U.S. patent application number 11/675732 was filed with the patent office on 2007-10-11 for nuclear matrix protein alterations associated with colon cancer and colon metastasis to the liver and uses thereof.
This patent application is currently assigned to University of Pittsburgh. Invention is credited to Anthony J. Bauer, Gisela Bruenagel, Robert H. Getzenberg, Robert E. Schoen.
Application Number | 20070238194 11/675732 |
Document ID | / |
Family ID | 27669038 |
Filed Date | 2007-10-11 |
United States Patent
Application |
20070238194 |
Kind Code |
A1 |
Bauer; Anthony J. ; et
al. |
October 11, 2007 |
NUCLEAR MATRIX PROTEIN ALTERATIONS ASSOCIATED WITH COLON CANCER AND
COLON METASTASIS TO THE LIVER AND USES THEREOF
Abstract
Proteins useful in the diagnosis of proliferative disorders of
the colon are present in nuclear matrix protein preparations and
can be characterized by molecular weight, isoelectric point, and
amino acid sequence. The proteins may be identified, for example,
by 2D-gel electrophoresis or by specific binding partners, such as
antibodies.
Inventors: |
Bauer; Anthony J.;
(Pittsburgh, PA) ; Bruenagel; Gisela; (Pittsburgh,
PA) ; Getzenberg; Robert H.; (Lutherville, MD)
; Schoen; Robert E.; (Pittsburgh, PA) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
University of Pittsburgh
|
Family ID: |
27669038 |
Appl. No.: |
11/675732 |
Filed: |
February 16, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11133177 |
May 20, 2005 |
7189823 |
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11675732 |
Feb 16, 2007 |
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10350367 |
Jan 24, 2003 |
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11133177 |
May 20, 2005 |
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60351819 |
Jan 25, 2002 |
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60412612 |
Sep 19, 2002 |
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Current U.S.
Class: |
436/501 |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 14/4748 20130101; G01N 33/57419 20130101 |
Class at
Publication: |
436/501 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT SPONSORED RESEARCH
[0002] This invention was made with government support from the
United States National Institutes of Health. The government may
have certain rights in this invention.
Claims
1. A method of analyzing a biological specimen for the presence of
an analyte indicative of a clinical condition, comprising the steps
of: contacting the specimen with an antibody which binds to the
analyte, detecting binding of the antibody in the specimen, and
correlating the detected binding to the clinical condition, wherein
the biological specimen is a body fluid, the antibody binds to
Ile-Leu-Leu-Phe-Asp-Tyr-Phe-Asn-Arg (SEQ ID NO: 28), and the
detected binding is correlated to a cell-proliferative disorder of
the colon.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/133,177, filed May 20, 205, which is a continuation of
U.S. patent application Ser. No. 10/350,367, filed Jan. 24, 2003,
which claims the benefit of U.S. Provisional Patent Appl. No.
60/351,819, filed Jan. 25, 2002, and U.S. Provisional Patent Appl.
No. 60/412,612, filed Sep. 19, 2002 each of which is hereby
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The present invention relates generally to proteins
associated with cell proliferative disorders of the colon including
primary colon cancer and colon cancer metastases to liver. The
invention also relates to proteins associated with normal colon
tissue.
[0004] Improvements in surgical procedures, adjuvant therapies, and
screening programs have facilitated an overall decline in the
mortality of colon cancer in the last 20 years (1). Colorectal
cancer nevertheless remains a significant disease accounting for
11% of all cancers in the United States with an estimated 130,200
new cases and 48,100 deaths expected in the year 2001 (2). When
diagnosed at an early localized stage, five-year survival of colon
cancer is 90%. Only 37% of incident cases are diagnosed at this
stage, the remainder often presenting to a physician when the tumor
has become metastatic.
[0005] More effective screening and prevention measures for
colorectal cancer are needed to address this public health problem.
Early detection procedures for colorectal cancer have included
tests for fecal blood or use of endoscopy. The fecal blood test
requires significant tumor size (sensitivity of 90% for small
polyps and 75% for diminutive polyps) and has a sensitivity of
about 26%, which means 74% of patients with malignant lesions will
remain undetected (3). The fecal occult blood test fails to detect
many early stage colon cancers because little if any blood is
released into the stool at that stage. Also the fecal occult test
is not very specific as a general screen, subjecting many to
needless discomfort and risk in subsequent full bowel
examination.
[0006] Visualization of precancerous and cancerous lesions by
endoscopy is effective in early detection but is an invasive method
with attendant significant risk of complications (4,5). For
example, the cecum is reached in 80%-95% of procedures (22) and
incomplete colonoscopies require either a repeat colonoscopy or
supplemental barium enema.
[0007] The complications and cost of the colonoscopy are
considerable and the appropriate frequently at which this procedure
should be used for cancer screening tool is unknown. Furthermore,
the procedural competence varies considerably among endoscopists.
Thus, colonoscopy is presently not useful for screening the general
population for colon cancer.
[0008] Much effort over the years has been directed to the
identification of improved diagnostic markers for colon cancer that
enable reliable early cancer detection or provide early prognostic
information. Carcinoembryonic antigen (CEA), which is a
tumor-associated glycoprotein, was found to be expressed at
increased levels in 95% of colorectal, gastric and pancreatic
cancers, and in the majority of breast, lung and head and neck
carcinomas (6). Diagnostic blood tests for CEA are in use for
following the course of therapy in the management of colorectal
cancer. In postoperative follow-up, CEA appears to be a useful
marker of recurrence (sensitivity, 77%; specificity 98%), mainly
for liver metastasis, but it has also been shown that only half of
colorectal cancers shed CEA levels sufficient for their detection
in monitoring therapy (7,8).
[0009] The utility of CEA in detecting recurrences is controversial
and has yet to be widely applied (9,10). Elevated CEA levels have
been reported in patients with nonmalignant disease and many
patients with colon cancer have normal CEA levels in the serum,
especially during the early stage of the disease (7,11). In light
of the currently available data, serum CEA determination possesses
neither the sensitivity or specificity needed as an early screening
test for colorectal cancer in the asymptomatic population (12).
[0010] Changes in nuclear shape, size and DNA organization
including major morphological transformation are unique
characteristics of cancer cells has been used to diagnose cancer.
Nuclear structure is determined by the scaffolding of the nucleus,
the nuclear matrix. The nuclear matrix consists of the peripheral
lamins, protein complexes, an internal ribonucleic protein network,
and residual nucleoli (23). The nuclear framework consists of
approximately 10% of the nuclear proteins, and is virtually devoid
of lipids, DNA, and histones (24). Most of the nuclear matrix
proteins identified to date are common to all cell types, but
several identified NMPs are tissue and cell line specific and NMPs
have been shown to undergo change with differentiation (25,
26).
[0011] Cell type-specific "fingerprinting" of aberrant nuclear
matrix proteins and their appearance in cancer development has led
to the analysis of nuclear matrix protein composition of a variety
of tumors in an effort to determine whether these proteins can be
developed as diagnostic and/or prognostic markers for cancer. By
means of high resolution, two-dimensional electrophoresis, specific
nuclear matrix protein alterations have been demonstrated to exist
in primary cancers of the prostate, bladder, renal and colon
(27-30). The detection of nuclear matrix proteins in the serum of
patients with various types of cancer has been accomplished
(31).
SUMMARY OF THE INVENTION
[0012] In accordance with one aspect of the invention, there is
provided a purified protein present in cancerous colon cells but
absent or reduced in amount in normal colonic epithelial cells. The
specified proteins include, CC2, CC3, CC4, CC5, CC6a, CC6b, L1, L2
and L5. The apparent molecular weight, isoelectric point and
partial amino acid sequence for these proteins are provided in the
context of the examples herein. In a preferred embodiment the
proteins are detected in nuclear matrix preparations ("NMPs").
[0013] In accordance with another aspect of the invention, proteins
are provided that are present in normal epithelial colonic cells
but that are absent or present in reduced amount in cancerous colon
cells. The specified proteins include N1-N6, the apparent molecular
weight, and isoelectric point of which are provided (see Examples,
infra). In a preferred embodiment, the N proteins are detected in
nuclear matrix preparations.
[0014] In accordance with yet another aspect of the invention,
there is provided purified proteins in cancerous colon cells and
liver metastasis derived therefrom which are useful as colon cancer
diagnostic markers. The specified proteins include L1, L2, L3, L4
and L5, the apparent molecular weight and isoelectric point of
which are provided below. In a preferred embodiment, proteins L1-L5
are detected in NMPs.
[0015] In accordance with a further aspect of the invention, there
is provided binding partners specific for the invention proteins.
The binding partners may be used, for example, for diagnostic or
therapeutic purposes. Methods of producing binding partners are
provided. The binding partner preferably is a monoclonal or
polyclonal antibody.
[0016] In accordance with yet a further aspect of the invention, a
method is provided for diagnosing in a patient a cell proliferative
disorder of the colon, preferably colon cancer, by analyzing
tissue, stool, or body fluid from the patient for the presence of
at least one protein that is present in cancerous colonic cells but
absent or reduced in amount in normal colonic epithelial cells, or
that is present in normal colonic epithelial cells but absent or
reduced in amount in cancerous colonic cells. Such diagnostic
proteins include, CC2, CC3, CC4, CC5, CC6a, CC6b, L1, L2 and L5 and
N1-N6. The proteins may be detected by any of a variety of means
including biochemical means such as 2-D gel electrophoresis,
detection with a specific binding partner or by determining the
level of encoding mRNA. In one embodiment, the proliferative
disorder is colorectal adenocarcinoma while in another embodiment,
the proliferative disorder is colonic adenoma.
[0017] In accordance with still yet a further aspect of the
invention, there is provided a method of diagnosing colon cancer in
a patient comprising analyzing tissue, stool or body fluid from the
patient for the presence of calreticulin. Calreticulin may be
detected by any of a variety of means including biochemical means
such as 2-D gel electrophoresis, detection with a specific binding
partner or by determining the level of encoding mRNA. In a
preferred embodiment, calreticulin is detected in NMP
preparations.
