U.S. patent application number 12/911617 was filed with the patent office on 2011-11-03 for colon disease targets and uses thereof.
This patent application is currently assigned to CELERA CORPORATION. Invention is credited to Tao HE, Yeounjin KIM, Steve RUBEN.
Application Number | 20110271357 12/911617 |
Document ID | / |
Family ID | 43216532 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110271357 |
Kind Code |
A1 |
KIM; Yeounjin ; et
al. |
November 3, 2011 |
COLON DISEASE TARGETS AND USES THEREOF
Abstract
The present invention provides a method for diagnosing and
detecting diseases associated with colon. The present invention
provides one or more proteins or fragments thereof, peptides or
nucleic acid molecules differentially expressed in colon diseases
(CCAT) and antibodies binds to CCAT. The present invention provides
that CCAT is used as targets for screening agents that modulates
the CCAT activities. Further the present invention provides methods
for treating diseases associated with colon.
Inventors: |
KIM; Yeounjin; (Germantown,
MD) ; HE; Tao; (Acton, MA) ; RUBEN; Steve;
(Brookville, MD) |
Assignee: |
CELERA CORPORATION
Alameda
CA
|
Family ID: |
43216532 |
Appl. No.: |
12/911617 |
Filed: |
October 25, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12070716 |
Feb 19, 2008 |
7842466 |
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12911617 |
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11490374 |
Jul 21, 2006 |
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12070716 |
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60701057 |
Jul 21, 2005 |
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60717208 |
Sep 16, 2005 |
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60763348 |
Jan 31, 2006 |
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60781313 |
Mar 13, 2006 |
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Current U.S.
Class: |
800/13 ;
424/139.1; 424/174.1; 435/235.1; 435/252.3; 435/252.33; 435/252.35;
435/254.2; 435/331; 435/344; 435/375; 435/419; 436/501; 506/9;
514/21.2; 530/350; 530/387.2; 530/387.3; 530/387.9; 530/388.8;
530/389.7; 530/391.3; 530/391.7; 536/23.5; 536/24.5 |
Current CPC
Class: |
C12N 2310/14 20130101;
A61P 35/00 20180101; C07K 14/47 20130101; G01N 33/5011 20130101;
G01N 33/57419 20130101; G01N 2500/00 20130101; A61K 39/0011
20130101; C07K 2317/734 20130101; C07K 2317/54 20130101; A61K
39/001102 20180801; A61K 2039/55 20130101; C07K 16/18 20130101;
C07K 2317/56 20130101; G01N 33/6893 20130101; C07K 2317/55
20130101; C12Q 1/6886 20130101; C12N 15/113 20130101; C07K 2317/732
20130101 |
Class at
Publication: |
800/13 ; 436/501;
435/235.1; 435/419; 435/331; 435/375; 506/9; 530/350; 514/21.2;
536/23.5; 536/24.5; 530/387.9; 530/389.7; 530/387.2; 530/388.8;
435/344; 530/387.3; 435/254.2; 435/252.3; 435/252.35; 435/252.33;
530/391.3; 530/391.7; 424/139.1; 424/174.1 |
International
Class: |
A01K 67/00 20060101
A01K067/00; G01N 33/53 20060101 G01N033/53; C12N 7/01 20060101
C12N007/01; C12N 5/10 20060101 C12N005/10; C12N 5/16 20060101
C12N005/16; C12N 5/09 20100101 C12N005/09; C40B 30/04 20060101
C40B030/04; C07K 14/47 20060101 C07K014/47; A61K 38/17 20060101
A61K038/17; C07H 21/00 20060101 C07H021/00; C07H 21/02 20060101
C07H021/02; C07K 16/18 20060101 C07K016/18; C07K 16/42 20060101
C07K016/42; C12N 1/19 20060101 C12N001/19; C12N 1/21 20060101
C12N001/21; A61K 39/395 20060101 A61K039/395; A61P 35/00 20060101
A61P035/00; G01N 33/566 20060101 G01N033/566 |
Claims
1. An isolated protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS:1-1233 and 2228-2638.
2. A composition comprising the protein of claim 1 and a
pharmaceutically acceptable carrier.
3. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: a) SEQ ID
NOS:1234-2227; b) nucleotide sequences that encode a protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:1-1233 and 2228-2638; and c) nucleotide
sequences that are completely complementary to the nucleotide
sequences of a) or b).
4. An isolated RNAi or antisense nucleic acid molecule that
selectively binds to the nucleic acid molecule of claim 3.
5. An isolated antibody that selectively binds to the protein of
claim 1.
6. The antibody of claim 5, wherein the antibody is at least one of
a monoclonal, polyclonal, fully human, humanized, chimeric,
single-chain, or anti-idiotypic antibody.
7. A cell line, hybridoma, phage, or transgenic organism that
produces the antibody of claim 5.
8. The antibody of claim 5, wherein the antibody is coupled to a
composition selected from the group consisting of detectable
substances and therapeutic agents.
9. A composition comprising the antibody of claim 5 and a
pharmaceutically acceptable carrier.
10. An isolated antibody fragment of the antibody of claim 5,
wherein the antibody fragment comprises a fragment selected from
the group consisting of: a) an Fab fragment; b) an F(ab').sub.2
fragment; and c) an Fv fragment.
11. A method of modulating cell proliferation or apoptosis, the
method comprising contacting a cell with the antibody of claim
5.
12. The method of claim 11, wherein the method comprises either
inhibiting proliferation of colon cancer cells or stimulating
apoptosis of colon cancer cells.
13. A method of modulating cell proliferation or apoptosis, the
method comprising contacting a cell with the RNAi or antisense
nucleic acid molecule of claim 4.
14. A method of detecting the protein of claim 1 in a sample, the
method comprising contacting the sample with an isolated antibody
that selectively binds to the protein and determining whether the
antibody binds to the protein.
15. A method of detecting the nucleic acid molecule of claim 3 in a
sample, the method comprising contacting the sample with an
oligonucleotide that specifically hybridizes to the nucleic acid
molecule and determining whether the oligonucleotide binds to the
nucleic acid molecule.
16. A method of diagnosing, prognosing, or determining risk of
colon cancer in a subject, the method comprising detecting at least
one molecule in a sample, wherein the presence or abundance of the
molecule is indicative of colon cancer, and wherein the molecule is
selected from the group consisting of: a) proteins comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOS:1-1233 and 2228-2638; b) antibodies that selectively bind to
the protein of a); c) nucleic acid molecules comprising a
nucleotide sequence selected from the group consisting of SEQ ID
NOS:1234-2227 and nucleotide sequences that encode the protein of
a); and d) nucleic acid molecules comprising a nucleotide sequence
that is completely complementary to the nucleic acid molecule of
c).
17. A method of treating colon cancer, the method comprising
administering a therapeutically effective amount of the antibody of
claim 5 to a subject.
18. A method of screening agents, the method comprising contacting
the protein of claim 1 or a cell that expresses the protein with an
agent, and assaying for whether the agent binds to the protein or
modulates the function, activity, or expression of the protein.
19. A composition comprising the agent identified by the method of
claim 18 and a pharmaceutically acceptable carrier.
20. A method of determining or predicting the effectiveness of a
treatment or selecting a treatment for administration to a subject
having colon cancer, the method comprising detecting the presence,
abundance, or activity of the protein of claim 1 in a sample and
determining or predicting the effectiveness of the treatment or
selecting the treatment for administration based on the presence,
abundance, or activity of the protein.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the fields of molecular biology
and oncology. Specifically, the invention provides a molecular
marker and a therapeutic agent for use in the diagnosis and
treatment of colon diseases.
BACKGROUND OF THE INVENTION
[0002] Cancer currently constitutes the second most common cause of
death in the United States. Carcinomas of the colon are the eighth
most prevalent form of cancer and fourth among the most common
causes of cancer deaths in this country. The incidence of colon
cancer has been increasing steadily in the past twenty years in
most industrialized countries, exhibiting the characteristics of a
growing epidemiological problem. In the year 2000, for example, an
estimated 28,600 deaths will be ascribed to this type of cancer and
approximately 28,600 new cases will be diagnosed.
[0003] Colon cancer is the second most frequently diagnosed
malignancy in the United States as well as the second most common
cause of cancer death. The five-year survival rate for patients
with colorectal cancer detected in an early localized stage is 92%;
unfortunately, only 37% of colorectal cancer is diagnosed at this
stage. The survival rate drops to 64% if the cancer is allowed to
spread to adjacent organs or lymph nodes, and to 7% in patients
with distant metastases.
[0004] The prognosis of colon cancer is directly related to the
degree of penetration of the tumor through the bowel wall and the
presence or absence of nodal involvement, consequently, early
detection and treatment are especially important. Currently,
diagnosis is aided by the use of screening assays for fecal occult
blood, sigmoidoscopy, colonoscopy and double contrast barium
enemas. Treatment regimens are determined by the type and stage of
the cancer, and include surgery, radiation therapy and/or
chemotherapy. Recurrence following surgery (the most common form of
therapy) is a major problem and is often the ultimate cause of
death. In spite of considerable research into therapies for the
disease, colon cancer remains difficult to diagnose and treat. In
spite of considerable research into therapies for these and other
cancers, colon cancer remains difficult to diagnose and treat
effectively. Accordingly, there is a need in the art for improved
methods for detecting and treating such cancers. The present
invention fulfills these needs and further provides other related
advantages.
[0005] In spite of considerable research into therapies for these
and other cancers, colon cancer remains difficult to diagnose and
treat effectively. Accordingly, there is a need in the art for
improved methods for detecting and treating such cancers. The
present invention fulfills these needs and further provides other
related advantages such as other colon diseases.
SUMMARY OF THE INVENTION
[0006] The present invention is based on the identification of
certain cell surface proteins, cytosolic proteins, and/or secreted
proteins that are differentially expressed in colon cancer. A
malignant cell often differs from a normal cell by a differential
expression of one or more proteins. These differentially expressed
proteins, and the fragments thereof, are important markers for the
diagnosis of colon disease. The differentially expressed proteins
of the present invention and the nucleic acids encoding said
proteins and the fragments of said proteins are referred to herein
as colon cancer associated target, CCAT proteins or CCAT nucleic
acids or CCAT peptides, respectively.
[0007] The present invention provides peptides and protein
differentially expressed in colon diseases (hereinafter CCAT).
Based on the site of protein localization, e.g., surface or
cytosolic, and protein characterization, e.g. receptor or enzyme,
specific uses of these CCATs are provided. Some of the CCATs of the
present invention serve as targets for one or more classes of
therapeutic agents, while others may be suitable for antibody
therapeutics.
