, or the like, as well as in other forms. Any number of data
processor structuring formats (e.g., text file or database) may be
employed in order to obtain or create a medium having recorded
thereon the markers of the present invention.
[0225] By providing the markers of the invention in readable form,
one can routinely access the marker sequence information for a
variety of purposes. For example, one skilled in the art can use
the nucleotide or amino acid sequences of the present invention in
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0226] The present invention therefore provides a medium for
holding instructions for performing a method for determining
whether a subject has endometriosis or a pre-disposition to
endometriosis, wherein the method comprises the steps of
determining the abundance of a marker and, based on the abundance
of the marker, determining whether the subject has endometriosis or
a pre-disposition to endometriosis and/or recommending a particular
treatment for endometriosis or pre-endometriosis condition.
[0227] The present invention further provides in an electronic
system and/or in a network, a method for determining whether a
subject has endometriosis or a pre-disposition to endometriosis
associated with a marker wherein the method comprises the steps of
determining the abundance of the marker, and based on the abundance
of the marker, determining whether the subject has endometriosis or
a pre-disposition to endometriosis, and/or recommending a
particular treatment for the endometriosis or pre-endometriosis
condition. The method may further comprise the step of receiving
phenotypic information associated with the subject and/or acquiring
from a network phenotypic information associated with the
subject.
[0228] The present invention also provides in a network, a method
for determining whether a subject has endometriosis or a
pre-disposition to endometriosis associated with a marker, said
method comprising the steps of receiving information associated
with the marker, receiving phenotypic information associated with
the subject, acquiring information from the network corresponding
to the marker and/or endometriosis, and based on one or more of the
phenotypic information, the marker, and the acquired information,
determining whether the subject has endometriosis or a
pre-disposition to endometriosis. The method may further comprise
the step of recommending a particular treatment for the
endometriosis or pre-endometriosis condition.
[0229] The present invention also provides a business method for
determining whether a subject has endometriosis or a
pre-disposition to endometriosis, said method comprising the steps
of receiving information associated with the marker, receiving
phenotypic information associated with the subject, acquiring
information from the network corresponding to the marker and/or
endometriosis, and based on one or more of the phenotypic
information, the marker, and the acquired information, determining
whether the subject has endometriosis or a pre-disposition to
endometriosis. The method may further comprise the step of
recommending a particular treatment for the endometriosis or
pre-endometriosis condition.
[0230] E. Surrogate Markers
[0231] The markers of the invention may serve as surrogate markers
for one or more disorders or disease states or for conditions
leading up to disease states, and in particular, endometriosis. As
used herein, a "surrogate marker" is an objective biochemical
marker which correlates with the absence or presence of a disease
or disorder, or with the progression of a disease or disorder
(e.g., with the presence or absence of a tumor). The presence or
quantity of such markers is independent of the disease. Therefore,
these markers may serve to indicate whether a particular course of
treatment is effective in lessening a disease state or disorder.
Surrogate markers are of particular use when the presence or extent
of a disease state or disorder is difficult to assess through
standard methodologies (e.g., early stage tumors), or when an
assessment of disease progression is desired before a potentially
dangerous clinical endpoint is reached (e.g., an assessment of
cardiovascular disease may be made using cholesterol levels as a
surrogate marker, and an analysis of HIV infection may be made
using HIV RNA levels as a surrogate marker, well in advance of the
undesirable clinical outcomes of myocardial infarction or
fully-developed AIDS). Examples of the use of surrogate markers in
the art include: Koomen et al. (2000) J. Mass. Spectrom. 35:
258-264; and James (1994) AIDS Treatment News Archive 209.
VI. Experimental Protocol
[0232] A. Identification of the Markers of the Invention
[0233] Serum samples were obtained from women with endometriosis
confirmed by laparoscopy and from women having no complaints
indicative of endometriosis. Samples were also obtained from women
diagnosed with polycystic ovarian syndrome (PCOS) and pelvic
inflammatory disease (PID). The serum samples were prepared for
analysis and analyzed using the following protocol.
[0234] 50 mL serum was diluted with 100 .mu.L 500 .mu.M aqueous
glycine, pH 2.0. The diluted serum was then ultrafiltered using a
10,000 dalton molecular weight cutoff membrane (Millipore; 14,000
RCF maximum) at 3,750 rpm on a swinging bucket for approximately
2.5 hours. The filtrate was transferred to a container for HPLC
analysis, which was performed using a Grace Vydac C-8 column (0.21
cm.times.15 cm; Catalog No. 208TP5215), and an injection volume of
0.1 mL. The column was eluted with a gradient of 95/4.9/0.1
acetonitrile/water/formic acid from 2% to 60% over 30 minutes. The
eluate was analyzed by mass spectrometry using a Waters
qudrupole-time-of-flight mass spectrometer with a Z-spray source
and an mass/charge range of 200 to 2000 daltons. Quantitation of a
particular retention time/mass to charge pair was taken as the ion
count from the mass spectrometer, which was assumed to be linear
with the concentration of the analyte.
[0235] The results for several of the markers of the invention are
set forth in FIGS. 1 to 6, each of which is a scatter plot showing
the concentrations (given as counts) of a given marker in the serum
of women diagnosed with endometriosis, the serum of healthy women,
and, in certain cases, the serum of women diagnosed with PCOS or
PID. The data set forth in FIGS. 1 and 2 indicate that the
fibrinopeptide B derivative (SEQ ID NO:28), the full length
fibrinopeptide A (SEQ ID NO:3) and the fibrinopeptide A fragment
(SEQ ID NO:7) are all present in higher concentrations in the serum
of women diagnosed with endometriosis than in the serum of healthy
women. FIG. 3 indicates that the fibrinopeptide A derivative (SEQ
ID NO:4) is present at higher concentrations in women diagnosed
with endometriosis than in the serum of healthy women or women
diagnosed with PCOS or PID. The data set forth in FIG. 4
demonstrate that the thymosin fragments (SEQ ID NOS:42 and 43) are
present at lower concentrations in the serum of women diagnosed
with endometriosis, PCOS and PID than in the serum of healthy
women. FIG. 5 shows that phosphoserine fibrinopeptide A (SEQ ID
NO:22) is present at lower concentrations in the serum of healthy
women than in the serum of women diagnosed with endometriosis, PCOS
or PID. The data in FIG. 6 demonstrate that the internal fibrinogen
fragment (SEQ ID NO:29) is present at lower concentrations in the
serum of women diagnosed with endometriosis than in the serum of
healthy women or women diagnosed with PCOS or PID.