[0018] In accordance with another aspect of the invention, there is
provided a method of evaluating colonic adenomas for potential to
become malignant. The method comprises analyzing the adenoma for
the presence of at least one protein that is present in cancerous
colonic cells but absent or reduced in amount in normal colonic
epithelial cells, or present in normal colonic epithelial cells but
absent or reduced in amount in cancerous colonic cells. The
proteins are CC2, CC3, CC4, CC5, CC6a, CC6b, L1, L2 and L5 and
N1-N6. In a preferred embodiment, the proteins are CC3, CC4, and
CC5. The proteins may be detected by any of a variety of means
including biochemical means such as 2-D gel electrophoresis,
detection with a specific binding partner or by determining the
level of encoding mRNA.
[0019] In accordance with a further aspect of the invention, there
is provided a method of diagnosing colon cancer to liver metastasis
in a patient. The method comprises analyzing a sample of liver for
the presence of at least one protein, wherein the protein is any of
L1-L5. The proteins may be detected by any of a variety of means
including biochemical means such as 2-D gel electrophoresis,
detection with a specific binding partner or by determining the
level of encoding mRNA.
DETAILED DESCRIPTION OF THE INVENTION
[0020] As noted, a key aspect of the present invention is the
inventor's provides a purified protein that is present in cancerous
colon cells but absent or reduced in amount in normal colonic
epithelial cells. These proteins along with their apparent
molecular weight (defined by SDS-PAGE) and isoelectric point are as
follows:
[0021] a) CC2 having a molecular weight of about 56 kD and a pI of
about 6.22;
[0022] b) CC3 having a molecular weight of about 43 kD and a pI of
about 6.3;
[0023] c) CC4 having a molecular weight of about 43 kD and a pI of
about 6.2;
[0024] d) CC5 having a molecular weight of about 42 kD and a pI of
about 6.2;
[0025] e) CC6a having a molecular weight of about 20 kD and a pI of
about 6.9;
[0026] f) CC6b having a molecular weight of about 20 kD and a pI of
about 6.8.
[0027] g) L1 having a molecular weight of about 50 kD and a pI of
about 6.01;
[0028] h) L2 having a molecular weight of about 20 kD and a pI of
about 5.73; and
[0029] i) L5 having a molecular weight of about 19 kD and a pI of
about 5.88.
[0030] These proteins also may be defined by amino acid sequence
shown in Tables 5, 6 and 9, and by presence in a NMP preparation.
For example, in the case of CC2, it is defined as comprising one or
more proteins having a molecular weight of about 56 kD and an
isoelectric point of about 6.22, wherein the protein comprises
amino acid sequence from any one or more of SEQ ID NOs: 14-17 and
23. Thus, CC2 can be defined by its molecular weight and pI, its
presence in an NMP preparation, and by SEQ ID NO: 14, SEQ ID NO:
15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 23, or any
combination of these sequences.
[0031] Also provided is a purified protein that is present in
normal epithelial colonic cells but absent or reduced in amount in
cancerous colon cells. These proteins along with their apparent
molecular weight (defined by SDS-PAGE) and isoelectric point are as
follows:
[0032] a) N1 having a molecular weight of about 40 kD and a pI of
about 5.5;
[0033] b) N2 having a molecular weight of about 30 kD and a pI of
about 5.94;
[0034] c) N3 having a molecular weight of about 30 kD and a pI of
about 5.88;
[0035] d) N4 having a molecular weight of about 30 kD and a pI of
about 5.80;
[0036] e) N5 having a molecular weight of about 30 kD and a pI of
about 5.73; and
[0037] f) N6 having a molecular weight of about 18 kD and a pI of
about 6.6.
Proteins N1-N5 can be defined by their molecular weight and pI, and
presence in an NMP preparation.
[0038] Further provided are various purified proteins useful as
diagnostic markers for colon cancer metastases to the liver. These
proteins along with their apparent molecular weight (defined by
SDS-PAGE) and isoelectric point are as follows:
[0039] a) L1 having a molecular weight of about 50 kD and a pI of
about 6.01;
[0040] b) L2 having a molecular weight of about 20 kD and a pI of
about 5.73;
[0041] c) L3 having a molecular weight of about 17 kD and a pI of
about 6.09;
[0042] d) L4 having a molecular weight of about 17 kD and a pI of
about 6.00; and
[0043] e) L5 having a molecular weight of about 19 kD and a pI of
about 5.88.
[0044] L1-L5 are useful as diagnostic markers of colon cancer
metastatic to liver because these proteins are more detectable in
samples of liver that contain colon cancer than in samples of
normal liver tissue. These proteins may be defined by their
molecular weight and pI, their presence in an NMP preparation and
by amino acid sequence as shown in Table 9.
[0045] As used herein, the term "absent or reduced in amount in
normal colonic epithelial cells" means that the protein is not
detectable in normal colonic epithelial cells or is detectable in
such cells but at a lower level than that for colorectal carcinoma
cells. In being more detectable in colorectal carcinoma versus
normal colonic epithelial cells, the protein can be detected in 80%
or more of cancer samples versus 50% or less for comparable
non-cancer samples. Similarly, in connection with proteins that are
markers of colon cancer to liver metastases, the term absent or
reduced in amount means that the proteins are more detectable in
samples of liver that contain colon cancer than in samples of
normal liver tissue. In being "more detectable, proteins L1-L5 are
detectable in 80% of colon to liver metastases versus 50% or less
for donor samples of normal liver tissue.
[0046] The increased detectability of L1-L5 in metastatic colon to
liver tissue versus normal liver tissue is believed to result from
an increased level of expression of these proteins in colon cancer
cells versus normal liver cells including hepatocytes. "Normal
liver tissue" includes "normal donor liver tissue," which refers to
liver tissue from a donor that does not have metastatic liver
cancer and "adjacent normal liver tissue," which refers to normal
liver tissue from a liver with colon cancer metastasis. The term
"adjacent" is used to identify the type of liver from which this
"normal" liver tissue is obtained (i.e. metastatic liver) rather
than to indicate any degree of proximity between such normal tissue
and the metastatic cancer.
[0047] The percentage of 2D gels in which L1-L5 were detected in
samples of liver containing colon cancer metastases and in samples
containing normal liver tissue is shown in Table 7, (Example 5).
Although L3 and L4 proteins were detected in normal adjacent liver
and in normal donor liver, the amount of protein detected was
qualitatively speaking lower in amount that in the cancer
metastatic tissue.
[0048] Protein identification by silver stained--high-resolution,
two-dimensional gel electrophoresis analysis of nuclear matrix
preparations is a preferred method for determining whether the
invention proteins are more detectable in a particular tissue than
in another tissue. Other approaches are possible and include
detection of the proteins in the tissue or in tissue extracts using
a binding partner specific for the protein or by detecting RNA
encoding the protein.
[0049] In this description, the phrase "nuclear matrix" refers to a
3-dimensional filamentous protein network that is present in the
interphase nucleus. The NMPs of the protein network provide a
framework to maintain the overall size and shape of the nucleus and
acts a structural attachment site for the DNA loops during
interphase.
[0050] An "NMP preparation" is a preparation from a biological
source (e.g., a cell, tissue or body fluid) that is enriched in
nuclear matrix proteins ("NMPs"). "Enriched" means that at least
some NMP are present at a higher frequency in the NMP that they are
in the natural state (e.g., in cell, tissue, or biological fluid).
NMP preparations may be prepared by well known methods in the art
such as detergent and urea extraction (see Getzenberg et al.
reference 29). An NMP preparation that is enriched in NMPs may
contain proteins that are not part of the nuclear matrix
[0051] The present invention provides binding partners specific for
the invention proteins. The binding partners are useful to detect
the presence of the protein in a sample. The protein and its
binding partner represent a binding pair of molecules, which
interact with each other through any of a variety of molecular
forces including, for example, ionic, covalent, hydrophobic, van
der waals, and hydrogen bonding, so that the pair have the property
of binding specifically to each other. Specific binding means that
the binding pair exhibit binding with each other under conditions
where they do not bind to another molecule. Examples of types of
specific binding pairs are antigen-antibody, biotin-avidin,
hormone-receptor, receptor-ligand, enzyme-substrate, IgG-protein A,
and the like. A preferred binding partner for the proteins of the
invention is an antibody, but other desirable binding partners may
comprise a nucleic acid (e.g. natural or synthetic DNA, RNA, gDNA,
cDNA, mRNA, tRNA, etc.), a lectin, an oligosaccharide, a
glycoprotein, a drug candidates (from, for example, a random
peptide library, a natural products library, a lectin library, a
combinatorial library, an oligosaccharide library or a phage
display library), a metabolite, a vitamin, a lipid, a steroid, a
metal, and the like.
[0052] In the present context, an "antibody" is a protein that is
made up of one or more polypeptides, substantially encoded by
immunoglobulin genes or fragments of such genes. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon and mu constant region genes, as well as a myriad of
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0053] A typical immunoglobulin (antibody) is a tetramer composed
of two identical pairs "light" chains (each about 25 kD) and two
identical pairs of "heavy" chains (each about 50-70 kD). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The terms "variable light chain" (VL) and "variable
heavy chain" (VH) refer to the variable portions of the light and
heavy chains, respectively. The antigen-recognition site or
ligand/substrate-binding site of an immunoglobulin molecule is
formed by three highly divergent stretches within the V regions of
the heavy and light chains known as the "hypervariable regions,"
which are interposed between more conserved flanking stretches
known as "framework regions." In an antibody molecule, the three
hypervariable regions of a light chain and the three hypervariable
regions of a heavy chain are disposed relative to each other in
three dimensional space to form an antigen binding surface. This
surface mediates recognition and binding of the target antigen or
ligand/substrate. The three hypervariable regions of each of the
heavy and light chains are referred to as "complementarily
determining regions" or "CDRs" and are discussed, for example, by
Kabat et al. (15). The portion of the antigen that interacts with
the CDRs of the antibody is referred to as an epitope.