[0008] Accordingly, the present invention provides a method for
diagnosing or detecting colon disease in a subject comprising:
determining the level of one or more CCAT proteins, or any
fragment(s) thereof, in a test sample from said subject, wherein
said CCAT protein comprises a sequence selected from a group
consisting of SEQ ID NOS:1-1233; wherein a differential level of
said CCAT protein(s) or fragment(s) in said sample relative to the
level of said protein(s) or fragment(s) in a test sample from a
healthy subject, or the level established for a healthy subject, is
indicative of colon disease.
[0009] The present invention also provides a method for detecting
colon cancer in a subject comprising: determining the level of one
or more CCAT peptide(s) comprising a peptide sequence selected from
a group consisting of SEQ ID NOS:2228-2638 in a test sample from
said subject, wherein a differential level of said CCAT peptide(s)
in said sample to the level of said CCAT peptide(s) in a test
sample from a healthy subject, or the level of said CCAT peptide(s)
established for a healthy subject, is indicative of colon
disease.
[0010] The present invention further provides a method for
detecting colon disease in a subject comprising: determining the
level of one or more CCAT nucleic acid(s), or any fragment(s)
thereof, in a test sample from said subject, wherein said CCAT
nucleic acid(s) encode a CCAT protein sequence selected from a
group consisting of SEQ ID NOS:1-1233; wherein a differential level
of said CCAT nucleic acids or fragment(s) in said sample relative
to the level of said protein(s) or fragment(s) in a test sample
from a healthy subject, or the level established for a healthy
subject, is indicative of colon disease.
[0011] The invention also provides methods for detecting the CCAT
peptides, gene or mRNA in a test sample for use in diagnosing the
presence, absence or progression of a disease. The test sample
includes but is not limited to a biological sample such as tissue,
blood, serum or biological fluid.
[0012] The present invention further provides a purified antibody
that binds specifically to a protein molecule, or any fragment
thereof, selected from a group consisting of SEQ ID NOS:1-1233.
[0013] The present invention further provides a composition
comprising an antibody that binds to a protein selected from a
group consisting of SEQ ID NOS:1-1233 and an acceptable
carrier.
[0014] The present invention further provides a method for treating
colon disease, comprising administering to a patient in need of
said treatment a therapeutically effective amount of one or more
antibody(ies) of this invention.
[0015] The present invention further provides a method for treating
colon disease comprising (i) identifying a subject having colon
disease and (ii) administering to a said patient a therapeutically
effective amount of one or more antibody(ies) of this
invention.
[0016] The present invention further provides a method to screen
for agents that modulate CCAT protein activity, comprising the
steps of (i) contacting a test agent with a CCAT protein and (ii)
assaying for CCAT protein activity, wherein a change in said
activity in the presence of said agent relative to CCAT protein
activity in the absence of said agent indicates said agent
modulates said CCAT protein activity.
[0017] The present invention further provides a method to screen
for agents that bind to CCAT protein, comprising the steps of (i)
contacting a test agent with a CCAT protein and (ii) measuring the
level of binding of agent to said CCAT protein.
[0018] The invention also provides diagnostic methods for human
disease, in particular for colon diseases, its metastatic stage,
and therapeutic potential.
[0019] The present invention further provides diagnostic method for
epithelial-cell related cancers. In particular pancreas, lung,
colon, prostate, ovarian, breast, bladder renal, hepatocellular,
pharyngeal, and gastric cancer.
[0020] The invention also provides a method for monitoring the
disease progression and the treatment progress.
[0021] The invention further provide a method of diagnosis by an
array, wherein the array is immobilized with two or more CCAT
proteins, peptides or nucleic acid molecules. The proteins,
peptides or nucleic acid molecules include but are not limited to
the SEQ ID NOS: 1-2638.
[0022] The invention also provides monoclonal or polyclonal
antibodies and composition thereof reactive with antigenic portion
of CCAT protein, peptides or fragments thereof in a form for use in
colon diseases diagnosis.
[0023] The invention further provides an immunogenic antibody for
treating colon diseases disease or diseases associated with colon
diseases.
[0024] The present invention provides a method for screening agents
that modulate CCAT activity, comprising the steps of (a) contacting
a sample comprising CCAT with an agent; and (b) assaying for CCAT
activity, wherein a change in said CCAT activity in the presence of
said agent relative to CCAT activity in the absence of said
compound indicates said agent modulates CCAT. The agents include
but are not limited to protein, peptide, antibody, nucleic acid
such as antisense RNA, RNAi fragments, small molecules.
[0025] The present invention further provides a method for treating
colon diseases, comprising: administering to a patient one or more
agents in a therapeutically effective amount to treat colon
diseases.
[0026] The present invention provides a method for treating colon
diseases, comprising: identifying a subject having colon diseases;
and administering to a patient to one or more antibodies in a
therapeutically effective amount to treat colon diseases.
[0027] The present invention further provides therapeutic potential
for epithelial-cell related cancers. In particular pancreas, lung,
colon, prostate, ovarian, breast, bladder renal, hepatocellular,
pharyngeal and gastric cancer.
Description of the Files Contained on the CD-R Named
CL001615CDR
[0028] The CD-R named CL001615CDR contains the following three text
(ASCII) files:
1) File SEQLIST.sub.--1615.txt provides the Sequence Listing. The
Sequence Listing provides the protein sequences (SEQ ID
NOS:1-1233); transcript sequences (SEQ ID NOS:1234-2227) and
peptide sequences (SEQ ID NOS:2228-2638) as shown in Table 1. File
SEQLIST.sub.--1615.txt is 11,250 KB in size. 2) File
TABLE1.sub.--1615.txt provides Table 1. File TABLE1.sub.--1615 is
402 KB in size. 3) File TABLE2.sub.--1615.txt provides Table 2.
File TABLE2.sub.--1615 is 5 KB in size.
[0029] The material contained on the CD-R labeled CL001615CDR is
hereby incorporated by reference pursuant to 37 CFR 1.77(b)(4).
TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110271357A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Description of Table 1
[0030] Table 1 (provided on the CD-R) discloses the peptides which
correspond to the protein in the colon cancer tumor, the expression
information, and the ratio compare to the control sample. The
expression is based on measuring the level of the peptides. "N/A"
represents the number of overexpression by more than two, whereas
numerical representation of overexpression is also indicated. "S"
which is overexpressed singleton indicates that the peptide peak in
diseased sample was detected and there was no peak detected in
control samples.
[0031] Table 1 also discloses the CCAT proteins, transcripts, and
peptides.
[0032] The transcript/protein information includes: [0033] a
protein number (1 through 1233) [0034] a Celera protein internal
identification number for the protein encoded by the Celera
transcript (hCP and/or UID) [0035] a public protein accession
number (Genbank e.g., RefSeq NP number, Swiss-prot, or Derwent) for
the protein [0036] an art-known gene/protein name [0037] a Celera
transcript internal identification number (hCT and/or UID) [0038] a
public transcript accession number (Genbank e.g., RefSeq NM number,
or Derwent) [0039] a Celera hCG and UID internal identification
numbers for the gene [0040] an art-known gene symbol [0041] Celera
genomic axis position (indicating start nucleotide position-stop
nucleotide position) [0042] the chromosome number of the chromosome
on which the gene is located [0043] an OMIM (Online Mendelian
Inheritance in Man; Johns Hopkins University/NCBI) public reference
number for obtaining further information regarding the medical
significance of each gene [0044] alternative gene/protein name(s)
and/or symbol(s) in the OMIM entry
Description of Table 2
[0045] Table 2 discloses tumor staging information as follows (all
tumor staging designations in Table 2 are those which are typically
used in the art): Sample ID Number, sample type (labeled "Sample"),
cancer type (labeled "Type"), Lymph Nodes (the "N" in the TNM
staging system, for Node, which designates the spread of a tumor to
the lymph nodes), Distant Metastasis (the "M" in the TNM staging
system, for Metastasis, which designates the extent of tumor
metastasis), Extent of Invasion (the "T" in the TNM staging system,
for Tumor, which designates the size and location of a primary
tumor), and AJCC Stage (which is assigned based on a combination of
the T, N, and M classifications).
[0046] The Lymph Nodes ("N") stages are as follows: "NX"=the
regional lymph nodes cannot be assessed, "N0"=the cancer has not
spread into the regional lymph nodes, "N1"=the cancer has spread to
one to three regional lymph nodes, and "N2"=the cancer has spread
to four or more regional lymph nodes.
[0047] The Distant Metastasis ("M") stages are as follows:
"MX"=distant spread cannot be assessed, "M0"=the disease has not
metastasized, and "M1"=there is metastasis to another part of the
body beyond the colon or rectum.
[0048] The Extent of Invasion ("T") stages are as follows: "TX"=the
primary tumor cannot be evaluated, "T0"=there is no evidence of a
primary tumor in the colon or rectum, "Tis"=carcinoma in situ
(cancer cells are present only in the epithelium or lamina
propria), "T1"=the tumor extends into the submucosa of the colon,
"T2"=the tumor has invaded the muscularis propria, "T3"=the tumor
has grown through the muscularis propria and into the subserosa or
into tissues surrounding the colon or rectum, and "T4"=the tumor
has invaded other organs beyond the colon or rectum or has caused a
perforation in the wall of the colon or rectum.
[0049] The AJCC Stages are as follows: "0"=carcinoma in situ,
"I"=the cancer has grown through the mucosa and has invaded the
muscular layer of the colon or rectum but has not spread into
nearby tissues or lymph nodes (T1 or T2, N0, M0), "IIA"=the cancer
has spread through the wall of the colon or rectum and may have
spread to nearby tissues but has not spread to the nearby lymph
nodes (T3, N0, M0), "IIB"=the cancer has spread through the colon
or rectum to nearby organs but has not spread to the nearby lymph
nodes (T4, N0, M0), "IIIA"=the cancer has grown through the inner
lining or into the muscle layers of the intestine and to one to
three lymph nodes, but has not spread to other parts of the body
(T1 or T2; N1, M0), "IIIB"=the cancer has grown through the bowel
wall or to surrounding organs and into one to three lymph nodes,
but has not spread to other parts of the body (T3 or T4, N1, M0),
"IIIC"=the cancer (which can be of any size) has spread to four or
more lymph nodes, but not to other distant parts of the body (any
T, N2, M0), "IV"=the cancer has metastasized to distant parts of
the body (e.g., liver or lungs) (any T, any N, M1), and
"Recurrent"=the cancer has returned to the colon or rectum or
another part of the body following treatment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
General Description
[0050] While the broadest definition of this invention is set forth
in the Summary of the Invention, certain nucleic acids, peptides or
proteins are preferred. For example a preferred method for
detecting colon disease by determining the level of one or more
CCAT protein(s) or any fragment(s) thereof is wherein the level of
CCAT protein(s) are determined by contacting one or more
antibody(ies) that specifically bind to the antigenic regions of
the CCAT protein(s). Further preferred is a method wherein the
level of two or more proteins are determined, more preferred
wherein the level of four or more proteins are determined and most
preferred wherein the level of eight or more proteins are
determined.