[0236] B. Summary of the Tables
[0237] Tables 1-3 list markers of the invention obtained using the
foregoing experimental protocol. Table 1 lists all of the markers
of the invention, which are over- or under-expressed in patient
samples compared to normal (i.e., a sample from a patient that does
not have endometriosis) samples. Table 2 lists markers whose
over-expression may be correlated with endometriosis as compared to
normal samples from patients that do not have endometriosis. Table
3 lists markers whose under-expression may be correlated with
endometriosis as compared to normal samples from patients that do
not have endometriosis.
[0238] The contents of all references, patents, published patent
applications, and database records, cited throughout this
application, are hereby incorporated by reference.
OTHER EMBODIMENTS
[0239] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
441866PRTHomo sapiens 1Met Phe Ser Met Arg Ile Val Cys Leu Val Leu
Ser Val Val Gly Thr 1 5 10 15Ala Trp Thr Ala Asp Ser Gly Glu Gly
Asp Phe Leu Ala Glu Gly Gly 20 25 30Gly Val Arg Gly Pro Arg Val Val
Glu Arg His Gln Ser Ala Cys Lys 35 40 45Asp Ser Asp Trp Pro Phe Cys
Ser Asp Glu Asp Trp Asn Tyr Lys Cys 50 55 60Pro Ser Gly Cys Arg Met
Lys Gly Leu Ile Asp Glu Val Asn Gln Asp65 70 75 80Phe Thr Asn Arg
Ile Asn Lys Leu Lys Asn Ser Leu Phe Glu Tyr Gln 85 90 95Lys Asn Asn
Lys Asp Ser His Ser Leu Thr Thr Asn Ile Met Glu Ile 100 105 110Leu
Arg Gly Asp Phe Ser Ser Ala Asn Asn Arg Asp Asn Thr Tyr Asn 115 120
125Arg Val Ser Glu Asp Leu Arg Ser Arg Ile Glu Val Leu Lys Arg Lys
130 135 140Val Ile Glu Lys Val Gln His Ile Gln Leu Leu Gln Lys Asn
Val Arg145 150 155 160Ala Gln Leu Val Asp Met Lys Arg Leu Glu Val
Asp Ile Asp Ile Lys 165 170 175Ile Arg Ser Cys Arg Gly Ser Cys Ser
Arg Ala Leu Ala Arg Glu Val 180 185 190Asp Leu Lys Asp Tyr Glu Asp
Gln Gln Lys Gln Leu Glu Gln Val Ile 195 200 205Ala Lys Asp Leu Leu
Pro Ser Arg Asp Arg Gln His Leu Pro Leu Ile 210 215 220Lys Met Lys
Pro Val Pro Asp Leu Val Pro Gly Asn Phe Lys Ser Gln225 230 235
240Leu Gln Lys Val Pro Pro Glu Trp Lys Ala Leu Thr Asp Met Pro Gln
245 250 255Met Arg Met Glu Leu Glu Arg Pro Gly Gly Asn Glu Ile Thr
Arg Gly 260 265 270Gly Ser Thr Ser Tyr Gly Thr Gly Ser Glu Thr Glu
Ser Pro Arg Asn 275 280 285Pro Ser Ser Ala Gly Ser Trp Asn Ser Gly
Ser Ser Gly Pro Gly Ser 290 295 300Thr Gly Asn Arg Asn Pro Gly Ser
Ser Gly Thr Gly Gly Thr Ala Thr305 310 315 320Trp Lys Pro Gly Ser
Ser Gly Pro Gly Ser Thr Gly Ser Trp Asn Ser 325 330 335Gly Ser Ser
Gly Thr Gly Ser Thr Gly Asn Gln Asn Pro Gly Ser Pro 340 345 350Arg
Pro Gly Ser Thr Gly Thr Trp Asn Pro Gly Ser Ser Glu Arg Gly 355 360
365Ser Ala Gly His Trp Thr Ser Glu Ser Ser Val Ser Gly Ser Thr Gly
370 375 380Gln Trp His Ser Glu Ser Gly Ser Phe Arg Pro Asp Ser Pro
Gly Ser385 390 395 400Gly Asn Ala Arg Pro Asn Asn Pro Asp Trp Gly
Thr Phe Glu Glu Val 405 410 415Ser Gly Asn Val Ser Pro Gly Thr Arg
Arg Glu Tyr His Thr Glu Lys 420 425 430Leu Val Thr Ser Lys Gly Asp
Lys Glu Leu Arg Thr Gly Lys Glu Lys 435 440 445Val Thr Ser Gly Ser
Thr Thr Thr Thr Arg Arg Ser Cys Ser Lys Thr 450 455 460Val Thr Lys
Thr Val Ile Gly Pro Asp Gly His Lys Glu Val Thr Lys465 470 475
480Glu Val Val Thr Ser Glu Asp Gly Ser Asp Cys Pro Glu Ala Met Asp
485 490 495Leu Gly Thr Leu Ser Gly Ile Gly Thr Leu Asp Gly Phe Arg
His Arg 500 505 510His Pro Asp Glu Ala Ala Phe Phe Asp Thr Ala Ser
Thr Gly Lys Thr 515 520 525Phe Pro Gly Phe Phe Ser Pro Met Leu Gly
Glu Phe Val Ser Glu Thr 530 535 540Glu Ser Arg Gly Ser Glu Ser Gly
Ile Phe Thr Asn Thr Lys Glu Ser545 550 555 560Ser Ser His His Pro
Gly Ile Ala Glu Phe Pro Ser Arg Gly Lys Ser 565 570 575Ser Ser Tyr
Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn Arg Gly 580 585 590Asp
Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met Ala Asp Glu Ala Gly 595 600
605Ser Glu Ala Asp His Glu Gly Thr His Ser Thr Lys Arg Gly His Ala
610 615 620Lys Ser Arg Pro Val Arg Asp Cys Asp Asp Val Leu Gln Thr
His Pro625 630 635 640Ser Gly Thr Gln Ser Gly Ile Phe Asn Ile Lys
Leu Pro Gly Ser Ser 645 650 655Lys Ile Phe Ser Val Tyr Cys Asp Gln
Glu Thr Ser Leu Gly Gly Trp 660 665 670Leu Leu Ile Gln Gln Arg Met
Asp Gly Ser Leu Asn Phe Asn Arg Thr 675 680 685Trp Gln Asp Tyr Lys
Arg Gly Phe Gly Ser Leu Asn Asp Glu Gly Glu 690 695 700Gly Glu Phe
Trp Leu Gly Asn Asp Tyr Leu His Leu Leu Thr Gln Arg705 710 715
720Gly Ser Val Leu Arg Val Glu Leu Glu Asp Trp Ala Gly Asn Glu Ala
725 730 735Tyr Ala Glu Tyr His Phe Arg Val Gly Ser Glu Ala Glu Gly
Tyr Ala 740 745 750Leu Gln Val Ser Ser Tyr Glu Gly Thr Ala Gly Asp
Ala Leu Ile Glu 755 760 765Gly Ser Val Glu Glu Gly Ala Glu Tyr Thr
Ser His Asn Asn Met Gln 770 775 780Phe Ser Thr Phe Asp Arg Asp Ala
Asp Gln Trp Glu Glu Asn Cys Ala785 790 795 800Glu Val Tyr Gly Gly
Gly Trp Trp Tyr Asn Asn Cys Gln Ala Ala Asn 805 810 815Leu Asn Gly
Ile Tyr Tyr Pro Gly Gly Ser Tyr Asp Pro Arg Asn Asn 820 825 830Ser
Pro Tyr Glu Ile Glu Asn Gly Val Val Trp Val Ser Phe Arg Gly 835 840
845Ala Asp Tyr Ser Leu Arg Ala Val Arg Met Lys Ile Arg Pro Leu Val
850 855 860Thr Gln8652491PRTHomo sapiens 2Met Lys Arg Met Val Ser
Trp Ser Phe His Lys Leu Lys Thr Met Lys 1 5 10 15His Leu Leu Leu
Leu Leu Leu Cys Val Phe Leu Val Lys Ser Gln Gly 20 25 30Val Asn Asp
Asn Glu Glu Gly Phe Phe Ser Ala Arg Gly His Arg Pro 35 40 45Leu Asp
Lys Lys Arg Glu Glu Ala Pro Ser Leu Arg Pro Ala Pro Pro 50 55 60Pro
Ile Ser Gly Gly Gly Tyr Arg Ala Arg Pro Ala Lys Ala Ala Ala65 70 75
80Thr Gln Lys Lys Val Glu Arg Lys Ala Pro Asp Ala Gly Gly Cys Leu
85 90 95His Ala Asp Pro Asp Leu Gly Val Leu Cys Pro Thr Gly Cys Gln
Leu 100 105 110Gln Glu Ala Leu Leu Gln Gln Glu Arg Pro Ile Arg Asn
Ser Val Asp 115 120 125Glu Leu Asn Asn Asn Val Glu Ala Val Ser Gln
Thr Ser Ser Ser Ser 130 135 140Phe Gln Tyr Met Tyr Leu Leu Lys Asp
Leu Trp Gln Lys Arg Gln Lys145 150 155 160Gln Val Lys Asp Asn Glu
Asn Val Val Asn Glu Tyr Ser Ser Glu Leu 165 170 175Glu Lys His Gln
Leu Tyr Ile Asp Glu Thr Val Asn Ser Asn Ile Pro 180 185 190Thr Asn
Leu Arg Val Leu Arg Ser Ile Leu Glu Asn Leu Arg Ser Lys 195 200
205Ile Gln Lys Leu Glu Ser Asp Val Ser Ala Gln Met Glu Tyr Cys Arg
210 215 220Thr Pro Cys Thr Val Ser Cys Asn Ile Pro Val Val Ser Gly
Lys Glu225 230 235 240Cys Glu Glu Ile Ile Arg Lys Gly Gly Glu Thr
Ser Glu Met Tyr Leu 245 250 255Ile Gln Pro Asp Ser Ser Val Lys Pro
Tyr Arg Val Tyr Cys Asp Met 260 265 270Asn Thr Glu Asn Gly Gly Trp
Thr Val Ile Gln Asn Arg Gln Asp Gly 275 280 285Ser Val Asp Phe Gly
Arg Lys Trp Asp Pro Tyr Lys Gln Gly Phe Gly 290 295 300Asn Val Ala
Thr Asn Thr Asp Gly Lys Asn Tyr Cys Gly Leu Pro Gly305 310 315
320Glu Tyr Trp Leu Gly Asn Asp Lys Ile Ser Gln Leu Thr Arg Met Gly
325 330 335Pro Thr Glu Leu Leu Ile Glu Met Glu Asp Trp Lys Gly Asp
Lys Val 340 345 350Lys Ala His Tyr Gly Gly Phe Thr Val Gln Asn Glu
Ala Asn Lys Tyr 355 360 365Gln Ile Ser Val Asn Lys Tyr Arg Gly Thr
Ala Gly Asn Ala Leu Met 370 375 380Asp Gly Ala Ser Gln Leu Met Gly
Glu Asn Arg Thr Met Thr Ile His385 390 395 400Asn Gly Met Phe Phe
Ser Thr Tyr Asp Arg Asp Asn Asp Gly Trp Leu 405 410 415Thr Ser Asp
Pro Arg Lys Gln Cys Ser Lys Glu Asp Gly Gly Gly Trp 420 425 430Trp
Tyr Asn Arg Cys His Ala Ala Asn Pro Asn Gly Arg Tyr Tyr Trp 435 440
445Gly Gly Gln Tyr Thr Trp Asp Met Ala Lys His Gly Thr Asp Asp Gly
450 455 460Val Val Trp Met Asn Trp Lys Gly Ser Trp Tyr Ser Met Arg
Lys Met465 470 475 480Ser Met Lys Ile Arg Pro Phe Phe Pro Gln Gln
485 490316PRTHomo sapiens 3Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala
Glu Gly Gly Gly Val Arg 1 5 10 15416PRTHomo sapiensVARIANT3Xaa =
Dehydroalanine 4Ala Asp Xaa Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly
Gly Val Arg 1 5 10 15515PRTHomo sapiens 5Asp Ser Gly Glu Gly Asp
Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 15614PRTHomo sapiens
6Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val 1 5
10714PRTHomo sapiens 7Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly
Gly Val Arg 1 5 10813PRTHomo sapiens 8Ser Gly Glu Gly Asp Phe Leu
Ala Glu Gly Gly Gly Val 1 5 10913PRTHomo sapiens 9Gly Glu Gly Asp
Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 101012PRTHomo sapiens 10Gly
Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val 1 5 101112PRTHomo
sapiens 11Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5
101211PRTHomo sapiens 12Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val
1 5 101311PRTHomo sapiens 13Gly Asp Phe Leu Ala Glu Gly Gly Gly Val
Arg 1 5 101410PRTHomo sapiens 14Gly Asp Phe Leu Ala Glu Gly Gly Gly
Val 1 5 101510PRTHomo sapiens 15Asp Phe Leu Ala Glu Gly Gly Gly Val
Arg 1 5 10169PRTHomo sapiens 16Asp Phe Leu Ala Glu Gly Gly Gly Val
1 5179PRTHomo sapiens 17Phe Leu Ala Glu Gly Gly Gly Val Arg 1
5188PRTHomo sapiens 18Phe Leu Ala Glu Gly Gly Gly Val 1 5197PRTHomo
sapiens 19Leu Ala Glu Gly Gly Gly Val 1 5206PRTHomo sapiens 20Ala
Glu Gly Gly Gly Val 1 5215PRTHomo sapiens 21Glu Gly Gly Gly Val 1
52216PRTHomo sapiensVARIANT3Xaa = Phosphoserine 22Ala Asp Xaa Glu
Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10
152315PRTHomo sapiensVARIANT2Xaa = Phosphoserine 23Asp Xaa Gly Glu
Gly Asp Phe Leu Ala Glu Gly Gly Gly Val Arg 1 5 10 152414PRTHomo
sapiensVARIANT2Xaa = Phosphoserine 24Asp Xaa Gly Glu Gly Asp Phe
Leu Ala Glu Gly Gly Gly Val 1 5 102514PRTHomo sapiensVARIANT1Xaa =
Phosphoserine 25Xaa Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val
Arg 1 5 102613PRTHomo sapiensVARIANT1Xaa = Phosphoserine 26Xaa Gly
Glu Gly Asp Phe Leu Ala Glu Gly Gly Gly Val 1 5 102714PRTHomo
sapiens 27Gln Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg 1
5 102814PRTHomo sapiensVARIANT1Xaa = Pyroglutamate 28Xaa Glu Gly
Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala 1 5 102915PRTHomo
sapiens 29Asp Glu Ala Gly Ser Glu Ala Asp His Glu Gly Thr His Ser
Thr 1 5 10 15301663PRTHomo sapiens 30Met Gly Pro Thr Ser Gly Pro
Ser Leu Leu Leu Leu Leu Leu Thr His 1 5 10 15Leu Pro Leu Ala Leu
Gly Ser Pro Met Tyr Ser Ile Ile Thr Pro Asn 20 25 30Ile Leu Arg Leu
Glu Ser Glu Glu Thr Met Val Leu Glu Ala His Asp 35 40 45Ala Gln Gly
Asp Val Pro Val Thr Val Thr Val His Asp Phe Pro Gly 50 55 60Lys Lys
Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr65 70 75
80Asn His Met Gly Asn Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe
85 90 95Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr Val Gln Ala Thr
Phe 100 105 110Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu
Gln Ser Gly 115 120 125Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr
Thr Pro Gly Ser Thr 130 135 140Val Leu Tyr Arg Ile Phe Thr Val Asn
His Lys Leu Leu Pro Val Gly145 150 155 160Arg Thr Val Met Val Asn
Ile Glu Asn Pro Glu Gly Ile Pro Val Lys 165 170 175Gln Asp Ser Leu
Ser Ser Gln Asn Gln Leu Gly Val Leu Pro Leu Ser 180 185 190Trp Asp
Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 195 200
205Tyr Tyr Glu Asn Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val
210 215 220Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val Glu Pro
Thr Glu225 230 235 240Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu
Glu Val Thr Ile Thr 245 250 255Ala Arg Phe Leu Tyr Gly Lys Lys Val
Glu Gly Thr Ala Phe Val Ile 260 265 270Phe Gly Ile Gln Asp Gly Glu
Gln Arg Ile Ser Leu Pro Glu Ser Leu 275 280 285Lys Arg Ile Pro Ile