[0054] Antibodies exist as intact immunoglobulins or as a number of
well-characterized fragments, such as those produced by digestion
with various peptidases and those that can be made by recombinant
DNA technology. Antibody fragments include Fab' monomer, Fab'2
dimer, Fv fragment, single chain Fv ("scFv") fragment, and the
like. See e.g., Huston et al., (16). Antibody fragments also
include antibody forms having a truncated or deleted segment of the
light and/or heavy chain constant region.
[0055] Antibodies of the present invention may be monoclonal or
polyclonal. In general polyclonal antibodies are present in the
sera of animals immunized by one or more injections of the
invention proteins or fragments thereof. In general, monoclonal
antibodies are prepared by obtaining a source of B cells from a
suitably immunized animal, immortalizing the B cells, cloning
populations of B cells from individual immortalized cells, and
selecting clones making an antibody of interest. Methods for making
polyclonal antibodies and monoclonal antibodies are well known in
the art. For example, see Harlow and Lane (17).
[0056] Accordingly, a method is provided for producing polyclonal
antibodies that differentiate primary colon cancer from normal
colon and/or colon cancer liver metastases from normal liver
tissue. The method comprises immunizing an animal with one or more
of the invention proteins and recovering the antibodies. There is
further provided a method of producing a monoclonal antibody that
differentiate primary colon cancer from normal colon and/or colon
cancer liver metastases from normal liver tissue. The method
comprises immunizing an animal with one or more of the invention
proteins, removing B cells from the immunized animal and
immortalizing and isolating the B cells that produce an antibody
specific for the protein. As an alternative to immortalization,
nucleic acid encoding antibody heavy and light chains may be
obtained from immune cells of the immunized animal. The nucleic
acid can then be cloned into appropriate expression vectors and
used to transform, transfect or otherwise transduce host cells so
that the host cells produce antibody derived from the heavy and
light chains present in B cells of the immunized animal. Host cells
producing antibodies of interest may be selected.
[0057] An immunogen suitable for eliciting specific antibodies to
the invention proteins can comprise a purified or a partially
purified preparation of the protein. A purified protein of the
invention is at least 20% pure, preferably at least 40% pure, more
preferably at least 60% pure, even more preferably at least 80%
pure and still even more preferably at least 90% and most
preferably at least about between 95 and 99% pure.
[0058] The terms "protein," "polypeptide," and "peptide" are used
interchangeably in this description to denote a polymer of amino
acid residues. The category of "protein" includes proteins
associated with other molecules, such as a glycoprotein, a
proteoglycan, a lipoprotein, a nucleic acid, and combinations
thereof. Unless otherwise specified, the terms "a," "an" or "the"
mean one or more. The proteins of the invention also include
conservatively substituted variants thereof. Minor modifications of
the primary amino acid sequence may result in proteins that have
substantially equivalent activity as compared to the natural
polypeptide described herein. Conservative substitutions in
sequence, which denote the replacement of an amino acid residue by
a structurally similar residue, are preferred. Examples of
conservative substitutions include the substitution of one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of argime for lysine,
glutamic for aspartic acids, or glutamine for asparagine, and the
like. Such modifications may be deliberate, as by site-directed
mutagenesis, or may be spontaneous, and can include deletion of
non-essential amino acids. Modification includes deletion of one or
more amino acids, which may be used to develop a smaller active
molecule that has broader utility. All of the polypeptides produced
by these modifications are included herein as long as the
biological activity of the native protein remains the same. The
proteins of the invention also include fragments of the protein
which are useful, for example, to generate binding partners for the
protein.
[0059] An NMP preparation can be used as a source of partially
purified protein for the immunogen. The invention proteins may be
purified by any of a number of well-known purification procedures,
including precipitation, chromatography, electrophoresis,
immunological separations involving monoclonal or polyclonal
antibodies, and the like. For example, a purified protein
preparation can be obtained by 2-D gel electrophoresis of an NMP
followed by excising the protein spot and using electrophoresis to
elute the protein from the gel (see Examples). Alternatively, the
proteins or their fragments may be synthesized by the well-known
solid phase peptide synthesis methods described, for example, by
Merrifield (13) or by Stewart and Young (14), based on protein
sequences.
[0060] Purified preparations of the invention proteins or fragments
thereof also may be prepared by recombinant expression using
vectors and host cells well known in the art and commercially
available such as are described in Goeddel et al. (18). Host cells
include, for example, mammalian, bacterial, yeast, and the like.
The host cell may be transformed with recombinant DNA by
conventional techniques known in the art. For example, where the
host is prokaryotic, such as E. coli, one can prepare competent
cells, which are capable of DNA uptake, from cells harvested after
the exponential growth phase and subsequently treated by the
CaCl.sub.2-method, by conventional procedures. Transformation also
can be performed by forming a protoplast of the host cell or by
electroporation. A eukaryotic host may be transformed with foreign
DNA using well known procedures such as by calcium phosphate
coprecipitation, microinjection, electroporation, encasement in
liposomes, virus vectors, and the like. Recombinant DNA also may be
introduced by transfection and transduction methods well known in
the art.
[0061] Eukaryotic cells can also be co-transformed with DNA
sequences encoding the proteins of the invention, and a second
foreign DNA molecule encoding a selectable phenotype, such as the
herpes simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (5V40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein.
[0062] The invention proteins or fragments thereof may be expressed
as a fusion to a foreign polypeptide, for example, a bacterial
ligand binding sequence such as GST or Staphylococcal Protein A.
Fusion to GST, for example, provides an efficient means to purify
the fusion product or increase its immunogenicity (see, e.g., Uhlen
and Moks, (19)). Immunogenicity may also be enhanced by chemically
coupling the protein or fragment to a suitable immunogenic carrier
protein. Carrier proteins useful for the present invention have
molecular weights of at least about 20,000 Daltons. Carrier
proteins useful in the present invention include, for example, GST,
hemocyanins such as from the keyhole limpet, serum albumin or
cationized serum albumin, thyroglobulin, ovalbumin, various toxoid
proteins such a tetanus toxoid or diphtheria toxoid,
immunoglobulins or heat shock proteins. Methods to chemically
couple a polypeptide to a carrier protein are well known in the art
and include conjugation by a water soluble carbodiimide such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,
conjugation by a homobifunctional cross-linker having, for example,
NHS ester groups or sulfo-NHS ester analogs, conjugation by a
heterobifunctional cross-linker having, for example, and NHS ester
and a maleimide group such as
sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
conjugation with gluteraldehyde, and the like. For example, see
Hermanson, (20) and U.S. Pat. No. 4,608,251 and No. 4,161,519.
[0063] Binding partners such as polyclonal or monoclonal antibodies
can be used to detect the invention proteins in immunoassays such
as RIA, EIA, and the like. Such assays may be competitive or
non-competitive and may based on a direct or an indirect format.
The binding partner can be used in liquid phase and/or bound to a
solid phase carrier. Carriers include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, natural and modified
cellulose, polyacrylamide, agarose, magnetite, and the like. The
nature of the carrier can be either soluble or insoluble. As is
typical of immunoassays, the binding partner or the invention
protein may be detectably labeled in any of various ways well known
in the art. Binding partners also may be used to detect the
invention proteins in electrophoretically dispersed gels (e.g., a
2-D gel) or attached to a solid phase membrane such as a Western
blot.
[0064] An immunogen comprising the invention proteins or fragments
thereof may also be administered with an adjuvant either by mixing
with the protein or fragment or by conjugating or otherwise linking
to the adjuvant. A variety of adjuvants are known including
Freund's (complete and incomplete), alum, muramyl dipeptide, BCG,
LPS, Ribi Adjuvant System.RTM., TiterMax.RTM., and the like. One
skilled in the art would know which type of adjuvant is appropriate
to use in a given circumstance.
[0065] The present invention also provides a method of diagnosing a
proliferative disorder of the colon in a patient, the method
comprising: analyzing tissue, stool or body fluid from the patient
for the presence of at least one protein that is present in
cancerous colonic cells but absent or reduced in amount in normal
colonic epithelial cells, or present in normal colonic epithelial
cells but absent or reduced in amount in cancerous colonic cells,
wherein the protein is selected from the group consisting of:
[0066] a) CC2 having a molecular weight of about 56 kD and a pI of
about 6.22;
[0067] b) CC3 having a molecular weight of about 43 kD and a pI of
about 6.3;
[0068] c) CC4 having a molecular weight of about 43 kD and a pI of
about 6.2;
[0069] d) CC5 having a molecular weight of about 42 kD and a pI of
about 6.2;
[0070] e) CC6a having a molecular weight of about 20 kD and a pI of
about 6.9;
[0071] f) CC6b having a molecular weight of about 20 kD and a pI of
about 6.8.
[0072] g) L1 having a molecular weight of about 50 kD and a pI of
about 6.01;
[0073] h) L2 having a molecular weight of about 20 kD and a pI of
about 5.73;
[0074] i) L5 having a molecular weight of about 19 kD and a pI of
about 5.88;
[0075] j) N1 having a molecular weight of about 40 kD and a pI of
about 5.5;
[0076] k) N2 having a molecular weight of about 30 kD and a pI of
about 5.94;
[0077] l) N3 having a molecular weight of about 30 kD and a pI of
about 5.88;
[0078] m) N4 having a molecular weight of about 30 kD and a pI of
about 5.80;
[0079] n) N5 having a molecular weight of about 30 kD and a pI of
about 5.73; and
[0080] o) N6 having a molecular weight of about 18 kD and a pI of
about 6.6.