[0051] A preferred method for detecting colon disease by
determining the level of one or more CCAT peptide(s) is wherein the
level of CCAT peptides(s) are determined by contacting one or more
antibody(ies) that specifically bind to the antigenic regions of
the CCAT peptide(s). Further preferred is a method wherein the
level of five or more peptides are determined, more preferred
wherein the level of ten or more peptides are determined and most
preferred wherein the level of fifteen or more peptides are
determined.
[0052] A preferred method for detecting colon disease by
determining the level of one or more CCAT nucleic acid(s) is
wherein the level of said CCAT nucleic acid(s) is determined by
contacting one or more probes that specifically hybridize to said
nucleic acid(s). Further preferred is a method wherein the level of
two or more nucleic acids are determined, more preferred wherein
the level of four or more nucleic acids are determined and most
preferred wherein the level of eight or more nucleic acids are
determined.
[0053] The methods for detecting colon disease provided by the
present invention may be used for diagnosing the presence of
disease in a patent, monitoring the presence of colon disease in
patients undergoing treatment and testing for the reoccurrence of
colon disease in patients that were successfully treated for colon
disease; preferably wherein the colon disease is colon cancer. The
test sample may be, but is not limited to, a biological sample such
as tissue, blood, serum or biological fluid.
[0054] The present invention is based on the discovery of
protein(s) and peptide(s) that are differentially expressed in
colon cancer samples versus normal colon diseases samples. These
proteins and peptide, and the encoding nucleic acid molecules are
associated with colon diseases, hereinafter the CCAT protein,
peptide or nucleic acids.
[0055] The discovery of disease specific target proteins is base on
discoveries made using proteomics techniques. The method uses on
MALDI-TOF TOF LC/MS analyses platform to generate protein
expression profiles from colon diseases tissues or cell lines in an
effort to discover and identify novel molecules associated with the
disease.
[0056] Based on these discoveries, the present invention provides
proteins, peptides, nucleic acids that are differential in colon
diseases, as well as antibodies binds to the proteins or peptides.
The present invention also provides methods for detection,
monitoring, diagnosis, prognosis, preventive and treatment of colon
diseases. The present invention provides a detection reagent,
markers for colon diseases at various stages, comprises CCAT
sequences isolated from human colon diseases tissue, sera, cell
lines, blood or biological fluids.
[0057] The present invention provides a method for treating colon
diseases targeting at CCAT. The treatment includes administration
of a therapeutically effective amount of composition comprise, but
not limit to, an antibody, an immunogenic peptide which induces T
cell response, a small molecule, a protein or a nucleic acid
molecule. The composition further comprises an agonist or
antigonist to CCAT. A "Colon or colorectal disease" includes but
not limited to colon cancer, colon tumor, diverticulosis,
diverticulitis, Crohn's disease, ulcerative colitis, irritable
bowel syndrome, inflammatory bowel disease, hemorrhoids, and anal
fissure.
[0058] The present invention may further provide a diagnostic or
therapeutic potential for epithelial-cell related cancers, which
include but are not limited to pancreas, lung, colon, prostate,
ovarian, breast, bladder renal, hepatocellular, pharyngeal and
gastric cancers.
[0059] The present invention further provides the target for
screening an agent for CCAT, wherein the agent is compounds of
small molecules, proteins, peptides, nucleic acids, antibodies or
other agonists or antigonists.
CCAT Peptide/Proteins and Peptides
[0060] The present invention provides isolated CCAT peptide and
protein molecules that consisting of, consisting essentially of, or
comprising the amino acid sequences of the CCAT peptides and
proteins disclosed in Table 1, (encoded by the nucleic acid
molecule shown in Table 1), as well as all obvious variants of
these peptides that are within the art to make and use. Some of
these variants are described in detail below.
[0061] In one embodiment CCAT peptides include, but are not limited
to, the amino acid sequence of SEQ ID NOS:2228-2638 and variants
thereof. A CCAT protein includes, but is not limited to, the amino
acid sequence of SEQ ID NOS:1-1233 and variants thereof. CCAT
proteins may be differentially expressed in colon cell line, blood,
tissue, serum or body fluids.
[0062] The peptide or protein or fragment thereof, to which the
invention pertains, however, are not to be construed as
encompassing peptide, protein or fragment that may be disclosed
publicly prior to the present invention.
[0063] The CCAT proteins and peptides of the present invention can
be purified to homogeneity or other degrees of purity. The level of
purification will be based on the intended use. The critical
feature is that the preparation allows for the desired function of
the peptide, even if in the presence of considerable amounts of
other components (the features of an isolated nucleic acid molecule
is discussed below).
[0064] As used herein, a "peptide" is defined as amino acid
sequences between 5-20 amino acids derived from CCAT proteins such
as SEQ ID NOS:1-1233 or variants thereof. The peptide
differentially expressed in either colon diseases cell line, blood,
tissue, serum or body fluids. In one embodiment peptides include,
but are not limited to, the amino acid sequence of SEQ ID
NOS:2228-2638, or variants thereof.
[0065] As used herein, a "protein" is full-length protein
differentially expressed in colon diseases cell line, tissue,
blood, serum or body fluids. A protein includes, but is not limited
to, the amino acid sequence of SEQ ID NOS:1-1233.
[0066] A peptide is said to be "isolated" or "purified" when it is
substantially free of cellular material or free of chemical
precursors or other chemicals. The peptides of the present
invention can be purified to homogeneity or other degrees of
purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components (the features of an
isolated nucleic acid molecule are discussed below).
[0067] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0068] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the CCAT peptide having less than about
30% (by dry weight) chemical precursors or other chemicals, less
than about 20% chemical precursors or other chemicals, less than
about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0069] The isolated CCAT proteins and peptide can be purified from
cells that naturally express it, purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods. Sambrook et al., Molecular Cloning: A
Laboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., (2001). Experimental data as provided in
Table 1 indicates expression in human colon tumor tissues. For
example, a nucleic acid molecule encoding the CCAT protein or
peptide is cloned into an expression vector, the expression vector
introduced into a host cell and the protein expressed in the host
cell. The protein or peptide can then be isolated from the cells by
an appropriate purification scheme using standard protein
purification techniques. Many of these techniques are described in
detail below.
[0070] A CCAT peptide or protein can be attached to heterologous
sequences to form chimeric or fusion proteins. Such Schimeric and
fusion proteins comprise a peptide operatively linked to a
heterologous protein having an amino acid sequence not
substantially homologous to the peptide. "Operatively linked"
indicates that the peptide and the heterologous protein are fused
in-frame. The heterologous protein can be fused to the N-terminus
or C-terminus of the peptide.
[0071] In some uses, the fusion protein does not affect the
activity of the peptide or protein per se. For example, the fusion
protein can include, but is not limited to, fusion proteins, for
example beta-galactosidase fusions, yeast two-hybrid GAL fusions,
poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion
proteins, particularly poly-His fusions, can facilitate the
purification of recombinant CCAT proteins or peptides. In certain
host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased by using a heterologous
signal sequence.
[0072] A chimeric or fusion CCAT protein or peptide can be produced
by standard recombinant DNA techniques. For example, DNA fragments
coding for the different protein sequences are ligated together
in-frame in accordance with conventional 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 Ausubel et
al., Current Protocols in Molecular Biology, 1992). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST protein). A CCAT-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the CCAT protein or peptide.
[0073] As mentioned above, the CCAT peptide or the CCAT protein has
obvious variants of the amino acid sequence, such as naturally
occurring mature forms of the CCAT, allelic/sequence variants of
the CCAT, non-naturally occurring recombinantly derived variants of
the CCATs, and orthologs and paralogs of the CCAT proteins or
peptides. Such variants can readily be generated using art-known
techniques in the fields of recombinant nucleic acid technology and
protein biochemistry.
[0074] It is understood, however, that CCAT and variants exclude
any amino acid sequences disclosed prior to the invention.
[0075] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the CCAT peptides
of the present invention. The degree of homology/identity present
will be based primarily on whether the peptide is a functional
variant or non-functional variant, the amount of divergence present
in the paralog family and the evolutionary distance between the
orthologs.
[0076] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment, at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of
a reference sequence is aligned for comparison purposes. 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 (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0077] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package, using either a
Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (Devereux, J., et al., Nucleic Acids Res.
12(1):387 (1984)), using a NWSgapdna.CMP matrix and a gap weight of
40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
In another embodiment, the percent identity between two amino acid
or nucleotide sequences is determined using the algorithm of E.
Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0078] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0079] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the CCAT peptides of the present invention as
well as being encoded by the same genetic locus as the CCAT peptide
provided herein (see Table 1).
[0080] Allelic variants of a CCAT peptide can readily be identified
as being a human protein having a high degree (significant) of
sequence homology/identity to at least a portion of the CCAT
peptide as well as being encoded by the same genetic locus as the
CCAT peptide provided herein. Genetic locus can readily be
determined based on the genomic information provided in Table 1,
such as the genomic sequence mapped to the reference human. As used
herein, two proteins (or a region of the proteins) have significant
homology when the amino acid sequences are typically at least about
70-80%, 80-90%, and more typically at least about 90-95% or more
homologous. A significantly homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence that will hybridize to a CCAT peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0081] Paralogs of a CCAT peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the CCAT peptide, as being encoded by a gene
from humans, and as having similar activity or function. Two
proteins will typically be considered paralogs when the amino acid
sequences are typically at least about 60% or greater, and more
typically at least about 70% or greater homology through a given
region or domain. Such paralogs will be encoded by a nucleic acid
sequence that will hybridize to a CCAT peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0082] Orthologs of a CCAT peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the CCAT peptide as well as being encoded by a
gene from another organism. Preferred orthologs will be isolated
from mammals, preferably primates, for the development of human
therapeutic targets and agents. Such orthologs will be encoded by a
nucleic acid sequence that will hybridize to a CCAT peptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins.
[0083] Non-naturally occurring variants of the CCAT peptides of the
present invention can readily be generated using recombinant
techniques. Such variants include, but are not limited to
deletions, additions and substitutions in the amino acid sequence
of the CCAT peptide. For example, one class of substitutions is
conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a CCAT peptide by another
amino acid of like characteristics. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr; exchange of the acidic residues Asp
and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues Lys and Arg; and replacements among
the aromatic residues Phe and Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al., Science 247:1306-1310 (1990).
[0084] Variant CCAT peptides can be fully functional or can lack
function in one or more activities, e.g. ability to bind substrate,
ability to phosphorylate substrate, ability to mediate signaling,
etc. Fully functional variants typically contain only conservative
variation or variation in non-critical residues or in non-critical
regions.
[0085] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0086] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity
such as CCAT activity or in assays such as an in vitro
proliferative activity. Sites that are critical for binding
partner/substrate binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992); de Vos et al. Science 255:306-312 (1992)).