Glu Asp Gly Ser Gly Glu Val Val Leu Ser Arg 290 295 300Lys Val Leu
Leu Asp Gly Val Gln Asn Leu Arg Ala Glu Asp Leu Val305 310 315
320Gly Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser
325 330 335Asp Met Val Gln Ala Glu Arg Ser Gly Ile Pro Ile Val Thr
Ser Pro 340 345 350Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr Phe
Lys Pro Gly Met 355 360 365Pro Phe Asp Leu Met Val Phe Val Thr Asn
Pro Asp Gly Ser Pro Ala 370 375 380Tyr Arg Val Pro Val Ala Val Gln
Gly Glu Asp Thr Val Gln Ser Leu385 390 395 400Thr Gln Gly Asp Gly
Val Ala Lys Leu Ser Ile Asn Thr His Pro Ser 405 410 415Gln Lys Pro
Leu Ser Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser 420 425 430Glu
Ala Glu Gln Ala Thr Arg Thr Met Gln Ala Leu Pro Tyr Ser Thr 435 440
445Val Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu
450 455 460Leu Arg Pro Gly Glu Thr Leu Asn Val Asn Phe Leu Leu Arg
Met Asp465 470 475 480Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr
Tyr Leu Ile Met Asn 485 490 495Lys Gly Arg Leu Leu Lys Ala Gly Arg
Gln Val Arg Glu Pro Gly Gln 500 505 510Asp Leu Val Val Leu Pro Leu
Ser Ile Thr Thr Asp Phe Ile Pro Ser 515 520 525Phe Arg Leu Val Ala
Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg 530 535 540Glu Val Val
Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Val545 550 555
560Gly Ser Leu Val Val Lys Ser Gly Gln Ser Glu Asp Arg Gln Pro Val
565 570 575Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly
Ala Arg 580 585 590Val Val Leu Val Ala Val Asp Lys Gly Val Phe Val
Leu Asn Lys Lys 595 600 605Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp
Val Val Glu Lys Ala Asp 610 615 620Ile Gly Cys Thr Pro Gly Ser Gly
Lys Asp Tyr Ala Gly Val Phe Ser625 630 635 640Asp Ala Gly Leu Thr
Phe Thr Ser Ser Ser Gly Gln Gln Thr Ala Gln 645 650 655Arg Ala Glu
Leu Gln Cys Pro Gln Pro Ala Ala Arg Arg Arg Arg Ser 660 665 670Val
Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys 675
680
685Glu Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met Arg
690 695 700Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu
Ala Cys705 710 715 720Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile
Thr Glu Leu Arg Arg 725 730 735Gln His Ala Arg Ala Ser His Leu Gly
Leu Ala Arg Ser Asn Leu Asp 740 745 750Glu Asp Ile Ile Ala Glu Glu
Asn Ile Val Ser Arg Ser Glu Phe Pro 755 760 765Glu Ser Trp Leu Trp
Asn Val Glu Asp Leu Lys Glu Pro Pro Lys Asn 770 775 780Gly Ile Ser
Thr Lys Leu Met Asn Ile Phe Leu Lys Asp Ser Ile Thr785 790 795
800Thr Trp Glu Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys
805 810 815Val Ala Asp Pro Phe Glu Val Thr Val Met Gln Asp Phe Phe
Ile Asp 820 825 830Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln
Val Glu Ile Arg 835 840 845Ala Val Leu Tyr Asn Tyr Arg Gln Asn Gln
Glu Leu Lys Val Arg Val 850 855 860Glu Leu Leu His Asn Pro Ala Phe
Cys Ser Leu Ala Thr Thr Lys Arg865 870 875 880Arg His Gln Gln Thr
Val Thr Ile Pro Pro Lys Ser Ser Leu Ser Val 885 890 895Pro Tyr Val
Ile Val Pro Leu Lys Thr Gly Leu Gln Glu Val Glu Val 900 905 910Lys
Ala Ala Val Tyr His His Phe Ile Ser Asp Gly Val Arg Lys Ser 915 920
925Leu Lys Val Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val
930 935 940Arg Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu Gly Val Gln
Lys Glu945 950 955 960Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val
Pro Asp Thr Glu Ser 965 970 975Glu Thr Arg Ile Leu Leu Gln Gly Thr
Pro Val Ala Gln Met Thr Glu 980 985 990Asp Ala Val Asp Ala Glu Arg
Leu Lys His Leu Ile Val Thr Pro Ser 995 1000 1005Gly Cys Gly Glu
Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile Ala 1010 1015 1020Val
His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe Gly Leu Glu1025
1030 1035 1040Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr
Thr Gln Gln 1045 1050 1055Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe
Ala Ala Phe Val Lys Arg 1060 1065 1070Ala Pro Ser Thr Trp Leu Thr
Ala Tyr Val Val Lys Val Phe Ser Leu 1075 1080 1085Ala Val Asn Leu
Ile Ala Ile Asp Ser Gln Val Leu Cys Gly Ala Val 1090 1095 1100Lys
Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val Phe Gln Glu1105
1110 1115 1120Asp Ala Pro Val Ile His Gln Glu Met Ile Gly Gly Leu
Arg Asn Asn 1125 1130 1135Asn Glu Lys Asp Met Ala Leu Thr Ala Phe
Val Leu Ile Ser Leu Gln 1140 1145 1150Glu Ala Lys Asp Ile Cys Glu
Glu Gln Val Asn Ser Leu Pro Gly Ser 1155 1160 1165Ile Thr Lys Ala
Gly Asp Phe Leu Glu Ala Asn Tyr Met Asn Leu Gln 1170 1175 1180Arg
Ser Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala Gln Met Gly1185
1190 1195 1200Arg Leu Lys Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr
Ala Lys Asp 1205 1210 1215Lys Asn Arg Trp Glu Asp Pro Gly Lys Gln
Leu Tyr Asn Val Glu Ala 1220 1225 1230Thr Ser Tyr Ala Leu Leu Ala
Leu Leu Gln Leu Lys Asp Phe Asp Phe 1235 1240 1245Val Pro Pro Val
Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly Gly 1250 1255 1260Gly
Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala Leu Ala1265
1270 1275 1280Gln Tyr Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn
Leu Asp Val 1285 1290 1295Ser Leu Gln Leu Pro Ser Arg Ser Ser Lys
Ile Thr His Arg Ile His 1300 1305 1310Trp Glu Ser Ala Ser Leu Leu
Arg Ser Glu Glu Thr Lys Glu Asn Glu 1315 1320 1325Gly Phe Thr Val
Thr Ala Glu Gly Lys Gly Gln Gly Thr Leu Ser Val 1330 1335 1340Val
Thr Met Tyr His Ala Lys Ala Lys Asp Gln Leu Thr Cys Asn Lys1345
1350 1355 1360Phe Asp Leu Lys Val Thr Ile Lys Pro Ala Pro Glu Thr
Glu Lys Arg 1365 1370 1375Pro Gln Asp Ala Lys Asn Thr Met Ile Leu
Glu Ile Cys Thr Arg Tyr 1380 1385 1390Arg Gly Asp Gln Asp Ala Thr
Met Ser Ile Leu Asp Ile Ser Met Met 1395 1400 1405Thr Gly Phe Ala
Pro Asp Thr Asp Asp Leu Lys Gln Leu Ala Asn Gly 1410 1415 1420Val
Asp Arg Tyr Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser Asp1425
1430 1435 1440Arg Asn Thr Leu Ile Ile Tyr Leu Asp Lys Val Ser His
Ser Glu Asp 1445 1450 1455Asp Cys Leu Ala Phe Lys Val His Gln Tyr
Phe Asn Val Glu Leu Ile 1460 1465 1470Gln Pro Gly Ala Val Lys Val
Tyr Ala Tyr Tyr Asn Leu Glu Glu Ser 1475 1480 1485Cys Thr Arg Phe
Tyr His Pro Glu Lys Glu Asp Gly Lys Leu Asn Lys 1490 1495 1500Leu
Cys Arg Asp Glu Leu Cys Arg Cys Ala Glu Glu Asn Cys Phe Ile1505
1510 1515 1520Gln Lys Ser Asp Asp Lys Val Thr Leu Glu Glu Arg Leu
Asp Lys Ala 1525 1530 1535Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys
Thr Arg Leu Val Lys Val 1540 1545 1550Gln Leu Ser Asn Asp Phe Asp
Glu Tyr Ile Met Ala Ile Glu Gln Thr 1555 1560 1565Ile Lys Ser Gly
Ser Asp Glu Val Gln Val Gly Gln Gln Arg Thr Phe 1570 1575 1580Ile
Ser Pro Ile Lys Cys Arg Glu Ala Leu Lys Leu Glu Glu Lys Lys1585
1590 1595 1600His Tyr Leu Met Trp Gly Leu Ser Ser Asp Phe Trp Gly
Glu Lys Pro 1605 1610 1615Asn Leu Ser Tyr Ile Ile Gly Lys Asp Thr
Trp Val Glu His Trp Pro 1620 1625 1630Glu Glu Asp Glu Cys Gln Asp
Glu Glu Asn Gln Lys Gln Cys Gln Asp 1635 1640 1645Leu Gly Ala Phe
Thr Glu Ser Met Val Val Phe Gly Cys Pro Asn 1650 1655
16603177PRTHomo sapiens 31Ser Val Gln Leu Thr Glu Lys Arg Met Asp
Lys Val Gly Lys Tyr Pro 1 5 10 15Lys Glu Leu Arg Lys Cys Cys Glu
Asp Gly Met Arg Glu Asn Pro Met 20 25 30Arg Phe Ser Cys Gln Arg Arg
Thr Arg Phe Ile Ser Leu Gly Glu Ala 35 40 45Cys Lys Lys Val Phe Leu
Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg 50 55 60Arg Gln His Ala Arg
Ala Ser His Leu Gly Leu Ala Arg65 70 7532935PRTHomo sapiens 32Asn
Tyr Ile Thr Glu Leu Arg Arg Gln His Ala Arg Ala Ser His Leu 1 5 10
15Gly Leu Ala Arg Ser Asn Leu Asp Glu Asp Ile Ile Ala Glu Glu Asn
20 25 30Ile Val Ser Arg Ser Glu Phe Pro Glu Ser Trp Leu Trp Asn Val