[0081] As already described, the protein may be detected by any of
a variety of means including biochemical means such as 2-D gel
electrophoresis, detection with a specific binding partner such as
a monoclonal or polyclonal antibody, and the like. The protein also
may be detected by determining the level of mRNA encoding the
protein through hybridization with an appropriate oligonucleotide
probe. A "patient" in this regard is a mammal, preferably a
human.
[0082] In this description, the phrase "proliferative disorder of
the colon" denotes malignant as well as non-malignant (or benign)
disorders of the colon, including but not limited to the colonic
epithelium. Such disorders include polyps such as with atypia or
dysplasia, sessile villous adenomas, pedunculated tubular adenomas,
and the like. The cells comprising these proliferative disorders
often appear morphologically and genotypically to differ from the
surrounding normal tissue. The proliferative disorder may be
associated, for example, with expression of the CC or L proteins of
the invention, in the latter case, particularly L1, L2 and L5.
Expression of a protein identified herein at an inappropriate time
during the cell cycle or in an incorrect cell type may result in a
cell-proliferative disorder. The protein-encoding polynucleotide in
the form of an antisense polynucleotide (or ribozyme) is useful in
treating hyperplasia and malignancies of the colon. In a preferred
embodiment, the proliferative disorder is colon cancer. Table 2
(Example 2) shows the percentage of 2-D gels in which L1, L2 and L5
were detected in nuclear matrix preparations of normal adjacent
colonic tissue and normal donor colonic tissue.
[0083] Also provided herein is a method of evaluating colonic
adenomas for potential to become malignant, the method comprising:
analyzing the adenoma for the presence of at least one protein that
is present in cancerous colonic cells but absent or reduced in
amount in normal colonic epithelial cells, or present in normal
colonic epithelial cells but absent or reduced in amount in
cancerous colonic cells, wherein the protein is selected from the
group consisting of:
[0084] a) CC2 having a molecular weight of about 56 kD and a pI of
about 6.22;
[0085] b) CC3 having a molecular weight of about 43 kD and a pI of
about 6.3;
[0086] c) CC4 having a molecular weight of about 43 kD and a pI of
about 6.2;
[0087] d) CC5 having a molecular weight of about 42 kD and a pI of
about 6.2;
[0088] e) CC6a having a molecular weight of about 20 kD and a pI of
about 6.9;
[0089] f) CC6b having a molecular weight of about 20 kD and a pI of
about 6.8.
[0090] g) L1 having a molecular weight of about 50 kD and a pI of
about 6.01;
[0091] h) L2 having a molecular weight of about 20 kD and a pI of
about 5.73;
[0092] i) L5 having a molecular weight of about 19 kD and a pI of
about 5.88;
[0093] j) N1 having a molecular weight of about 40 kD and a pI of
about 5.5;
[0094] k) N2 having a molecular weight of about 30 kD and a pI of
about 5.94;
[0095] l) N3 having a molecular weight of about 30 kD and a pI of
about 5.88;
[0096] m) N4 having a molecular weight of about 30 kD and a pI of
about 5.80;
[0097] n) N5 having a molecular weight of about 30 kD and a pI of
about 5.73; and
[0098] o) N6 having a molecular weight of about 18 kD and a pI of
about 6.6; wherein detection of said proteins indicates an
increased potential to become malignant. In a preferred embodiment,
the proteins are CC3, CC4 and CC5.
[0099] The invention also contemplates the diagnosing of colon
cancer to liver metastasis in a patient, by analyzing tissue or
body fluid from the patient for the presence of at least one
protein selected from the group consisting of:
[0100] a) L1 having a molecular weight of about 50 kD and a pI of
about 6.01;
[0101] b) L2 having a molecular weight of about 20 kD and a pI of
about 5.73;
[0102] c) L3 having a molecular weight of about 17 kD and a pI of
about 6.09;
[0103] d) L4 having a molecular weight of about 17 kD and a pI of
about 6.00; and
[0104] e) L5 having a molecular weight of about 19 kD and a pI of
about 5.88.
[0105] Detection of proteins according to the present invention can
allow differentiation among colonic cell proliferative disorders
that have a potential to become malignant or to metastasize,
respectively. The protein compositions described herein also are
useful as markers for early diagnosis of a colonic cell
proliferative disorder such as colorectal cancer and the early
detection of recurrence, and its metastastic diseases derived
therefrom, knowledge that is central to the effective treatment of
this disease. In addition to their use individually, detection of
any two or more invention proteins can be combined to diagnose
proliferative disorders of the colon including colon cancer and its
liver metastases. Detection of the invention proteins also may be
combined with detecting other colon cancer markers, such as CEA or
previously reported colon cancer NMPs (32), to diagnose the colon
cancer and its liver metastases.
[0106] The particular proteins or fragments described herein may be
detected indirectly, for example, by detecting the level of
encoding mRNA. Thus, the present invention provides a purified
polynucleotide sequence encoding the above-identified protein or
fragments of the preceding embodiments. Also provided is a nucleic
acid probe that hybridizes to the polynucleotide sequence encoding
the above-mentioned protein or fragments thereof (or by
hybridization to the complementary sequence). The term "nucleic
acid" as used herein refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and also encompasses known analogs of natural nucleotides that can
function in a similar manner as naturally occurring nucleotides.
The probe is preferably single stranded and preferably comprises at
least about 14 nucleotides in length, more preferably at least
about 18 nucleotides in length, and most preferably about 25
nucleotides in length. The nucleic acid probe can be detectably
labeled such as with a radioisotope, a bioluminescent compound, a
chemiluminescent compound, a fluorescent compound, a metal chelate,
an enzyme, and the like.
[0107] Also provided is a kit for detecting a colonic cell
proliferative disorder. The kit may include a binding partner or
nucleic acid probe that is specific for any one or more of the
invention proteins. The binding partner or probe can be labeled for
ease of detection. The kit may have reagents in different vials and
may include positive and negative controls, buffers such as for
conducting the reactions, and a directional insert. The kit also
may have an oligonucleotide primer that permits amplification of a
target polynucleotide sequence encoding one of the invention
proteins, for example, by polymerase chain reaction (PCR)
amplification.
[0108] The present invention also provides methods to treat colon
cancer and its liver metastases, by reducing the level of
expression or activity of a protein which is present primary colon
cancer cells and liver metastases derived therefrom. Such proteins
include any of the CC or L proteins disclosed herein. The level of
expression of the protein can be reduced, for example, by treating
the individual with an antisense nucleic acid or a ribozyme.
[0109] An antisense nucleic acid is a short DNA or RNA
oligonucleotide designed to be complementary to a specific gene
sequence. The purpose in using antisense to alter specific gene
expression resulting from the binding of the antisense
oligonucleotide to the gene sequence. Antisense molecules that bind
to a specific region of the DNA helix may result in triplex
formation since a third strand is formed at the site of binding to
the two stands comprising the helix. Antisense oligonucleotides
that target RNA may be either non-catalytic or catalytic (i.e.,
ribozymes). Binding of non-catalytic antisense molecules to the
target RNA can block further RNA processing or transport through
any of several possible mechanisms, including, for example: 1)
transient inhibition by the prevention of ribosome binding to the
RNA by masking its binding site on the mRNA; 2) permanent
inhibition of the process by which RNase H degrades RNA in RNA/DNAs
hybrids, an enzyme present in most cells; and 3) permanent
inhibition by cross-linking the oligonucleotide to the target
RNA.
[0110] The antisense compounds preferably comprise between about 8
to about 30 nucleobases (i.e., from about 8 to about 30 linked
nucleosides), and more preferably from about 12 to about 25
nucleobases. The antisense oligonucleotide can be linear or
circular in configuration. Antisense compounds useful herein may
include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. Preferred modified
oligonucleotide backbones include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Extensive citation to methods of preparing antisense
oligonucleotides can be found, for example, in U.S. Pat. No.
6,210,892 to Bennett.
[0111] Where the cell proliferative disorder is characterized as
having under-expression of an invention protein (e.g., N1 or N6) a
polynucleotide sequence encoding the missing under-expressed
protein (such as for a growth or tumor suppressor protein) can be
administered to increase the level of expression. This can be
achieved by introducing into the cells of a host subject an
expression vector comprising a polynucleotide sequence encoding any
one or more of the invention proteins. Preferably, the expression
vector is introduced into the cells of the host subject ex vivo,
yielding transformed cells, and the transformed cells are then
reintroduced into the subject. This can be achieved, for example,
with an RNA virus vector such as a retroviral vector. Cells
transformed to express the proteins are also useful, for example,
as a tumor vaccine. Methods of preparing vectors for delivering
sense or antisense nucleic acid into cells (in vivo or in vitro)
and methods of formulating these delivery vehicles for
administration are well known in the art. See, e.g., U.S. Pat. No.
5,824,490 to Coffey et al. The level of expression of the invention
protein also can be reduced by treating the individual with a
compound that effects a regulatory region controlling expression
such a promoter or an enhancer or that modulates the activity of
the proteins. Such compounds may be identified by a) incubating the
proliferative cells (e.g., colorectal cancer cells) expressing the
invention protein with a test composition under conditions that
allow the cells and test composition to interact, and (b) measuring
whether the test composition blocks or enhances the function of the
protein. Caco-2 cells are an example of a colorectal cancer cell
line that is useful in this regard.
[0112] The invention will be described in greater detail by
reference to the following non-limiting examples.
EXAMPLES
Example 1
Methods for Isolation and Purification of NMPs
[0113] A. Tissue Processing:
[0114] Colon adenocarcinoma liver metastases samples (N=12) and
matched adjacent normal liver tissues (N=12) were collected through
the Early Detection Research Network (EDRN) of the University of
Pittsburgh Medical Center under institutional IRB approval. The
patients ranged in age from 44-75 with a mean age of 62.4 years.
Sixty percent of the sample population was female. The carcinomas,
staged according the standard TNM system, were categorized as shown
in Table 1.