[0087] The present invention further provides fragments of the
CCATs, in addition to proteins and peptides that comprise and
consist of such fragments, particularly those comprising the
residues identified in Table 1. As used herein, a fragment
comprises at least 8, 10, 12, 14, 16, 18, 20 or more contiguous
amino acid residues from a CCAT. Such fragments can be chosen based
on the ability to retain one or more of the biological activities
of the CCAT or could be chosen for the ability to perform a
function, e.g. bind a substrate or act as an immunogen.
Particularly important fragments are biologically active fragments,
peptides that are, for example, about 8 or more amino acids in
length. Such fragments will typically comprise a domain or motif of
the CCAT, e.g., active site, a transmembrane domain or a
substrate-binding domain. Further, possible fragments include, but
are not limited to, domain or motif containing fragments, soluble
peptide fragments, and fragments containing immunogenic structures.
Predicted domains and functional sites are readily identifiable by
computer programs well known and readily available to those of
skill in the art (e.g., PROSITE analysis).
[0088] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in CCATs are described in basic
texts, detailed monographs, and the research literature, and they
are well known to those of skill in the art.
[0089] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0090] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[0091] Accordingly, the CCATs of the present invention also
encompass derivatives or analogs in which a substituted amino acid
residue is not one encoded by the genetic code, in which a
substituent group is included, in which the mature CCAT is fused
with another compound, such as a compound to increase the half-life
of the CCAT (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature CCAT, such as a
leader or secretory sequence or a sequence for purification of the
mature CCAT or a pro-protein sequence.
Protein/Peptide Uses
[0092] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in Table 1; to raise antibodies or to elicit
another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein (or its binding partner or ligand) in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a CCAT-effector
protein interaction or CCAT-ligand interaction), the protein can be
used to identify the binding partner/ligand so as to develop a
system to identify inhibitors of the binding interaction. Any or
all of these uses are capable of being developed into reagent grade
or kit format for commercialization as commercial products.
[0093] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", Sambrook, J., E.
F. Fritsch and T. Maniatis eds., 3rd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (2001) and "Methods in
Enzymology: Guide to Molecular Cloning Techniques", Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987.
[0094] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, CCATs isolated from
humans and their human/mammalian orthologs serve as targets for
identifying agents for use in mammalian therapeutic applications,
e.g. a human drug, particularly in modulating a biological or
pathological response in a cell or tissue that expresses the CCAT.
Experimental data as provided in Table 1 indicate that the CCAT of
the present invention are overexpressed in colon tumor tissue. A
large percentage of pharmaceutical agents are being developed that
modulate the activity of CCAT proteins, particularly members of the
CCAT subfamily (see Background of the Invention). The structural
and functional information provided in the Background and Figures
provide specific and substantial uses for the molecules of the
present invention, particularly in combination with the expression
information provided in Table 1. Experimental data as provided in
Table 1 indicates expression in human colon tumor tissues. Such
uses can readily be determined using the information provided
herein, that which is known in the art, and routine
experimentation.
[0095] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to CCATs that
are related to members of the CCAT subfamily. Such assays involve
any of the known CCAT functions or activities or properties useful
for diagnosis and treatment of CCAT-related conditions that are
specific for the subfamily of CCATs that the one of the present
invention belongs to, particularly in cells and tissues that
express the CCAT. Experimental data as provided in Table 1 indicate
that the CCAT of the present invention are overexpressed in colon
tumor tissue
[0096] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the CCAT, as a biopsy or expanded in cell culture. Experimental
data as provided in Table 1 indicates expression in human colon
tumor tissues. In an alternate embodiment, cell-based assays
involve recombinant host cells expressing the CCAT protein.
[0097] The polypeptides can be used to identify compounds or agents
that modulate CCAT activity of the protein in its natural state or
an altered form that causes a specific disease or pathology
associated with the CCAT. Both the CCATs of the present invention
and appropriate variants and fragments can be used in
high-throughput screens to assay candidate compounds for the
ability to bind to the CCAT. These compounds can be further
screened against a functional CCAT to determine the effect of the
compound on the CCAT activity. Further, these compounds can be
tested in animal or invertebrate systems to determine
activity/effectiveness. Compounds can be identified that activate
(agonist) or inactivate (antagonist) the CCAT to a desired
degree.
[0098] Further, the proteins of the present invention can be used
to screen a compound or an agent for the ability to stimulate or
inhibit interaction between the CCAT protein and a molecule that
normally interacts with the CCAT protein, e.g. a substrate or or an
extracellular binding ligand or a component of the signal pathway
that the CCAT protein normally interacts (for example, a cytosolic
signal protein or another CCAT). Such assays typically include the
steps of combining the CCAT protein with a candidate compound under
conditions that allow the CCAT protein, or fragment, to interact
with the target molecule, and to detect the formation of a complex
between the protein and the target or to detect the biochemical
consequence of the interaction with the CCAT protein and the
target, such as any of the associated effects of signal
transduction such as protein phosphorylation, cAMP turnover, and
adenylate cyclase activation, etc.
[0099] Candidate compounds or agents include, for example, 1)
peptides such as soluble peptides, including Ig-tailed fusion
peptides and members of random peptide libraries (see, e.g., Lam et
al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86
(1991)) and combinatorial chemistry-derived molecular libraries
made of D- and/or L-configuration amino acids; 2) phosphopeptides
(e.g., members of random and partially degenerate, directed
phosphopeptide libraries, see, e.g., Songyang et al., Cell
72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric, and single chain antibodies as
well as Fab, F(ab')2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0100] One candidate compound or agent is a soluble fragment of the
CCAT that competes for substrate binding. Other candidate compounds
include mutant CCATs or appropriate fragments containing mutations
that affect CCAT function and thus compete for substrate.
Accordingly, a fragment that competes for substrate, for example
with a higher affinity, or a fragment that binds substrate but does
not allow release, is encompassed by the invention.
[0101] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) CCAT
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate CCAT activity. Thus, the
phosphorylation of a substrate, activation of a protein, a change
in the expression of genes that are up- or down-regulated in
response to the CCAT protein dependent signal cascade can be
assayed.
[0102] Any of the biological or biochemical functions mediated by
the CCAT can be used as an endpoint assay. These include all of the
biochemical or biochemical/biological events described herein, in
the references cited herein, incorporated by reference for these
endpoint assay targets, and other functions known to those of
ordinary skill in the art or that can be readily identified using
the information provided in Table 1. Specifically, a biological
function of a cell or tissues that expresses the CCAT can be
assayed. Experimental data as provided in Table 1 indicate that the
CCAT of the present invention are overexpressed in colon tumor
tissue.
[0103] Binding and/or activating compounds can also be screened by
using chimeric CCAT proteins in which the amino terminal
extracellular domain, or parts thereof, the entire transmembrane
domain or subregions, such as any of the seven transmembrane
segments or any of the intracellular or extracellular loops and the
carboxy terminal intracellular domain, or parts thereof, can be
replaced by heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate then that which is recognized by the native
CCAT. Accordingly, a different set of signal transduction
components is available as an end-point assay for activation. This
allows for assays to be performed in other than the specific host
cell from which the CCAT is derived.
[0104] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the CCAT (e.g. binding partners and/or
ligands). Thus, a compound is exposed to a CCAT polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble CCAT polypeptide is also added to the
mixture. If the test compound interacts with the soluble CCAT
polypeptide, it decreases the amount of complex formed or activity
from the CCAT. This type of assay is particularly useful in cases
in which compounds are sought that interact with specific regions
of the CCAT. Thus, the soluble polypeptide that competes with the
target CCAT region is designed to contain peptide sequences
corresponding to the region of interest.
[0105] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the CCAT protein, or fragment, or
its target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0106] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of CCAT-binding protein found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques. For example, either the polypeptide or its target
molecule can be immobilized utilizing conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively,
antibodies reactive with the protein but which do not interfere
with binding of the protein to its target molecule can be
derivatized to the wells of the plate, and the protein trapped in
the wells by antibody conjugation. Preparations of a CCAT-binding
protein and a candidate compound are incubated in the CCAT
protein-presenting wells and the amount of complex trapped in the
well can be quantitated. Methods for detecting such complexes, in
addition to those described above for the GST-immobilized
complexes, include immunodetection of complexes using antibodies
reactive with the CCAT protein target molecule, or which are
reactive with CCAT protein and compete with the target molecule, as
well as CCAT-linked assays which rely on detecting an enzymatic
activity associated with the target molecule.
[0107] Agents that modulate one of the CCATs of the present
invention can be identified using one or more of the above assays,
alone or in combination. It is generally preferable to use a
cell-based or cell free system first and then confirm activity in
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0108] Modulators of CCAT protein activity identified according to
these drug screening assays can be used to treat a subject with a
disorder mediated by the CCAT pathway, by treating cells or tissues
that express the CCAT. Experimental data as provided in Table 1
indicates expression in human colon tumor tissues. These methods of
treatment include the steps of administering a modulator of CCAT
activity in a pharmaceutical composition to a subject in need of
such treatment, the modulator being identified as described
herein.
[0109] In yet another aspect of the invention, the CCAT proteins
can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993)
[0110] Oncogene 8:1693-1696; and Brent WO94/10300), to identify
other proteins, which bind to or interact with the CCAT and are
involved in CCAT activity. Such CCAT-binding proteins are also
likely to be involved in the propagation of signals by the CCAT
proteins or CCAT targets as, for example, downstream elements of a
CCAT-mediated signaling pathway. Alternatively, such CCAT-binding
proteins are likely to be CCAT inhibitors.
[0111] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a CCAT
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences that encode an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a CCAT-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the CCAT protein.
[0112] 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 CCAT-modulating
agent, an antisense CCAT nucleic acid molecule, an CCAT-RNAi
fragment, a CCAT-specific antibody, or a CCAT-binding partner) can
be used in an animal or other 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 or other 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.
[0113] The CCAT proteins of the present invention are also useful
to provide a target for diagnosing a disease or predisposition to
disease mediated by the peptide. Accordingly, the invention
provides methods for detecting the presence, or levels of, the
protein (or encoding mRNA) in a cell, tissue, or organism.
Experimental data as provided in Table 1 indicates expression in
human colon tumor tissues. The method involves contacting a
biological sample with a compound capable of interacting with the
CCAT protein such that the interaction can be detected. Such an
assay can be provided in a single detection format or a
multi-detection format such as an antibody chip array.
[0114] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject.
[0115] The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered CCAT activity in cell-based or
cell-free assay, alteration in substrate or antibody-binding
pattern, altered isoelectric point, direct amino acid sequencing,
and any other of the known assay techniques useful for detecting
mutations in a protein. Such an assay can be provided in a single
detection format or a multi-detection format such as an antibody
chip array.