Glu 35 40 45Asp Leu Lys Glu Pro Pro Lys Asn Gly Ile Ser Thr Lys Leu
Met Asn 50 55 60Ile Phe Leu Lys Asp Ser Ile Thr Thr Trp Glu Ile Leu
Ala Val Ser65 70 75 80Met Ser Asp Lys Lys Gly Ile Cys Val Ala Asp
Pro Phe Glu Val Thr 85 90 95Val Met Gln Asp Phe Phe Ile Asp Leu Arg
Leu Pro Tyr Ser Val Val 100 105 110Arg Asn Glu Gln Val Glu Ile Arg
Ala Val Leu Tyr Asn Tyr Arg Gln 115 120 125Asn Gln Glu Leu Lys Val
Arg Val Glu Leu Leu His Asn Pro Ala Phe 130 135 140Cys Ser Leu Ala
Thr Thr Lys Arg Arg His Gln Gln Thr Val Thr Ile145 150 155 160Pro
Pro Lys Ser Ser Leu Ser Val Pro Tyr Val Ile Val Pro Leu Lys 165 170
175Thr Gly Leu Gln Glu Val Glu Val Lys Ala Ala Val Tyr His His Phe
180 185 190Ile Ser Asp Gly Val Arg Lys Ser Leu Lys Val Val Pro Glu
Gly Ile 195 200 205Arg Met Asn Lys Thr Val Ala Val Arg Thr Leu Asp
Pro Glu Arg Leu 210 215 220Gly Arg Glu Gly Val Gln Lys Glu Asp Ile
Pro Pro Ala Asp Leu Ser225 230 235 240Asp Gln Val Pro Asp Thr Glu
Ser Glu Thr Arg Ile Leu Leu Gln Gly 245 250 255Thr Pro Val Ala Gln
Met Thr Glu Asp Ala Val Asp Ala Glu Arg Leu 260 265 270Lys His Leu
Ile Val Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile 275 280 285Gly
Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Glu Thr Glu 290 295
300Gln Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Gly Ala Leu Glu
Leu305 310 315 320Ile Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg
Gln Pro Ser Ser 325 330 335Ala Phe Ala Ala Phe Val Lys Arg Ala Pro
Ser Thr Trp Leu Thr Ala 340 345 350Tyr Val Val Lys Val Phe Ser Leu
Ala Val Asn Leu Ile Ala Ile Asp 355 360 365Ser Gln Val Leu Cys Gly
Ala Val Lys Trp Leu Ile Leu Glu Lys Gln 370 375 380Lys Pro Asp Gly
Val Phe Gln Glu Asp Ala Pro Val Ile His Gln Glu385 390 395 400Met
Ile Gly Gly Leu Arg Asn Asn Asn Glu Lys Asp Met Ala Leu Thr 405 410
415Ala Phe Val Leu Ile Ser Leu Gln Glu Ala Lys Asp Ile Cys Glu Glu
420 425 430Gln Val Asn Ser Leu Pro Gly Ser Ile Thr Lys Ala Gly Asp
Phe Leu 435 440 445Glu Ala Asn Tyr Met Asn Leu Gln Arg Ser Tyr Thr
Val Ala Ile Ala 450 455 460Gly Tyr Ala Leu Ala Gln Met Gly Arg Leu
Lys Gly Pro Leu Leu Asn465 470 475 480Lys Phe Leu Thr Thr Ala Lys
Asp Lys Asn Arg Trp Glu Asp Pro Gly 485 490 495Lys Gln Leu Tyr Asn
Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu 500 505 510Leu Gln Leu
Lys Asp Phe Asp Phe Val Pro Pro Val Val Arg Trp Leu 515 520 525Asn
Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr 530 535
540Phe Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Ala Pro
Asp545 550 555 560His Gln Glu Leu Asn Leu Asp Val Ser Leu Gln Leu
Pro Ser Arg Ser 565 570 575Ser Lys Ile Thr His Arg Ile His Trp Glu
Ser Ala Ser Leu Leu Arg 580 585 590Ser Glu Glu Thr Lys Glu Asn Glu
Gly Phe Thr Val Thr Ala Glu Gly 595 600 605Lys Gly Gln Gly Thr Leu
Ser Val Val Thr Met Tyr His Ala Lys Ala 610 615 620Lys Asp Gln Leu
Thr Cys Asn Lys Phe Asp Leu Lys Val Thr Ile Lys625 630 635 640Pro
Ala Pro Glu Thr Glu Lys Arg Pro Gln Asp Ala Lys Asn Thr Met 645 650
655Ile Leu Glu Ile Cys Thr Arg Tyr Arg Gly Asp Gln Asp Ala Thr Met
660 665 670Ser Ile Leu Asp Ile Ser Met Met Thr Gly Phe Ala Pro Asp
Thr Asp 675 680 685Asp Leu Lys Gln Leu Ala Asn Gly Val Asp Arg Tyr
Ile Ser Lys Tyr 690 695 700Glu Leu Asp Lys Ala Phe Ser Asp Arg Asn
Thr Leu Ile Ile Tyr Leu705 710 715 720Asp Lys Val Ser His Ser Glu
Asp Asp Cys Leu Ala Phe Lys Val His 725 730 735Gln Tyr Phe Asn Val
Glu Leu Ile Gln Pro Gly Ala Val Lys Val Tyr 740 745 750Ala Tyr Tyr
Asn Leu Glu Glu Ser Cys Thr Arg Phe Tyr His Pro Glu 755 760 765Lys
Glu Asp Gly Lys Leu Asn Lys Leu Cys Arg Asp Glu Leu Cys Arg 770 775
780Cys Ala Glu Glu Asn Cys Phe Ile Gln Lys Ser Asp Asp Lys Val
Thr785 790 795 800Leu Glu Glu Arg Leu Asp Lys Ala Cys Glu Pro Gly
Val Asp Tyr Val 805 810 815Tyr Lys Thr Arg Leu Val Lys Val Gln Leu