[0115] Colon adenocarcinoma samples (N=10) and matched adjacent
normal liver tissues (N=10) also were collected through the Early
Detection Research Network (EDRN) of the University of Pittsburgh
Medical Center under institutional IRB approval. The patients
ranged in age from 36-82 with a mean age of 71 years. Sixty percent
of the sample population was female. Staging for these carcinomas
categorized as shown in Table 1. TABLE-US-00001 TABLE 1 Stage and
UI Grade of Colon Carcinoma Tumors Used for NMP Preparation
Clinical Parameter Result (number of samples) Tumor location Right
hemicolon (n = 5) Left heimcolon (n = 5) Tumor Stage (UICC) I:
T.sub.1-2N.sub.0M.sub.0 (n = 0) II: T.sub.3-4N.sub.0M.sub.0 (n = 5)
III: T.sub.1-4N.sub.1-2M.sub.0 (n = 4) IV:
T.sub.1-4N.sub.1-2M.sub.1 (n = 1) Tumor Grade G1 (n = 0) G2 (n = 9)
G3 (n = 1)
[0116] Normal liver tissue was obtained from trauma victims
including one sample from a gunshot victim and two from an
automobile accident victim (N=3). The patients ranged in age from
36-48 with a mean age of 40.3 years. Two of these normal
individuals were male and one female. None of the patients had
liver cirrhosis. Thirty percent of the patients had mild steatosis.
Diagnosis was obtained from pathology reports, which accompanied
each specimen and was confirmed histologically. Tissues were stored
at -80.degree. C. prior to processing. Sporadic colon
adenocarcinoma samples and matched adjacent normal tissues and
normal donor colon tissues were collected as previously described
(30).
[0117] Normal colon tissue was obtained from trauma victims; Two of
these individuals had gunshot wounds, one suffered from automobile
trauma and one was an organ donor (N=4). The patients ranged in age
from 20-59 with a mean age of 47.2 years. The normal colon donors
were all male. Diagnosis was obtained from pathology reports, which
accompanied each specimen and was confirmed histologically.
[0118] The colon cancer cell lines, SW480 and Caco-2, were obtained
from the American Type Culture Collection (Manassas, Va.). Both
cell lines were established from primary human colon cancer cells.
The SW480 cell line was grown in Leibovitz medium with 10% fetal
bovine serum at 37.degree. C. without CO.sub.2. The Caco-2 cell
line was grown in Dulbecco's minimal essential medium (DMEM)
supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mM),
1% penicillin/streptomycin, 1% sodium pyruvate (100 mM), 1% MEN
non-essential amino acids, 1.5% HEPES Buffer (1M) at 37.degree. C.
in a 5% CO.sub.2 atmosphere.
[0119] The colon cancer cell line CX-1 was a kind gift from Lee YJ
Ph.D. University of Pittsburgh. The cell line has been established
from primary human colon cancer cells. The cell line was grown in
RPMI-1640 media with 10% fetal bovine serum and 1%
penicillin/streptomycin at 37.degree. C. in a 5% CO.sub.2
atmosphere.
[0120] Normal human hepatocytes (50.times.10.sup.6), obtained from
a 63 year-old female organ donor, were a gift from Dr. Stephen
Strom (University of Pittsburgh, Pa.). Human primary liver cancer
cell lines, huh 7, HepG 2, were a kind of gift of Dr. George
Michalopoulos, University of Pittsburgh, Pa. Both cell lines were
grown in DMEM with 10% fetal bovine serum and 1%
penicillin/streptomycin at 37.degree. C. in a 5% CO.sub.2
atmosphere.
[0121] B. Nuclear Matrix Preparation:
[0122] Nuclear matrix proteins were extracted from various cells
and tissues according to the method of Getzenberg et al (29). In
brief, tissues were finely minced into small pieces and homogenized
with a Teflon.RTM. pestle on ice with 0.5% Triton X-100 in a
solution containing 2 mM vanadyl ribonucleoside (Rnase inhibitor)
to release the lipids and soluble proteins. The homogenate was
filtered through a 350 .mu.m nylon mesh. Treatment with DNAse and
RNAse was performed to remove the soluble chromatin, resulting in a
remaining fraction comprising intermediate filaments and NMPs. This
fraction then was disassembled with 8 M urea, and the insoluble
components consisting of carbohydrates and extracellular matrix
were removed by centrifugation. After dialysis to remove the urea,
intermediate filaments were allowed to reassemble and were
subsequently removed by centrifugation.
[0123] The NMPs then were precipitated with ethanol and resuspended
in 2D sample buffer consisting 9 M urea, 65 nM
3-((3-cholamidopropyl)-dimethyl-ammonio)-1-propane-sulfonate, 2.2%
ampholytes and 140 mM DTT, and quantitated by Coomassie Plus
protein assay (Pierce Chemical Co., Rockford, Ill.) with bovine
serum albumin as a standard. The pellet following ethanol
precipitation containing NMPs represented <1% of the total
starting cellular protein.
[0124] C. High Resolution, Two-Dimensional Electrophoresis:
[0125] This procedure was performed using the Investigator 2-D gel
system (Genomic Solution, Ann Arbor, Mich.) as described previously
(29, 33-34). One hundred .mu.g of protein were loaded per gel onto
a capillary size IEF column. One dimensional isoelectric focusing
was carried out for 18,000 volt-hours using 1 mm.times.18 inch tube
gels after 1.5 hours of prefocusing. The tube gels were extruded
and placed on top of 1 mm SDS Duracryl (Genomic Solution, Ann
Arbor, Mich.) high tensile strength PAGE slab gels. The gels were
electrophoresed at 12.degree. C. constant temperature for 4.5 to 5
hours. Gels were fixed with 50% methanol and 10% acetic acid. After
thorough rinsing and rehydration, gels were treated with 5%
glutaraldehyde and 5 mM DTT after buffering with 50 mM Phosphate
(pH 7.2). The gels were stained with silver stain using the method
of Wray et al (Accurate Chemical Co., Westbury, N.Y.) (21).
Molecular weights of colon NMPs were identified using standards
provided by Genomic Solutions. Isoelectric points (PI's) were
determined using carbamylated standards; BDH-distributed by
Gallard-Schlessinger (Carle Place, N.Y.) and Sigma Chemical Co.
(St. Louis, Mo.).
[0126] Multiple gels were run for each sample and multiple samples
were run at different times. The gels were analyzed using the
BioImage 2D Electrophoresis Analysis System (BioImage, Ann Arbor,
Mich.), that matches protein spots between gels and sorts the gels
and protein spots into a database. Only protein spots clearly and
reproducibly identical in all gels of a sample type were taken into
account as those representing the described NMP's.
Example 2
Identification of NMPs Characteristic of Colon Carcinoma or Normal
Colonic Epithelium
[0127] A. Analysis of NMPs from Colorectal Cancer and Normal
Colonic Epithelium
[0128] Evaluation of NMPs separated by high-resolution
two-dimensional gel electrophoresis identified seven NMPs of
clinical value that were expressed primarily in colorectal
adenocarcinomas were identified and designated as shown in Table 2.
CC2 to CC6a,b were strongly expressed in all colon tumors, but were
undetectable in adjacent normal tissue. In contrast, CC1 was
expressed in carcinoma extracts and adjacent normal or donor tissue
but the signal was stronger for colon cancers than either source of
normal tissue.
[0129] Six NMPs of clinical value that were expressed primarily in
normal colonic epithelium were designated as shown in Table 2.
N2-N6 were expressed in a minority of the colon cancers (about
20%), but were expressed in all adjacent normal and donor extracts.
In contrast, N1 was expressed in most cancer extracts (about 70%)
and in all normal extracts, but was more strongly but the signal
was stronger for the normal extracts. The characteristics of the
identified colon carcinoma NMPs are summarized in Table 2.
TABLE-US-00002 TABLE 2 Characteristics of Colorectal Adenocarcinoma
and Normal Colonic Epithelial NMPs Molecular Marker Weight No. (kD)
PI Tissue Expression CC1 59 4.40 Cancer and normal epithelium but
stronger for cancer (10/10 cancer; 10/10 normal adjacent; and 4/4
normal donor) CC2 56 6.22 Cancer only (8/10 cancer; 0/10 adjacent
normal; and 0/4 donor normal) CC3 43 6.27 Cancer only (10/10
cancer; 0/10 adjacent normal; and 0/4 donor normal) CC4 43 6.22
Cancer only (10/10 cancer; 0/10 adjacent normal; and 0/4 donor
normal) CC5 42 6.25 Cancer only (10/10 cancer; 0/10 adjacent
normal; and 0/4 donor normal) CC6a 20 6.86 Cancer and donor
epithelium but not adjacent normal epithelium (8/10 cancer; 0/10
adjacent normal; and 4/4 donor normal) CC6b 20 6.81 Cancer and
donor epithelium but not adjacent normal epithelium. (8/10 cancer;
0/10 adjacent normal; and 4/4 donor normal) N1 40 5.50 Normal
epithelium (adjacent and donor) and cancer (about 70% of samples)
but stronger for normal (7/10 cancer; 10/10 adjacent normal; and
4/4 donor normal) N2 30 5.94 Normal epithelium mainly** (2/10
cancer; 10/10 adjacent normal; and 4/4 donor normal) N3 30 5.88
Normal epithelium mainly (2/10 cancer; 10/10 adjacent normal; and
4/4 donor normal) N4 30 5.80 Normal epithelium mainly (2/10 cancer;
10/10 adjacent normal; and 4/4 donor normal) N5 30 5.73 Normal
epithelium mainly (2/10 cancer; 10/10 adjacent normal; and 4/4
donor normal) N6 18 6.58 Normal epithelium mainly (1/10 cancer;
10/10 adjacent normal; and 4/4 donor normal) *NC means that the
reference describes nothing close in size or pI. **Refers to an
expression frequency of about 10-20% for cancer samples.