[0116] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0117] The peptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes affects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
CCAT protein in which one or more of the CCAT functions in one
population are different from those in another population. The
peptides thus allow a target to ascertain a genetic predisposition
that can affect treatment modality. Thus, in a ligand-based
treatment, polymorphism may give rise to amino terminal
extracellular domains and/or other substrate-binding regions that
are more or less active in substrate binding, and CCAT activation.
Accordingly, substrate dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing a polymorphism. As an alternative to genotyping,
specific polymorphic peptides could be identified.
[0118] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in Table 1
indicates expression in human colon tumor tissues. Accordingly,
methods for treatment include the use of the CCAT protein or
fragments.
Antibodies
[0119] The present invention provides antibodies specifically bind
to CCAT proteins or fragments thereof, peptides, or antigenic
portion thereof.
[0120] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof as describe above.
[0121] The antibody of present invention selectively binds a target
CCAT when it binds the target domain and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0122] The term "antibody" is used in the broadest sense, and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), humanized antibody and
antibody fragments (e.g., Fab, F(ab').sub.2 and Fv) so long as they
exhibit the desired biological activity. Antibodies (Abs) and
immunoglobulins (Igs) are glycoproteins having the same structural
characteristics. While antibodies exhibit binding specificity to a
specific antigen, immunoglobulins include both antibodies and other
antibody-like molecules that lack antigen specificity.
[0123] As used herein, antibodies are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies between the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (VH) followed by a
number of constant domains. Each light chain has a variable domain
at one end (VL) and a constant domain at its other end. The
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light and heavy chain variable domains. Chothia et al.,
J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, Proc. Natl.
Acad. Sci. USA 82 4592-4596 (1985).
[0124] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of the environment in
which is produced. Contaminant components of its production
environment are materials that would interfere with diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
preferred embodiments, the antibody will be purified as measurable
by at least three different methods: 1) to greater than 95% by
weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight; 2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequentator; or 3) to homogeneity
by SDS-PAGE under reducing or non-reducing conditions using
Coomasie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0125] An "antigenic region" or "antigenic determinant" or an
"epitope" includes any protein determinant capable of specific
binding to an antibody. This is the site on an antigen to which
each distinct antibody molecule binds. Epitopic determinants
usually consist of active surface groupings of molecules such as
amino acids or sugar side chains and usually have specific
three-dimensional structural characteristics, as well as charge
characteristics.
[0126] "Antibody specificity," is an antibody, which has a stronger
binding affinity for an antigen from a first subject species than
it has for a homologue of that antigen from a second subject
species. Normally, the antibody "bind specifically" to a human
antigen (i.e., has a binding affinity (Kd) value of no more than
about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 M and most preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second subject species which is at least about
50 fold, or at least about 500 fold, or at least about 1000 fold,
weaker than its binding affinity for the human antigen. The
antibody can be of any of the various types of antibodies as
defined above, but preferably is a humanized or human antibody
(Queen et al., U.S. Pat. Nos. 5,530,101, 5,585,089; 5,693,762; and
6,180,370).
[0127] The present invention provides an "antibody variant," which
refers to an amino acid sequence variant of an antibody wherein one
or more of the amino acid residues have been modified. Such variant
necessarily have less than 100% sequence identity or similarity
with the amino acid sequence having at least 75% amino acid
sequence identity or similarity with the amino acid sequence of
either the heavy or light chain variable domain of the antibody,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, and most preferably at least 95%. Since
the method of the invention applies equally to both polypeptides,
antibodies and fragments thereof, these terms are sometimes
employed interchangeably.
[0128] The term "variable" in the context of variable domain of
antibodies refers to the fact that certain portions of the variable
domains differ extensively in sequence among antibodies and are
used in the binding and specificity of each particular antibody for
its particular antigen. However, the variability is not evenly
distributed through the variable domains of antibodies. It is
concentrated in three segments called complementarity determining
regions (CDRs) also known as hypervariable regions both in the
light chain and the heavy chain variable domains. There are at
least two techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al., Sequences
of Proteins of Immunological Interest (National Institute of
Health, Bethesda, Md. 1987); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Chothia, C.
et al. (1989), Nature 342: 877). The more highly conserved portions
of variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four FR
regions, largely adopting a .beta.-Sheet configuration, connected
by three CDRs, which form loops connecting, and in some cases
forming part of, the .beta.-sheet structure. The CDRs in each chain
are held together in close proximity by the FR regions and, with
the CDRs from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see Kabat et al.) The constant
domains are not involved directly in binding an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0129] The term "antibody fragment" refers to a portion of a
full-length antibody, generally the antigen binding or variable
region. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2 and Fv fragments. Papain digestion of antibodies
produces two identical antigen binding fragments, called the Fab
fragment, each with a single antigen binding site, and a residual
"Fc" fragment, so-called for its ability to crystallize readily.
Pepsin treatment yields an F(a').sub.2 fragment that has two
antigen binding fragments which are capable of crosslinking
antigen, and a residual other fragment (which is termed pFc').
Additional fragments can include diabodies, linear antibodies,
single-chain antibody molecules, and multispecific antibodies
formed from antibody fragments. As used herein, "functional
fragment" with respect to antibodies, refers to Fv, F(ab) and
F(ab').sub.2 fragments.
[0130] An "Fv" fragment is the minimum antibody fragment that
contains a complete antigen recognition and binding site. This
region consists of a dimer of one heavy and one light chain
variable domain in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). It is in this configuration that the three
CDRs of each variable domain interact to define an antigen-binding
site on the surface of the V.sub.H-V.sub.L dimer. Collectively, the
six CDRs confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three CDRs specific for an antigen) has the ability to
recognize and bind antigen, although at a lower affinity than the
entire binding site.
[0131] The Fab fragment [also designated as F(ab)] also contains
the constant domain of the light chain and the first constant
domain (CH1) of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxyl
terminus of the heavy chain CH1 domain including one or more
cysteines from the antibody hinge region. Fab'-SH is the
designation herein for Fab' in which the cysteine residue(s) of the
constant domains have a free thiol group. F(ab') fragments are
produced by cleavage of the disulfide bond at the hinge cysteines
of the F(ab').sub.2 pepsin digestion product. Additional chemical
couplings of antibody fragments are known to those of ordinary
skill in the art.
[0132] The present invention further provides monoclonal antibody,
polyclonal antibody as well as humanized antibody. In general, to
generate antibodies, an isolated peptide is used as an immunogen
and is administered to a mammalian organism, such as a rat, rabbit
or mouse. The full-length protein, an antigenic peptide fragment or
a fusion protein of the CCAT protein can be used. Particularly
important fragments are those covering functional domains, some but
not all the examples of the domains are identified in Table 1. Many
methods are known for generating and/or identifying antibodies to a
given target peptide. Several such methods are described by Harlow,
Antibodies, Cold Spring Harbor Press, (1989).
[0133] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In additional to their specificity, the
monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" antibody indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein, Nature 256, 495 (1975), or may be made by
recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.
The monoclonal antibodies for use with the present invention may
also be isolated from phage antibody libraries using the techniques
described in Clackson et al. Nature 352: 624-628 (1991), as well as
in Marks et al., J. Mol. Biol. 222: 581-597 (1991). For detailed
procedure for making a monoclonal antibody, see the Example
below.
[0134] "Humanized" forms of non-human (e.g. murine or rabbit)
antibodies are chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementary determining region (CDR) of
the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibody
may comprise residues, which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. These
modifications are made to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see: Jones et al., Nature 321, 522-525 (1986); Reichmann
et al., Nature 332, 323-327 (1988) and Presta, Curr. Op. Struct.
Biol. 2, 593-596 (1992).
[0135] Polyclonal antibodies may be prepared by any known method or
modifications of these methods including obtaining antibodies from
patients. For example, a complex of an immunogen such as CCAT
protein, peptides or fragments thereof and a carrier protein is
prepared and an animal is immunized by the complex according to the
same manner as that described with respect to the above monoclonal
antibody preparation and the description in the Example. A serum or
plasma containing the antibody against the protein is recovered
from the immunized animal and the antibody is separated and
purified. The gamma globulin fraction or the IgG antibodies can be
obtained, for example, by use of saturated ammonium sulfate or DEAE
Sephadex, or other techniques known to those skilled in the
art.
[0136] The antibody titer in the antiserum can be measured
according to the same manner as that described above with respect
to the supernatant of the hybridoma culture. Separation and
purification of the antibody can be carried out according to the
same separation and purification method of antibody as that
described with respect to the above monoclonal antibody and in the
Example.
[0137] The protein used here in as the immunogen is not limited to
any particular type of immunogen. In one aspect, antibodies are
preferably prepared from regions or discrete fragments of the CCAT
proteins. Antibodies can be prepared from any region of the peptide
as described herein. In particular, they are selected from a group
consisting of SEQ ID NOS:2228-2638 and fragments of SEQ ID
NOS:1-1233. An antigenic fragment will typically comprise at least
8 contiguous amino acid residues. The antigenic peptide can
comprise, however, at least 10, 12, 14, 16 or more amino acid
residues. Such fragments can be selected on a physical property,
such as fragments correspond to regions that are located on the
surface of the protein, e.g., hydrophilic regions or can be
selected based on sequence uniqueness.
[0138] Antibodies may also be produced by inducing production in
the lymphocyte population or by screening antibody libraries or
panels of highly specific binding reagents as disclosed in Orlandi
et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al.
(1991; Nature 349:293-299). A protein may be used in screening
assays of phagemid or B-lymphocyte immunoglobulin libraries to
identify antibodies having a desired specificity. Numerous
protocols for competitive binding or immunoassays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Smith G. P., 1991, Curr. Opin.
Biotechnol. 2: 668-673.
[0139] The antibodies of the present invention can also be
generated using various phage display methods known in the art. In
phage display methods, functional antibody domains are displayed on
the surface of phage particles which carry the polynucleotide
sequences encoding them. In a particular, such phage can be
utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0140] Antibody can be also made recombinantly. When using
recombinant techniques, the antibody variant can be produced
intracellularly, in the periplasmic space, or directly secreted
into the medium. If the antibody variant is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10: 163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
minutes. Cell debris can be removed by centrifugation. Where the
antibody variant is secreted into the medium, supernatants from
such expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0141] The antibodies or antigen binding fragments may also be
produced by genetic engineering. The technology for expression of
both heavy and light cain genes in E. coli is the subject the
following PCT patent applications; publication number WO 901443,
WO901443, and WO 9014424 and in Huse et al., 1989 Science
246:1275-1281. The general recombinant methods are well known in
the art.
[0142] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .delta.1, .delta.2 or .delta.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .delta.3 (Guss et
al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0143] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
Antibody Uses
[0144] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Experimental data as provided in Table 1 indicate that the CCAT of
the present invention are overexpressed in colon tumor tissue.