Ser Asn Asp Phe Asp Glu 820 825 830Tyr Ile Met Ala Ile Glu Gln Thr
Ile Lys Ser Gly Ser Asp Glu Val 835 840 845Gln Val Gly Gln Gln Arg
Thr Phe Ile Ser Pro Ile Lys Cys Arg Glu 850 855 860Ala Leu Lys Leu
Glu Glu Lys Lys His Tyr Leu Met Trp Gly Leu Ser865 870 875 880Ser
Asp Phe Trp Gly Glu Lys Pro Asn Leu Ser Tyr Ile Ile Gly Lys 885 890
895Asp Thr Trp Val Glu His Trp Pro Glu Glu Asp Glu Cys Gln Asp Glu
900 905 910Glu Asn Gln Lys Gln Cys Gln Asp Leu Gly Ala Phe Thr Glu
Ser Met 915 920 925Val Val Phe Gly Cys Pro Asn 930 93533343PRTHomo
sapiens 33Ser Glu Glu Thr Lys Glu Asn Glu Gly Phe Thr Val Thr Ala
Glu Gly 1 5 10 15Lys Gly Gln Gly Thr Leu Ser Val Val Thr Met Tyr
His Ala Lys Ala 20 25 30Lys Asp Gln Leu Thr Cys Asn Lys Phe Asp Leu
Lys Val Thr Ile Lys 35 40 45Pro Ala Pro Glu Thr Glu Lys Arg Pro Gln
Asp Ala Lys Asn Thr Met 50 55 60Ile Leu Glu Ile Cys Thr Arg Tyr Arg
Gly Asp Gln Asp Ala Thr Met65 70 75 80Ser Ile Leu Asp Ile Ser Met
Met Thr Gly Phe Ala Pro Asp Thr Asp 85 90 95Asp Leu Lys Gln Leu Ala
Asn Gly Val Asp Arg Tyr Ile Ser Lys Tyr 100 105 110Glu Leu Asp Lys
Ala Phe Ser Asp Arg Asn Thr Leu Ile Ile Tyr Leu 115 120 125Asp Lys
Val Ser His Ser Glu Asp Asp Cys Leu Ala Phe Lys Val His 130 135
140Gln Tyr Phe Asn Val Glu Leu Ile Gln Pro Gly Ala Val Lys Val
Tyr145 150 155 160Ala Tyr Tyr Asn Leu Glu Glu Ser Cys Thr Arg Phe
Tyr His Pro Glu 165 170 175Lys Glu Asp Gly Lys Leu Asn Lys Leu Cys
Arg Asp Glu Leu Cys Arg 180 185 190Cys Ala Glu Glu Asn Cys Phe Ile
Gln Lys Ser Asp Asp Lys Val Thr 195 200 205Leu Glu Glu Arg Leu Asp
Lys Ala Cys Glu Pro Gly Val Asp Tyr Val 210 215 220Tyr Lys Thr Arg
Leu Val Lys Val Gln Leu Ser Asn Asp Phe Asp Glu225 230 235 240Tyr
Ile Met Ala Ile Glu Gln Thr Ile Lys Ser Gly Ser Asp Glu Val 245 250
255Gln Val Gly Gln Gln Arg Thr Phe Ile Ser Pro Ile Lys Cys Arg Glu
260 265 270Ala Leu Lys Leu Glu Glu Lys Lys His Tyr Leu Met Trp Gly
Leu Ser 275 280 285Ser Asp Phe Trp Gly Glu Lys Pro Asn Leu Ser Tyr
Ile Ile Gly Lys 290 295 300Asp Thr Trp Val Glu His Trp Pro Glu Glu
Asp Glu Cys Gln Asp Glu305 310 315 320Glu Asn Gln Lys Gln Cys Gln
Asp Leu Gly Ala Phe Thr Glu Ser Met 325 330 335Val Val Phe Gly Cys
Pro Asn 3403416PRTHomo sapiens 34Ser Glu Glu Thr Lys Glu Asn Glu
Gly Phe Thr Val Thr Ala Glu Gly 1 5 10 153515PRTHomo sapiens 35Glu
Glu Thr Lys Glu Asn Glu Gly Phe Thr Val Thr Ala Glu Gly 1 5 10
153617PRTHomo sapiens 36Ser Ser Lys Ile Thr His Arg Ile His Trp Glu
Ser Ala Ser Leu Leu 1 5 10 15Arg378PRTHomo sapiens 37His Trp Glu
Ser Ala Ser Leu Leu 1 53843PRTHomo sapiens 38Ser Asp Lys Pro Asp
Met Ala Glu Met Glu Lys Phe Asp Lys Ser Lys 1 5 10 15Leu Lys Lys
Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro Ser Lys Glu 20 25 30Thr Ile
Glu Gln Glu Lys Gln Ala Gly Glu Ser 35 403943PRTHomo sapiens 39Ser
Asp Lys Pro Asp Met Ala Glu Ile Glu Lys Phe Asp Lys Pro Lys 1 5 10
15Leu Lys Lys Thr Glu Thr
Gln Glu Lys Asn Pro Leu Pro Ser Lys Glu 20 25 30Thr Ile Glu Gln Glu
Lys Gln Ala Gly Glu Ser 35 404044PRTHomo sapiens 40Met Ser Asp Lys
Pro Asp Met Ala Glu Ile Glu Lys Phe Asp Lys Ser 1 5 10 15Lys Leu
Lys Lys Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro Ser Lys 20 25 30Glu
Thr Ile Glu Gln Glu Lys Gln Ala Gly Glu Ser 35 404143PRTHomo
sapiens 41Ser Asp Lys Ser Asp Met Ala Glu Ile Glu Lys Phe Asp Lys
Ser Lys 1 5 10 15Leu Lys Lys Thr Glu Thr Gln Glu Lys Asn Pro Leu
Pro Ser Lys Glu 20 25 30Thr Ile Glu Gln Glu Lys Gln Ala Gly Glu Ser
35 404222PRTHomo sapiens 42Thr Gln Glu Lys Asn Pro Leu Pro Ser Lys
Glu Thr Ile Glu Gln Glu 1 5 10 15Lys Gln Ala Gly Glu Ser
204321PRTHomo sapiensVARIANT1Xaa = Pyroglutamate 43Xaa Glu Lys Asn
Pro Leu Pro Ser Lys Glu Thr Ile Glu Gln Glu Lys 1 5 10 15Gln Ala
Gly Glu Ser 204413PRTHomo sapiens 44Glu Gly Leu Asn Asp Asn Glu Glu
Gly Phe Phe Ser Ala 1 5 10
* * * * *
References