[0130] B. Analysis of CC NMPs from Colorectal Tumor Cell Lines
[0131] NMP preparations were isolated from two human colon cancer
cell lines, Caco-2 and SW480, essentially as described for NMP
preparation from human tissue. These NMPs were analyzed by 2
dimensional gel electrophoresis as described above for human tissue
NMPs. Both cell lines expressed none of the proteins, which are
expressed in the adjacent or donor gels (N1-N6). The cell line
SW480 expressed the colon cancer associated proteins, CC1 and
CC6a/b; the Caco-2 cell line expressed CC1, CC3, CC4, CC6a,b. CC2
was not detectable in NMP preparations of Caco2, for SW480 but was
detectable in the cancer cell line CX-1. CC3, CC4 but not CC5 was
detected in CX-1 NMP.
[0132] These studies demonstrate that nuclear matrix proteins
isolated from human colon cancer are distinct from normal adjacent
and donor tissue, indicating both loss and gain from specific
proteins. The presence or absence of unique NMPs in cancer cells is
useful for diagnosing disease and provide novel information about
the function of NMPs in carcinogenesis.
[0133] C. Analysis of CC NMPs in Adenomatous Polyps of the
Colon
[0134] Colon polyps (n=20) were collected through the Early
Detection Research Network (EDRN) of the University of Pittsburgh
Medical Center under institutional IRB approval. One juvenile
polyp, six tubular adenoma (TA), seven tubulovillous adenoma (TVA)
and six tubulovillous adenoma with high grade dysplasia (TVA with
HGD) were examined. The patients ranged in age from 18 to 77 with a
mean age of 58 years. 55 percent of the sample population was
female. Diagnosis was obtained from pathology reports, which
accompanied each specimen and was confirmed histologically. The
histological partition of the colon polyps is shown in Table 3. The
tissues were stored at -80.degree. C. prior to processing.
TABLE-US-00003 TABLE 3 Characteristics of patients with colon
polyps juvenile polyp N = 1 Age = 18 Male 1 Tubular adenoma N = 6
Average age = 58 male 4; female 2 Tubulovillous N = 7 Average age =
56 male 1; female 7 adenoma Tubulovillous N = 6 Average age = 62
male 3; female 3 adenoma with high grade dysplasia
[0135] The results for detecting the presence of CC2, CC3, CC4 and
CC5 NMPs in various adenomas of the colon is shown in Table 4.
TABLE-US-00004 TABLE 4 Presence of CC NMPs in adenomatous colonic
polyps Colonic abnormality CC2 CC3 CC4 CC5 Tubulovillus adenoma 0%
83% 100% 33% with focal high grade dysplasia (n = 6) Tubulovillus
adenoma 0% 86% 86% 0% (n = 7) Tubular adenoma 0% 83% 100% 17% (n =
6) Juvenile polyps 0% 0% 0% 0% (n = 1)
[0136] CC2 was not seen in any of the pre-malignant polyps. CC5 was
present in only two (33%) pre-malignant TVA with HGD and in one
(17%) TA. CC3 and CC4 were present in 83%-100% in TA, in 86% in TVA
and in 83%-100% in TVA with HGD. None of the nuclear matrix
proteins were seen in the juvenile polyp, which is not a precursor
of colon cancer.
[0137] CC5, which was present in only 2 pre-malignant TVA with HGD
and in one TA, but was present in all colon cancer tissues, is
expressed at the junction of an advanced adenoma and invasive colon
cancer. CC5 is a promising marker for malignant potential of colon
polyps because it is expressed in the advanced polyps and in the
colon cancer epithelia. CC3 and CC4 were present in most adenomas,
regardless of advancement. Both proteins are expressed earlier in
the development of adenomatous polyps and are also expressed in all
colon cancer (Table: 2). The combination of all four CC markers
tested also provides diagnostic value for early detection of
malignant progression in colon polyps.
Example 3
Partial Amino Acid Sequence of Colon Carcinoma NMPs
[0138] A. Method for Polypeptide Isolation and Sequencing
[0139] A partial amino acid sequence was determined for particular
CC markers isolated two-dimensional gels using an adaptation of a
technique developed by Gevaert et al. (35). Briefly,
two-dimensional gels run with NMP preparations were negatively
stained by incubating in 0.2M Imidazole for 15 minutes, washing
several times with deionized water, staining with a warmed solution
of 0.3M zinc chloride and terminating the staining reaction by
washing in deionized water. Protein spots were excised from the gel
and frozen at -80.degree. C. The gel spots were thawed, pooled and
stained for 20 min with 0.25% Coomassie blue in 45% methanol/9%
acetic acid. The isolated gel spots were destained by agitation in
destaining solution (5% methanol/7.5% acetic acid) for 1 hour,
washed with deionized water for 1 hour, and equilibrated in SDS
polyacrylamide sample buffer (1% SDS/10% glycerol/50 mM DTT/12 mM
Tris-HCl pH 7.1) for 1 hour before loading into an SDS
acrylamide/agarose gel.
[0140] Following destaining, the protein from each gel spots was
concentrated on a mini-agarose/acrylamide gel. The gel was formed
between two pre-warmed (60.degree. C.) glass plates (10 cm.times.9
cm), separated by spacers 1 cm wide and 1.5 mm thick. A strip of
Whatman 3 MM paper was applied to the bottom to serve as a support
for the lower agarose gel, preventing the gel from slippage during
electrophoresis. A 2 cm wide.times.1.5 cm thick spacer was inserted
between the two parallel spacers to form a sample well for
receiving the gel spots. The sample well was formed by a 2 cm
wide.times.1.5 cm thick spacer set between two parallel spacers
each 1 cm wide.times.1.5 cm thick inserted at the center of the
glass plates and attached with adhesive tape at the top edge of the
back plate.
[0141] The running gel portion of the mini-gel consisted of a 2 cm
deep agarose (1.45% agarose in 0.36 M Tris-HCl pH 8.7/0.1% SDS)
which was poured and allowed to set. A polyacrylamide stacking gel
(5.45% acrylamide/0.13% bisacrylamide/0.12 M Tris-HCl pH 6.8/0.1%
SDS) was applied over the agarose. After the stacking gel
polymerized, the central well-forming spacer was removed, leaving a
loading well with dimensions 2 cm high, 2 cm wide and 1.5 mm thick.
The mini concentration gel was then mounted on a small
electrophoresis tank (BioRad, Hercules, Calif.), and the loading
slot filled with the SDS sample buffer-equilibrated gel spots. The
remaining volume was filled with blank gel pieces.
[0142] Mini-gels were run at 100 V, allowing the proteins to elute
out of the combined gel pieces and into the acrylamide. At this
time, the central spacer was re-inserted into the sample well until
the dye front passed the two parallel 1 cm wide spacers. At that
point, the central spacer was removed and electrophoresis continued
until the dye front entered the agarose and reached the filter
paper.
[0143] The agarose running gel was removed and fixed in fresh 50%
methanol/10% acetic acid shaking, at room temperature for 30
minutes. The gel was stained with 0.05% Coomassie blue stain (50%
methanol/10% acetic acid) for 5 minutes and then destained in 5%
methanol/7% acetic acid for 2 hours with constant agitation. The
protein band was then excised in a minimal volume of agarose gel,
transferred to a sterile tube, and digested thoroughly with
trypsin. Trypsin fragments were subjected to preparative reverse
phase C18 HPLC and fractions containing different peptides were
isolated. An automated sequencer was used to determine a sequence
of fragments in different fractions.
[0144] B. Peptide Sequencing Results
[0145] N-terminal amino acid sequences for trypsin fragments of
particular isolated CC polypeptides isolated from NMP preparations
of Caco-2 cells is shown is Table 5 while the sequences for
particular CC polypeptides isolated from NMP preparations of human
colorectal cancer specimens are shown in Table 6. TABLE-US-00005
TABLE 5 Partial Amino Acid Sequence of CC3 and CC6 Proteins
Isolated from Caco-2 NMP Preparation Marker No. Amino Acid Sequence
(5'-3') CC6a PXVKPNSYVDGVEV (peak 31) (SEQ ID NO:1) EGDLIEDY (peak
17) (SEQ ID NO:2) CC3 YPVEAFN (Peak 9) (SEQ ID NO:3) TVAPLFIVIPN
(Peak 35) (SEQ ID NO:4) XVTGLTQIETLFAAPGVD (Peak 47) (SEQ ID NO:5)
SMTEAEQQQLIDDHIFLFDKPVSP (Peak 52) (SEQ ID NO:6) SLPQNIPPLTQTPV
(Peak No:31) (SEQ ID NO:7) VLPGEIVEYSR (Peak No:15) (SEQ ID
NO:8)
[0146] TABLE-US-00006 TABLE 6 Partial Amino Acid Sequence of CC
Proteins Isolated from Human Colorectal Tissue NMP Preparations
Marker No. Amino Acid Sequence (5'-3') CC1 YTIFDNFLITNDEAYAEEFG
(Peak 30) (SEQ ID NO:9) QIDNPDYKGTXIHPE (Peak 34) (SEQ ID NO:10)
PAVYFKEQFLDGDGW (Peak 24) (SEQ ID NO:11) TLIVRPDNTYEVK (Peak 18)
(SEQ ID NO:12) YAVLITVLQDS (Peak 13) (SEQ ID NO:13) AKTDFATFLYT
(Peak 28) (SEQ ID NO:22) CC2 NLPQE (Peak 07) (SEQ ID NO:14)
TEPELQDKIHQ (Peak 18) (SEQ ID NO:15) TDAPSFSDIPNL (Peak No:20) (SEQ
ID NO:16) LKYENEVALR (Peak No:20) (SEQ ID NO:23) XQKEDVPSE (Peak
No:02) (SEQ ID NO:17) CC3 VYOEPLVFR (Peak 31) (SEQ ID NO:24)
RAPFQELYND (Peak 31) (SEQ ID NO:25) XFYQLDAYPSGAXY (Peak 35) (SEQ
ID NO:26) CC4 VIEAFNR (Peak 17) (SEQ ID NO:27) ILLFDYFNR (Peak 28)
(SEQ ID NO:28) VLVALEPLS (Peak 29) (SEQ ID NO:29) CC6a NAFNDGLK
(Peak 12) (SEQ ID NO:18) YFDSFGDLSSASAIMGN (Peak 19) (SEQ ID NO:19)
TYFSHIDVSPGSAQVK (Peak 18) (SEQ ID NO:20) CC6b SLDEQEQTK (Peak 06)
(SEQ ID NO:21)
[0147] The amino acid sequences were run against public amino acid
sequence databases to determine if they represent previously known
proteins. The sequence of amino acids fragments generated from CC1
(SEQ ID NOS: 9, 10 and 11) were consistent with the protein
calreticulin, while CC1 sequences with SEQ ID NO: 12 and 13 were
consistent with a precursor of calreticulin. Calreticulin is an
important multifunctional calcium-binding protein and is the major
calcium binding protein found in the membranes of smooth muscle
sarcoplasmic reticulum and non-muscle endoplasmic reticulum (ER).