Further, such antibodies can be used to detect protein in situ, in
vitro, or in a cell lysate or supernatant in order to evaluate the
abundance and pattern of expression. Also, such antibodies can be
used to assess abnormal tissue distribution or abnormal expression
during development or progression of a biological condition.
Antibody detection of circulating fragments of the full length
protein can be used to identify turnover.
[0145] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in Table 1 indicates expression in human colon tumor tissues. If a
disorder is characterized by a specific mutation in the protein,
antibodies specific for this mutant protein can be used to assay
for the presence of the specific mutant protein.
[0146] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in Table 1 indicates
expression in human colon tumor tissues. The diagnostic uses can be
applied, not only in genetic testing, but also in monitoring a
treatment modality. Accordingly, where treatment is ultimately
aimed at correcting expression level or the presence of aberrant
sequence and aberrant tissue distribution or developmental
expression, antibodies directed against the protein or relevant
fragments can be used to monitor therapeutic efficacy. More
detection and diagnosis methods are described in detail below.
[0147] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0148] The antibodies are also useful for tissue typing.
Experimental data as provided in Table 1 indicates expression in
human colon tumor tissues. Thus, where a specific protein has been
correlated with expression in a specific tissue, antibodies that
are specific for this protein can be used to identify a tissue
type.
[0149] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the CCAT peptide to
a binding partner such as a substrate or another antibody. These
uses can also be applied in a therapeutic context in which
treatment involves inhibiting the protein's function. An antibody
can be used, for example, to block binding, thus modulating
(agonizing or antagonizing) the peptides activity. Antibodies can
be prepared against specific fragments containing sites required
for function or against intact protein that is associated with a
cell or cell membrane. More therapeutics methods are described in
detail below.
[0150] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a standard;
and instructions for use. Such a kit can be supplied to detect a
single protein or epitope or can be configured to detect one of a
multitude of epitopes, such as in an antibody detection array.
Arrays are described in detail below for nucleic acid arrays and
similar methods have been developed for antibody arrays.
Nucleic Acid Molecules
[0151] The present invention further provides isolated nucleic acid
molecules that encode a CCAT peptide or protein of the present
invention. Such nucleic acid molecules will consist of, consist
essentially of, or comprise a nucleotide sequence that encodes one
of the CCAT peptides of the present invention, an allelic variant
thereof, or an ortholog or paralog thereof. The nucleic acid
molecules and the fragments thereof of the present invention
pertains, however, are not to be construed as encompassing
fragments that may be disclosed publicly prior to the present
invention.
[0152] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0153] Moreover, an "isolated" nucleic acid molecule, such as a
transcript/cDNA molecule, can be substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0154] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0155] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in Table 1,
(SEQ ID NOS:1234-2227), or any nucleic acid molecule that encodes a
protein provided in Table 1, (SEQ ID NOS:1-1233). A nucleic acid
molecule comprises a nucleotide sequence when the nucleotide
sequence is at least part of the final nucleotide sequence of the
nucleic acid molecule. In such a fashion, the nucleic acid molecule
can be only the nucleotide sequence or have additional nucleic acid
residues, such as nucleic acid residues that are naturally
associated with it or heterologous nucleotide sequences. Such a
nucleic acid molecule can have a few additional nucleotides or can
comprise several hundred or more additional nucleotides. A brief
description of how various types of these nucleic acid molecules
can be readily made/isolated is provided below.
[0156] In Table 1, human transcript sequences are provided. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0157] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0158] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the CCAT
peptide alone, the sequence encoding the mature peptide and
additional coding sequences, such as a leader or secretory sequence
(e.g., a pre-pro or pro-protein sequence), the sequence encoding
the mature peptide, with or without the additional coding
sequences, plus additional non-coding sequences, for example
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding and stability of mRNA. In addition, the nucleic
acid molecule may be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0159] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0160] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention as well
as nucleic acid molecules that encode obvious variants of the CCAT
proteins of the present invention that are described above. Such
nucleic acid molecules may be naturally occurring, such as allelic
variants (same locus), paralogs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to nucleic acid molecules, cells, or organisms.
Accordingly, as discussed above, the variants can contain
nucleotide substitutions, deletions, inversions and insertions.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[0161] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in Table 1. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence.
[0162] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0163] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive nucleotides.
[0164] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in Table 1 or a fragment of this
sequence. Such nucleic acid molecules can readily be identified as
being able to hybridize under moderate to stringent conditions, to
the nucleotide sequence shown in the Figure sheets or a fragment of
the sequence. Allelic variants can readily be determined by genetic
locus of the encoding gene.
[0165] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous 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 Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45 C, followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
Nucleic Acid Molecule Uses
[0166] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in Table 1 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in Table 1.
[0167] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in Table 1.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0168] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0169] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0170] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0171] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods.
[0172] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0173] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein. The nucleic acid
molecules are also useful for making vectors that express part, or
all, of the peptides.
[0174] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0175] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0176] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form and distribution
of nucleic acid expression. Experimental data as provided in Table
1 indicate that the CCAT of the present invention are overexpressed
in colon tumor tissue. Accordingly, the probes can be used to
detect the presence of, or to determine levels of, a specific
nucleic acid molecule in cells, tissues, and in organisms. The
nucleic acid whose level is determined can be DNA or RNA.
Accordingly, probes corresponding to the peptides described herein
can be used to assess expression and/or gene copy number in a given
cell, tissue, or organism. These uses are relevant for diagnosis of
disorders involving an increase or decrease in CCAT protein
expression relative to normal results.
[0177] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA include Southern hybridizations and in situ
hybridization.
[0178] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a CCAT protein, such as
by measuring a level of a CCAT-encoding nucleic acid in a sample of
cells from a subject e.g., mRNA or genomic DNA, or determining if a
CCAT gene has been mutated. Experimental data as provided in Table
1 indicate that the CCAT of the present invention are overexpressed
in colon tumor tissue. More detection and diagnosis methods are
described in detail below.
[0179] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate CCAT nucleic acid
expression.
[0180] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the CCAT gene, particularly biological
and pathological processes that are mediated by the CCAT in cells
and tissues that express it. Experimental data as provided in Table
1 indicates expression in human colon tumor tissues. The method
typically includes assaying the ability of the compound to modulate
the expression of the CCAT nucleic acid and thus identifying a
compound that can be used to treat a disorder characterized by
undesired CCAT nucleic acid expression. The assays can be performed
in cell-based and cell-free systems. Cell-based assays include
cells naturally expressing the CCAT nucleic acid or recombinant
cells genetically engineered to express specific nucleic acid
sequences.
[0181] The assay for CCAT nucleic acid expression can involve
direct assay of nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in the signal pathway. Further, the
expression of genes that are up- or down-regulated in response to
the CCAT protein signal pathway can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0182] Thus, modulators of CCAT gene expression can be identified
in a method wherein a cell is contacted with a candidate compound
or agent and the expression of mRNA determined. The level of
expression of CCAT mRNA in the presence of the candidate compound
or agent is compared to the level of expression of CCAT mRNA in the
absence of the candidate compound or agent. The candidate compound
can then be identified as a modulator of nucleic acid expression
based on this comparison and be used, for example to treat a
disorder characterized by aberrant nucleic acid expression. When
expression of mRNA is statistically significantly greater in the
presence of the candidate compound than in its absence, the
candidate compound is identified as a stimulator of nucleic acid
expression. When nucleic acid expression is statistically
significantly less in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of nucleic acid expression.
[0183] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound or an agent
identified through drug screening as a gene modulator to modulate
CCAT nucleic acid expression in cells and tissues that express the
CCAT. Experimental data as provided in Table 1 indicate that the
CCAT of the present invention are overexpressed in colon tumor
tissue. Modulation includes both up-regulation (i.e. activation or
agonization) or down-regulation (suppression or antagonization) or
nucleic acid expression.
[0184] Alternatively, a modulator for nucleic acid expression can
be a small molecule or drug identified using the screening assays
described herein as long as the drug or small molecule inhibits the
CCAT nucleic acid expression in the cells and tissues that express
the protein. Experimental data as provided in Table 1 indicates
expression in human colon tumor tissues.
[0185] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds or agents on the
expression or activity of the CCAT gene in clinical trials or in a
treatment regimen. Thus, the gene expression pattern can serve as a
barometer for the continuing effectiveness of treatment with the
compound, particularly with compounds to which a patient can
develop resistance. The gene expression pattern can also serve as a
marker indicative of a physiological response of the affected cells
to the compound. Accordingly, such monitoring would allow either
increased administration of the compound or the administration of
alternative compounds to which the patient has not become
resistant. Similarly, if the level of nucleic acid expression falls
below a desirable level, administration of the compound could be
commensurately decreased.
[0186] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in CCAT nucleic acid expression, and
particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in CCAT
genes and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally
occurring genetic mutations in the CCAT gene and thereby to
determine whether a subject with the mutation is at risk for a
disorder caused by the mutation. Mutations include deletion,
addition, or substitution of one or more nucleotides in the gene,
chromosomal rearrangement, such as inversion or transposition,
modification of genomic DNA, such as aberrant methylation patterns
or changes in gene copy number, such as amplification. Detection of
a mutated form of the CCAT gene associated with a dysfunction
provides a diagnostic tool for an active disease or susceptibility
to disease when the disease results from overexpression,
underexpression, or altered expression of a CCAT protein.
[0187] Individuals carrying mutations in the CCAT gene can be
detected at the nucleic acid level by a variety of techniques.
Genomic DNA can be analyzed directly or can be amplified by using
PCR prior to analysis. RNA or cDNA can be used in the same way. In
some uses, detection of the mutation involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.
Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,
or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,
Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et
al., PNAS 91:360-364 (1994)), the latter of which can be
particularly useful for detecting point mutations in the gene (see
Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method
can include the steps of collecting a sample of cells from a
patient, isolating nucleic acid (e.g., genomic, mRNA or both) from
the cells of the sample, contacting the nucleic acid sample with
one or more primers which specifically hybridize to a gene under
conditions such that hybridization and amplification of the gene
(if present) occurs, and detecting the presence or absence of an
amplification product, or detecting the size of the amplification
product and comparing the length to a control sample. Deletions and
insertions can be detected by a change in size of the amplified
product compared to the normal genotype. Point mutations can be
identified by hybridizing amplified DNA to normal RNA or antisense
DNA sequences.
[0188] Alternatively, mutations in a CCAT gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0189] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0190] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant CCAT gene and a wild-type gene can be determined
by direct DNA sequencing. A variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
(Naeve, C. W., (1995) Biotechniques 19:448), including sequencing
by mass spectrometry (see, e.g., PCT International Publication No.
WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and
Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).
[0191] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0192] The nucleic acid molecules are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the CCAT gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment.