Calreticulin has an immunological function in the folding and
peptide-loading of newly synthesized molecules of the major
histocompatability complex (MHC) class I protein. A complete MHC
class I molecule consists of a 3-domain alpha-chain and a smaller
.beta.2-microglobulin bound to a short peptide fragment. When the
.beta.2-microglobulin first binds to the alpha-chain, this
partially folded heterodimer binds to a complex of proteins
including calreticulin. Calreticulin's binding of the MHC molecule
is regulated by glucose trimming of nascent N-linked
oligosaccharides--the oligosaccharide moiety in the alpha1 domain
and a residue within the alpha3 domain of the MHC class I molecule
are critical for intersection with calreticulin. After peptide
loading and deglucosylation of N-linked glycans, calreticulin
dissociates from the heterodimer.
Example 4
Identification of Calreticulin in Colon Cancer NMP Preparations
[0148] The presence of calreticulin in colon cancer was evaluated
using NMP preparations as well as nuclear and cytoplasmic
extracts.
[0149] A. Methods
[0150] Nuclear and Cytoplasmic Extraction: NE-PER nuclear and
cytoplasmic extraction reagents (Pierce Chemical Co., Rockford,
Ill.) were used for the preparation of nuclear and cytoplasmic
extracts. The protein concentration was quantitated by Coomassie
Plus protein assay (Pierce Chemical Co., Rockford, Ill.) with
bovine serum albumin as a standard.
[0151] One-Dimensional Immunoblot: One-dimensional immunoblot
analysis was performed according to standard established protocols.
Ten .mu.g of each sample of extracted NMPs was suspend in PBS
(phosphatase buffered salt solution) and nuclear and cytoplasmic
extracts suspend in nuclear extraction reagent (NER) or in
cytoplasmic extraction reagent (CERII) were separated by 12%
SDS-PAGE. Ten .mu.l of Rainbow markers (Amersham Life Sciences,
Arlington Heights, Ill.) were also loaded. Proteins were then
transferred to a polyvinylidene difluoride membrane (Millipore,
Bedford, Mass.), and the membrane was blocked overnight in 4%
nonfat dry milk in PBS with 0.2% Tween.RTM. 20 at 4.degree. C. The
membrane was then washed with PBS and 0.2% Tween.RTM. 20, followed
by a 1-h incubation with a 1:2000 dilution of anti-calreticulin IgG
(Research Diagnostics, NC, Flanders, N.J.) and 2% nonfat dry milk
with 0.2% Tween.RTM. 20 in PBS. The membrane was further washed
with PBS and 0.2% Tween.RTM. 20 and incubated for 1 h in a 1:5000
dilution of goat anti-rabbit IgG (Amersham Life Sciences, Arlington
Heights, Ill.) secondary antibody conjugated with horseradish
peroxidase (Amersham Life Sciences, Arlington Heights, Ill.). For
determination of the relative purity of each protein extraction,
the membranes were probed with a monoclonal .alpha.-tubulin mouse
antibody (1:500) (specific for cytoplasmic tubulin) and 2% nonfat
dry milk with 0.2% Tween.RTM. 20 in PBS. These membranes were
further washed with PBS and 0.2% Tween.RTM. 20 and incubated for 1
hour in a 1:5000 dilution of goat anti-mouse IgG (Amersham Life
Sciences, Arlington Heights, Ill.) conjugated with horseradish
peroxidase. The membranes were washed again with PBS and 0.2%
Tween.RTM. 20 and the proteins detected by chemiluminescence
reaction using the ECL immunoblot kit (Amersham Life Sciences,
Arlington Heights, Ill.).
[0152] Two-dimensional immunoblot: After performing 2-D
electrophoresis, the area of the gel where the spot was located and
which the peptide sequence resulted in identification as
calreticulin was removed and was transferred to a polyvinylidene
difluoride membrane (Millipore, Bedford, Mass.). This area of the
gel was utilized instead of the entire gel, because the large gel
format makes blotting and processing difficult. Thereafter, the
same procedure described above for the one-dimensional immunoblot
was followed by a 1-h incubation with a 1:2000 dilution of
anti-calreticulin IgG (Research Diagnostics, Inc., Flanders, N.J.)
with 2% nonfat dry milk with 0.2% Tween.RTM. 20 in PBS. The
membrane was further washed with PBS and 0.2% Tween.RTM. 20 and
incubated for 1 h in a 1:5000 dilution of goat anti-rabbit IgG
(Amersham Life Sciences, Arlington Heights, Ill.) secondary
antibody conjugated with horseradish peroxidase (Amersham Life
Sciences, Arlington Heights, Ill.). The membrane washed again with
PBS and 0.2% Tween.RTM. 20 and proteins were detected by a
chemiluminescence reaction using the ECL immunoblot kit (Amersham
Life Sciences, Arlington Heights, Ill.).
[0153] B. Results
[0154] The two-dimensional immunoblot of CC1 probed positive with
the anti-calreticulin antibody. A one-dimensional immunoblot of
colon cancer, normal adjacent and normal donor colon tissue
cytoplasmic, nuclear and nuclear matrix protein fractions was
probed with antibodies specific for calreticulin and
.alpha.-tubulin (cytoplasmic 51 kD protein). Tubulin was not
detectable in nuclear and the nuclear matrix protein fractions
indicating the that lack of contamination of cytoplasmic material
in these fractions. Calreticulin was detected (about a 60 kD band)
in human colon cancer tissue, adjacent normal tissue and normal
donor tissue in all three different protein fractions. These
results show that calreticulin is present in the nuclear protein
fraction and more specifically in the nuclear matrix protein
fraction of colon cancer cells, and that such presence is not due
to contamination by cytoplasmic calreticulin. Calreticulin was less
detectable in normal adjacent and normal donor colon tissue NMP
preparations by one dimensional immunoblotting.
Example 5
Identification of NMPs Characteristic of Liver Metastases and
Primary Colon Cancer Cells
[0155] A. Expression of L1-L5 Proteins in NMPs from Various
Sources
[0156] High-resolution two-dimensional gel electrophoresis of
various NMP preparations identified five proteins (L1-L5) of
clinical value (Table 7). NMPs L1-L5 were detectable in about 83%
to 100% of 2D gels for NMP preparations of the various colon cancer
to liver metastases. L1-L5 were undetectable in NMP preparations of
normal hepatocytes. Three proteins, L1, L2 and L5, were
undetectable in NMP preparations of normal donor liver tissue, and
two proteins, L3 and L4, were detectable in a lower percentage of
gels prepared from NMP preparations of normal donor liver tissue as
compared to that of colon to liver metastasis. Four of the proteins
(L1, L2, L4 and L5) were detectable in less than 40% in the
adjacent normal liver tissue samples (Table 7), while protein L3
was detectable in 92% of the adjacent liver tissue samples (Table
7).
[0157] Proteins L1, L2, L3 and L4 also were detectable in 60% or
more of gels prepared using NMP preparations of primary colon
cancer cells (Table 7). Protein L5, on the other hand, was
detectable in only 20% of gels prepared using NMP preparations of
in primary colon cancer cells (Table 7). Proteins L3 and L4 were
expressed in 100% of gels prepared using NMP preparations of the
normal adjacent colon and 100% and 75% respectively for donor colon
tissue. Protein L2 was expressed in 10% of the adjacent normal
colon tissue, while L1 was not detectable in the adjacent normal
colon, but was detectable in one of the four donor colon samples
(25% in Table 7). Electrophoretic characteristics of the identified
proteins are shown in Table 7.
[0158] Thus, L1-L5 are differentially expressed in normal liver
metastases versus normal hepatocytes. L2 and L2 are the most
differential of all proteins, the difference being seen in both
colon liver metastases versus adjacent liver tissue and in primary
colon cancer versus adjacent colon tissue. L3 is differential for
liver metastases versus adjacent liver tissue or for primary colon
cancer versus normal adjacent colon. TABLE-US-00007 TABLE 7
Characteristics of NMPs in Liver Metastases and Other Liver and
Colon Tissues 2D Gel Tissue Expression Colon Ca. to Liver Normal
Normal Normal Normal Normal Markers/ Mr. mets. Adj. liver donor
liver Hepato- Colon Ca. Adj. Colon donor colon NMPs (kD) PI (n =
12) (n = 12) (n = 3) cytes (n = 10) (n = 10) (n = 4) L.sub.1 50
6.01 92% 42% 0% 0% 70% 0% 25 L.sub.2 20 5.73 100% 17% 0% 0% 100%
10% 0% L.sub.3 17 6.09 100% 92% 33% 0% 90% 100% 100 L.sub.4 17 6.00
100% 17% 33% 0% 60% 100% 75% L.sub.5 18 5.88 83% 8% 0% 0% 20% 0% 0%
Adj. = adjacent; Ca--cancer; mets = metastasis
[0159] B. Analysis of NMPs from Liver and Colon Cancer Tissue and
Cell Lines
[0160] Tissue samples are complex mixtures of epithelial, stromal,
immunological and other cell types. To determine whether the
nuclear matrix changes detected actually represented changes that
were occurring in the neoplastic cells, as well as to identify
potential models, the NMP composition of two human primary liver
cancer cell lines were examined. While the NMP fingerprints from
the pure cell lines would be expected to be distinct from the three
dimensional complex of liver metastasis and colon cancer specimens
they serve as tools for generating reagents as well as examining a
single cell type. The human liver cancer cell lines Huh 7 and HepG
2 and the human colon cancer cell lines Caco-2 and SW480 were grown
and their NMPs were isolated. These NMPs were then separated and
analyzed as described above. The cell line HepG 2 expressed none of
the liver metastasis associated proteins, and the Huh 7 cell line
expressed one of the liver metastasis associated proteins (L1). The
cell line Caco-2 expressed two of the proteins (L3, L2) and the
cell line SW480 expressed just one protein (L2). Electrophoretic
characteristics of the identified proteins in these cell lines are
shown in Table 8. TABLE-US-00008 TABLE 8 Characteristics of NMPs in
Colon Cancer Cell Lines Markers/ Mr. 2D-Gel Cell Expression NMPs
(kD) PI Hep G.sub.2 Huh 7 Caco-2 SW 480 L.sub.1 50 6.01 0% 0% 0% 0%
L.sub.2 20 5.73 0% 0% 100% 100% L.sub.3 17 6.09 0% 0% 100% 0%
L.sub.4 17 6.00 0% 0% 0% 0% L.sub.5 19 5.88 0% 0% 0% 0%
[0161] These studies demonstrate that nuclear matrix proteins
uniformly present in NMP preparations from human liver metastasis
are not readily present in NMP preparations of normal donor tissue
or isolated hepatocytes. In addition, in some cases, the normal
adjacent liver tissue contains alterations in the nuclear matrix
pattern similar to those found in liver metastasis. These five
proteins were also found to varying degrees in primary colon
cancer, but were not found in other cancer types.
[0162] The functional identification of these proteins and their
detection through the generation of NMP antibodies could be used to
develop tests for colon cancer prognosis and early detection of
metastases. Antibodies are generated to detect specific nuclear
matrix proteins in the blood or tissue samples. Assay is developed
that include the detection and combination of individuals proteins
identified herewith. Development of assays with these antibodies
potentially serve as tumor marker with high sensitivity and
specificity.
[0163] Additionally the presence of unique NMPs in liver metastasis
or the up regulation in cancer cells could provide novel
information about their function in development of metastasis and
provide us with additional targets for anticancer therapies.
Example 6
Isolation of "L" Proteins from 2-D Gels and Sequencing
[0164] Methods for isolating and sequencing L proteins were as
described for the CC proteins in Example 3. N-terminal amino acid
sequences for trypsin fragments of particular isolated L
polypeptides isolated from NMP preparations of colorectal
metastases to liver cells is shown is Table 9. TABLE-US-00009 TABLE
9 Partial Amino Acid Sequence of L2 and L5 Proteins Isolated from
Human Colorectal Metastases to Liver NMP Preparations Marker No.
Amino Acid Sequence (5'-3') L2 QFYQLDAYPSGAWYYVP (Peak *) (SEQ ID
NO:30) FFVALFPEVF (Peak 23) (SEQ ID NO:31) QFYQLDAYPSGAWYYVP (Peak
31) (SEQ ID NO:32) L5 TDAPSFSDIPNL (Peak No:20) (SEQ ID NO:33)
FFVALFPEVFGK (Peak No:26) (SEQ ID NO:34) QFYQL (Peak No:28) (SEQ ID
NO:35) AVPYPQRDMPI (Peak 38) (SEQ ID NO:36)
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[0200] The invention thus has been disclosed broadly and
illustrated in reference to representative embodiments described
above. Those skilled in the art will recognize that various
modifications can be made to the present invention without
departing from the spirit and scope thereof.
Sequence CWU 1
1
36 1 14 PRT Homo sapiens MOD_RES (2) variable amino acid 1 Pro Xaa
Val Lys Phe Asn Ser Tyr Val Asp Gly Val Glu Val 1 5 10 2 8 PRT Homo
sapiens 2 Glu Gly Asp Leu Ile Glu Asp Tyr 1 5 3 7 PRT Homo sapiens
3 Tyr Pro Val Glu Ala Phe Asn 1 5 4 11 PRT Homo sapiens 4 Thr Val
Ala Pro Leu Phe Ile Val Ile Pro Asn 1 5 10 5 18 PRT Homo sapiens
MOD_RES (1) variable amino acid 5 Xaa Val Thr Gly Leu Thr Gln Ile
Glu Thr Leu Phe Ala Ala Pro Gly 1 5 10 15 Val Asp 6 23 PRT Homo
sapiens 6 Ser Met Thr Glu Ala Glu Gln Gln Gln Leu Ile Asp Asp His
Phe Leu 1 5 10 15 Phe Asp Lys Pro Val Ser Pro 20 7 14 PRT Homo
sapiens 7 Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr Pro Val 1
5 10 8 11 PRT Homo sapiens 8 Val Leu Pro Gly Glu Ile Val Glu Tyr
Ser Arg 1 5 10 9 20 PRT Homo sapiens 9 Tyr Thr Ile Phe Asp Asn Phe
Leu Ile Thr Asn Asp Glu Ala Tyr Ala 1 5 10 15 Glu Glu Phe Gly 20 10
15 PRT Homo sapiens MOD_RES (11) variable amino acid 10 Gln Ile Asp
Asn Pro Asp Tyr Lys Gly Thr Xaa Ile His Pro Glu 1 5 10 15 11 15 PRT
Homo sapiens 11 Pro Ala Val Tyr Phe Lys Glu Gln Phe Leu Asp Gly Asp
Gly Trp 1 5 10 15 12 13 PRT Homo sapiens 12 Thr Leu Ile Val Arg Pro
Asp Asn Thr Tyr Glu Val Lys 1 5 10 13 11 PRT Homo sapiens 13 Tyr
Ala Val Leu Ile Thr Val Leu Gln Asp Ser 1 5 10 14 5 PRT Homo
sapiens 14 Asn Leu Pro Gln Glu 1 5 15 11 PRT Homo sapiens 15 Thr
Glu Pro Glu Leu Gln Asp Lys Ile His Gln 1 5 10 16 12 PRT Homo
sapiens 16 Thr Asp Ala Pro Ser Phe Ser Asp Ile Pro Asn Leu 1 5 10
17 9 PRT Homo sapiens MOD_RES (1) variable amino acid 17 Xaa Gln
Lys Glu Asp Val Pro Ser Glu 1 5 18 8 PRT Homo sapiens 18 Asn Ala
Phe Asn Asp Gly Leu Lys 1 5 19 17 PRT Homo sapiens 19 Tyr Phe Asp
Ser Phe Gly Asp Leu Ser Ser Ala Ser Ala Ile Met Gly 1 5 10 15 Asn
20 16 PRT Homo sapiens 20 Thr Tyr Phe Ser His Ile Asp Val Ser Pro
Gly Ser Ala Gln Val Lys 1 5 10 15 21 9 PRT Homo sapiens 21 Ser Leu
Asp Glu Gln Glu Gln Thr Lys 1 5 22 11 PRT Homo sapiens 22 Ala Lys
Thr Asp Phe Ala Thr Phe Leu Tyr Thr 1 5 10 23 10 PRT Homo sapiens
23 Leu Lys Tyr Glu Asn Glu Val Ala Leu Arg 1 5 10 24 9 PRT Homo
sapiens 24 Val Tyr Leu Glu Pro Leu Val Phe Arg 1 5 25 10 PRT Homo
sapiens 25 Arg Ala Pro Phe Gln Glu Leu Tyr Asn Asp 1 5 10 26 14 PRT
Homo sapiens MOD_RES (1) variable amino acid MOD_RES (13) variable
amino acid 26 Xaa Phe Tyr Gln Leu Asp Ala Tyr Pro Ser Gly Ala Xaa
Tyr 1 5 10 27 7 PRT Homo sapiens 27 Val Ile Glu Ala Phe Asn Arg 1 5
28 9 PRT Homo sapiens 28 Ile Leu Leu Phe Asp Tyr Phe Asn Arg 1 5 29
9 PRT Homo sapiens 29 Val Leu Val Ala Leu Glu Pro Leu Ser 1 5 30 17
PRT Homo sapiens 30 Gln Phe Tyr Gln Leu Asp Ala Tyr Pro Ser Gly Ala
Trp Tyr Tyr Val 1 5 10 15 Pro 31 10 PRT Homo sapiens 31 Phe Phe Val
Ala Leu Phe Pro Glu Val Phe 1 5 10 32 17 PRT Homo sapiens 32 Gln
Phe Tyr Gln Leu Asp Ala Tyr Pro Ser Gly Ala Trp Tyr Tyr Val 1 5 10
15 Pro 33 12 PRT Homo sapiens 33 Thr Asp Ala Pro Ser Phe Ser Asp
Ile Pro Asn Leu 1 5 10 34 12 PRT Homo sapiens 34 Phe Phe Val Ala
Leu Phe Pro Glu Val Phe Gly Lys 1 5 10 35 5 PRT Homo sapiens 35 Gln
Phe Tyr Gln Leu 1 5 36 11 PRT Homo sapiens 36 Ala Val Pro Tyr Pro
Gln Arg Asp Met Pro Ile 1 5 10
* * * * *