[0193] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0194] The nucleic acid molecules are thus useful as antisense
constructs to control CCAT gene expression in cells, tissues, and
organisms. A DNA antisense nucleic acid molecule is designed to be
complementary to a region of the gene involved in transcription,
preventing transcription and hence production of CCAT protein. An
antisense RNA or DNA nucleic acid molecule would hybridize to the
mRNA and thus block translation of mRNA into CCAT protein.
[0195] The nucleic acid of the present invention may also be used
to specifically suppress gene expression by methods such as RNA
interference (RNAi), which may also include cosuppression and
quelling. This and antisense RNA or DNA of gene suppression are
well known in the art. A review of this technique is found in
Science 288:1370-1372, 2000. RNAi also operates on a
post-transcriptional level and is sequence specific, but suppresses
gene expression far more efficiently than antisense RNA. RNAi
fragments, particularly double-stranded (ds) RNAi, can be also used
to generate loss-of-function phenotypes.
[0196] The present invention relates to isolated RNA molecules
(double-stranded; single-stranded) of from about 21 to about 25
nucleotides which mediate RNAi. As used herein, about 21 to about
25 nt includes nucleotides 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28 and 29 nucleotides in length. The isolated RNAs of the
present invention mediate degradation of mRNA, the transcriptional
product of a gene. Such mRNA is also referred to herein as mRNA to
be degraded. As used herein, the terms RNA, RNA molecule(s), RNA
segment(s) and RNA fragment(s) are used interchangeably to refer to
RNA that mediates RNA interference. These terms include
double-stranded RNA, single-stranded RNA, isolated RNA (partially
purified RNA, essentially pure RNA, synthetic RNA, recombinantly
produced RNA), as well as altered RNA that differs from naturally
occurring RNA by the addition, deletion, substitution and/or
alteration of one or more nucleotides. Such alterations can include
addition of non-nucleotide material, such as to the end(s) of the
21-25 nt RNA or internally (at one or more nucleotides of the RNA).
Nucleotides in the RNA molecules of the present invention can also
comprise non-standard nucleotides, including non-naturally
occurring nucleotides or deoxyribonucleotides. Collectively, all
such altered RNAs are referred to as analogs or analogs of
naturally-occurring RNA. RNA of 21-25 nucleotides of the present
invention need only be sufficiently similar to natural RNA that it
has the ability to mediate RNAi. As used herein the phrase
"mediates RNAi" refers to the ability to distinguish which RNAs are
to be degraded by the RNAi machinery or process. RNA that mediates
RNAi interacts with the RNAi machinery such that it directs the
degradation of particular mRNAs. Such RNA may include RNAs of
various structure, including short hairpin RNA.
[0197] In one embodiment, the present invention relates to RNA
molecules of about 21 to about 25 nucleotides that direct cleavage
of specific mRNA to which their sequence corresponds. It is not
necessary that there be perfect correspondence of the sequences,
but the correspondence must be sufficient to enable the RNA to
direct RNAi cleavage of the target mRNA (Holen et al. (2005)
Nucleic Acids Res. 33, 4704-4710). In a particular embodiment, the
21-25 nt RNA molecules of the present invention comprise a 3'
hydroxyl group.
[0198] The present invention relates to 21-25 nt RNAs of specific
genes, produced by chemical synthesis or recombinant DNA
techniques, that mediate RNAi. As used herein, the term isolated
RNA includes RNA obtained by any means, including processing or
cleavage of dsRNA; production by chemical synthetic methods; and
production by recombinant DNA techniques. The invention further
relates to uses of the 21-25 nt RNAs, such as for therapeutic or
prophylactic treatment and compositions comprising 21-25 nt RNAs
that mediate RNAi, such as pharmaceutical compositions comprising
21-25 nt RNAs and an appropriate carrier.
[0199] The present invention also relates to a method of mediating
RNA interference of genes of a patient. In one embodiment, RNA of
about 21 to about 25 nt which targets the specific mRNA to be
degraded is introduced into a patient's cells. The cells are
maintained under conditions allowing degradation of the mRNA,
resulting in RNA-mediated interference of the mRNA of the gene in
the cells of the patient. Treatment of patients with cancer with
the RNAi will inhibit the growth and spread of the cancer and
reduce the tumor. Treatment of patients using RNAi can also be in
combination with other anti-cancer compounds. The RNAi may be used
in combination with other treatment modalities, such as
chemotherapy, cryotherapy, hyperthermia, radiation therapy, and
other similar treatments. In one embodiment, a chemotherapy agent
was combined with the RNAi. In another embodiment, a chemotherapy
named Gemzar was used.
[0200] Treatment of cancer or tumors in patients requires
introduction of the RNA into the cancer or tumor cells. RNA may be
directly introduced into the cell, or introduced extracellularly
into a cavity, interstitial space, into the circulation of a
patient, or introduced orally. Methods for oral introduction
include direct mixing of the RNA with food, as well as engineered
approaches in which a species that is used as food is engineered to
express the RNA and then ingested. Physical methods of introducing
nucleic acids, for example, injection directly into the cell or
extracellular injection into the patient, may also be used.
Vascular or extravascular circulation, the blood or lymph system,
and the cerebrospinal fluid are sites where the RNA may be
introduced. RNA may be introduced into an embryonic stem cell, or
another multipotent cell derived from the patient. Physical methods
of introducing nucleic acids include injection of a solution
containing the RNA, bombardment by particles covered by the RNA,
soaking cells or tissue in a solution of the RNA, or
electroporation of cell membranes in the presence of the RNA. A
viral construct packaged into a viral particle may be used to
introduce an expression construct into the cell, with the construct
expressing RNA. Other methods known in the art for introducing
nucleic acids to cells may be used, such as lipid-mediated carrier
transport, chemical-mediated transport, and the like. Thus the RNA
may be introduced along with components that perform one or more of
the following activities: enhance RNA uptake by the cell, promote
annealing of the duplex strands, stabilize the annealed strands, or
otherwise increase inhibition of the target gene. The RNAi may be
used in combination with other treatment modalities, such as
chemotherapy, cryotherapy, hyperthermia, radiation therapy, and the
like.
[0201] The present invention may be used alone or as a component of
a kit having at least one of the reagents necessary to carry out
the in vitro or in vivo introduction of RNA to tissue or patients.
Preferred components are the dsRNA and a vehicle that promotes
introduction of the dsRNA. Such a kit may also include instructions
to allow a user of the kit to practice the invention.
[0202] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of CCAT nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired CCAT nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the CCAT protein, such as substrate
binding.
[0203] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in CCAT gene
expression. Thus, recombinant cells, which include the patient's
cells that have been engineered ex vivo and returned to the
patient, are introduced into an individual where the cells produce
the desired CCAT protein to treat the individual.
[0204] The invention also encompasses kits for detecting the
presence of a CCAT nucleic acid in a biological sample.
Experimental data as provided in Table 1 indicate that the CCAT of
the present invention are overexpressed in colon tumor tissue. For
example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting CCAT nucleic
acid in a biological sample; means for determining the amount of
CCAT nucleic acid in the sample; and means for comparing the amount
of CCAT nucleic acid in the sample with a standard. The compound or
agent can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect CCAT protein mRNA
or DNA.
Vectors/Host Cells
[0205] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0206] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the nucleic acid molecules. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the nucleic acid molecules when the host cell
replicates.
[0207] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[0208] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
[0209] The regulatory sequences to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage, the lac, TRP, and TAC
promoters from E. coli, the early and late promoters from SV40, the
CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[0210] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0211] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 3rd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (2001).
[0212] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (2001).
[0213] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0214] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0215] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0216] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein; increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enteroenzyme. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), 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. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)). Recombinant protein
expression can be maximized in host bacteria by providing a genetic
background wherein the host cell has an impaired capacity to
proteolytically cleave the recombinant protein. (Gottesman, S.,
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, Calif. (1990) 119-128). Alternatively, the
sequence of the nucleic acid molecule of interest can be altered to
provide preferential codon usage for a specific host cell, for
example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118
(1992)).
[0217] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0218] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell. Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0219] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840 (1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0220] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the nucleic acid molecules described
herein. These are found for example in Sambrook, J., Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 3rd. ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(2001).
[0221] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0222] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0223] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(2001).
[0224] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the nucleic acid molecule
vector.
[0225] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0226] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0227] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0228] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as CCATs, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0229] Where the peptide is not secreted into the medium, which is
typically the case with CCATs, the protein can be isolated from the
host cell by standard disruption procedures, including freeze thaw,
sonication, mechanical disruption, use of lysing agents and the
like. The peptide can then be recovered and purified by well-known
purification methods including ammonium sulfate precipitation, acid
extraction, anion or cationic exchange chromatography,
phosphocellulose chromatography, hydrophobic-interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography, lectin chromatography, or high performance liquid
chromatography.
[0230] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
Uses of Vectors and Host Cells
[0231] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a CCAT protein or peptide that can be further purified to
produce desired amounts of CCAT protein or fragments. Thus, host
cells containing expression vectors are useful for peptide
production.
[0232] Host cells are also useful for conducting cell-based assays
involving the CCAT protein or CCAT protein fragments, such as those
described above as well as other formats known in the art. Thus, a
recombinant host cell expressing a native CCAT protein is useful
for assaying compounds that stimulate or inhibit CCAT protein
function.
[0233] Host cells are also useful for identifying CCAT protein
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant CCAT protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native CCAT protein.
[0234] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA which is integrated into
the genome of a cell from which a transgenic animal develops and
which remains in the genome of the mature animal in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a CCAT protein and identifying
and evaluating modulators of CCAT protein activity. Other examples
of transgenic animals include non-human primates, sheep, dogs,
cows, goats, chickens, and amphibians.
[0235] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the CCAT
protein nucleotide sequences can be introduced as a transgene into
the genome of a non-human animal, such as a mouse.
[0236] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the CCAT
protein to particular cells.
[0237] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0238] In another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0239] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter Go phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0240] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
effect substrate binding, CCAT protein activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo CCAT protein function,
including substrate interaction, the effect of specific mutant CCAT
proteins on CCAT protein function and substrate interaction, and
the effect of chimeric CCAT proteins. It is also possible to assess
the effect of null mutations, that is, mutations that substantially
or completely eliminate one or more CCAT protein functions.
Detection and Diagnosis
[0241] The present invention provides a method for detecting CCAT
nucleic acids, proteins, peptides and fragments thereof that are
differentially expressed in colon diseases in a test sample,
preferably in a biological sample.
[0242] The present invention further provides a method for
diagnosing the colon diseases, by detecting the nucleic acids,
proteins, peptides and fragments thereof. The further embodiment
includes but is not limited to, monitoring the disease prognosis
(recurrance), diagnosing disease stage, preventing the disease and
treating the disease.
[0243] As used herein, a "biological sample" can be collected from
tissues, blood, sera, cell lines or biological fluids such as,
plasma, interstitial fluid, urine, cerebrospinal fluid, and the
like, containing cells. In preferred embodiments, a biological
sample comprises cells or tissues suspected of having diseases
(e.g., cells obtained from a biopsy).
[0244] As used herein, a "differential level" is defined as the
level of CCAT protein or nucleic acids in a test sample either
above or below the level of the ones in control samples, wherein
the level of control samples is obtained either from a control cell
line, a normal tissue or body fluids, or combination thereof, from
a healthy subject.
[0245] As used herein, a "subject" can be a mammalian subject or
non mammalian subject, preferably, a mammalian subject. A mammalian
subject can be human or non-human, preferably human. A healthy
subject is defined as a subject without detectable colon diseases
or colon associated diseases by using conventional diagnostic
methods.
[0246] As used herein the "diseases" include colon diseases and
colon associated disease.
[0247] As used herein, "cancer" includes epithelial-cell related
cancers, for example pancreatic, lung, colon, prostate, ovarian,
breast, bladder renal, hepatocellular, pharyngeal and gastric
cancers.
Nucleic Acid Detections
[0248] The present invention is not limited to the detection
methods described above. Any suitable detection method that allows
for the specific detection of colon diseases cells, tissues or
organs may be utilized. For example, in some embodiments, the
expression of RNA corresponding to a CCAT gene is detected by
hybridization to an antisense oligonucleotide (described below). In
other embodiments, RNA expression is detected by hybridization
assays such as Northern blots, RNase assays, reverse transcriptase
PCR amplification, and the like. One preferred detection method is
using RT PCR by using TaqMan technology (ABI, Foster City,
Calif.).
[0249] In another embodiment, the present invention provides a
method for diagnosing or detecting colon diseases in a subject
comprising: determining the level of one or more CCAT nucleic acid
molecules or any fragment(s) thereof in a test sample from said
subject, wherein said CCAT nucleic acid molecule(s) comprises a
sequence selected from a group consisting of SEQ ID NOS:1234-2227
and a combination thereof;
[0250] wherein a differential level of said CCAT nucleic acid
molecule(s) relative to the level of said nucleic acid molecule(s)
in a test sample from a healthy subject, or the level established
for a healthy subject, is indicative of colon diseases.
[0251] In another embodiment, the detecting or diagnosing method
comprises determining level of differential expression of 2, 4, 8,
10, 20 or more nucleic acid molecules, preferably, the nucleic acid
molecules comprise or consists of a sequence selected from the
group consisting of SEQ ID NOS:1234-2227 and combination
thereof.
[0252] In further embodiments of the present invention, the
presence of particular sequences in the genome of a subject is
detected. Such sequences include CCAT sequences associated with
abnormal expression of CCAT (e.g., overexpression or expression at
a physiological inappropriate time). These sequences include
polymorphisms, including polymorphisms in the transcribed sequence
(e.g., that effect CCAT processing and/or translation) and
regulatory sequences such as promoters, enhances, repressors, and
the like. These sequences may also include polymorphisms in genes
or control sequences associated with factors that affect expression
such as transcription factors, and the like. Any suitable method
for detecting and/or identifying these sequences is within the
scope of the present invention including, but not limited to,
nucleic acid sequencing, hybridization assays (e.g., Southern
blotting), single nucleotide polymorphism assays (See e.g., U.S.
Pat. No. 5,994,069, herein incorporated by reference in its
entirety), and the like.
Protein Detections
[0253] The present invention provides methods for diagnosing or
detecting the differential presence of CCAT protein. In some
embodiments (e.g., where CCATs are overexpressed in diseased
cells), CCAT proteins are detected directly. In other embodiments
(e.g., where the presence of a CCATs are underexpressed), CCAT to
the disease antigens are detected non-existence.
[0254] The diagnostic methods of the present invention find utility
in the diagnosis and characterization of diseases. For example, the
presence of an antibody to a specific protein may be indicative of
a cancer or disease. In addition, certain CCAT may be indicative of
a specific stage or sub-type of the same cancer or disease.
[0255] The information obtained is also used to determine prognosis
and appropriate course of treatment. For example, it is
contemplated that individuals with a specific CCAT expression or
stage of colon diseases may respond differently to a given
treatment that individuals lacking the CCAT expression. The
information obtained from the diagnostic methods of the present
invention thus provides for the personalization of diagnosis and
treatment.
[0256] In one embodiment, the present invention provides a method
for monitoring colon diseases treatment in a subject comprising:
determining the level of one or more CCAT proteins or any
fragment(s) or peptide(s) thereof in a test sample from said
subject, wherein said CCAT protein(s) comprises a sequence selected
from a group consisting of SEQ ID NOS:1-1233, SEQ ID NOS:2228-2638
and a combination thereof; wherein an level of said CCAT protein(s)
similar to the level of said protein(s) in a test sample from a
healthy subject, or the level established for a healthy subject, is
indicative of successful treatment.
[0257] In another embodiment, the present invention provides a
method for diagnosing recurrence of colon diseases following
successful treatment in a subject comprising: determining the level
of one or more CCAT proteins or any fragment(s) or peptide(s)
thereof in a test sample from said subject, wherein said CCAT
protein(s) comprises a sequence selected from a group consisting of
SEQ ID NOS:1-1233, SEQ ID NOS:2228-2638 or a combination thereof;
wherein a changed level of said CCAT protein(s) relative to the
level of said protein(s) in a test sample from a healthy subject,
or the level established for a healthy subject, is indicative of
recurrence of colon diseases.
[0258] In yet another embodiment, the present invention provides a
method for diagnosing or detecting colon diseases in a subject
comprising: determining the level of one or more CCAT proteins or
any fragment(s) or peptides thereof in a test sample from said
subject, wherein said CCAT protein(s) comprises a sequence selected
from a group consisting of SEQ ID NOS:1-1233, SEQ ID NOS:2228-2638
and a combination thereof; wherein a differential level of said
CCAT protein(s) relative to the level of said protein(s) in a test
sample from a healthy subject, or the level established for a
healthy subject, is indicative of colon diseases.
[0259] The detecting or diagnosing method comprises determining
level of differential expression of 2, 4, 8, 10, 20 or more
proteins, preferably, the proteins are selected from a group
consisting of SEQ ID NOS:1-1233 and combination thereof.
[0260] Further, the detecting or diagnosing method comprises
determining level of differential expression of 5, 10, 15, 20, 40,
60, 80, 100 or more CCAT peptides, preferably the peptides are
selected from the group consisting of SEQ ID NOS:2228-2638 and
combination thereof.
[0261] These methods are also useful for diagnosing diseases that
show differential protein expression. As describe earlier, normal,
control or standard values or level established from a healthy
subject for protein expression are established by combining body
fluids or tissue, cell extracts taken from a normal healthy
mammalian or human subject with specific antibodies to a protein
under conditions for complex formation. Standard values for complex
formation in normal and diseased tissues are established by various
methods, often photometric means. Then complex formation as it is
expressed in a subject sample is compared with the standard values.
Deviation from the normal standard and toward the diseased standard
provides parameters for disease diagnosis or prognosis while
deviation away from the diseased and toward the normal standard may
be used to evaluate treatment efficacy.
[0262] In yet another embodiment, the present invention provides a
detection or diagnostic method of CCATs by using LC/MS. The
proteins from cells are prepared by methods known in the art (R.
Aebersold Nature Biotechnology Volume 21 Number 6 Jun. 2003). The
differential expression of proteins in disease and healthy samples
are quantitated using Mass Spectrometry and ICAT (Isotope Coded
Affinity Tag) labeling, which is known in the art. ICAT is an
isotope label technique that allows for discrimination between two
populations of proteins, such as a healthy and a disease sample.
The LC/MS spectra are collected for the labeled samples. The raw
scans from the LC/MS instrument are subjected to peak detection and
noise reduction software. Filtered peak lists are then used to
detect `features` corresponding to specific peptides from the
original sample(s). Features are characterized by their
mass/charge, charge, retention time, isotope pattern and
intensity.
[0263] The intensity of a peptide present in both healthy and
disease samples can be used to calculate the differential
expression, or relative abundance, of the peptide. The intensity of
a peptide found exclusively in one sample can be used to calculate
a theoretical expression ratio for that peptide (singleton).
Expression ratios are calculated for each peptide of each replicate
of the experiment (Table 1). Thus overexpression or under
expression of CCAT protein or peptide are similar to the expression
pattern in Table 1 in a test subject indicates the likelihood of
having colon diseases or diseases associated with colon.
[0264] Immunological methods for detecting and measuring complex
formation as a measure of protein expression using either specific
polyclonal or monoclonal antibodies are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELISAs), radioimmunoassays (RIAs), fluorescence-activated cell
sorting (FACS) and antibody arrays. Such immunoassays typically
involve the measurement of complex formation between the protein
and its specific antibody. These assays and their quantitation
against purified, labeled standards are well known in the art
(Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-based
immunoassay utilizing antibodies reactive to two non-interfering
epitopes is preferred, but a competitive binding assay may be
employed (Pound (1998) Immunochemical Protocols, Humana Press,
Totowa N.J.). More immunological detections are described in
section below.
Antibody Detections
[0265] Antibodies are useful to detect the presence of one of the
proteins or fragments thereof, peptides of the present invention in
cells or tissues to determine the pattern of expression of the
protein among various tissues in an organism and over the course of
normal development.
[0266] Further, as described above, the antibodies can be used to
assess expression in disease states such as in active stages of the
disease or in an individual with a predisposition toward disease
related to the protein's function. The antibodies can also be used
to assess normal and aberrant subcellular localization of cells in
the various tissues in an organism.
[0267] Detection on a protein by an antibody can be facilitated by
coupling (i.e., physically linking) the antibody to a detectable
substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials (see
below). The antibodies may also be useful in diagnostic assays,
e.g., for detecting expression of an antigen, for example CCAT
protein, peptide or fragment thereof, in specific cells, tissues,
blood, serum or body fluids.
[0268] For diagnostic applications, the antibody or its variant
typically will be labeled with a detectable moiety. Numerous labels
are available which can be generally grouped into the following
categories:
[0269] (a) Radioisotopes, such as .sup.36S, .sup.14C, .sup.125H,
and .sup.131I. The antibody variant can be labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, vol 1-2, Coligen et al., Ed., Wiley-Interscience, New
York, Pubs. (1991) for example and radioactivity can be measured
using scintillation counting.
[0270] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
variant using the techniques disclosed in Current Protocols in
Immunology, supra, for example. Fluorescence can be quantified
using a fluorimeter.
[0271] (c) Various enzyme-substrate labels are available and U.S.
Pat. Nos. 4,275,149, 4,318,980 provides a review of some of these.
The enzyme generally catalyzes a chemical alteration of the
chromogenic substrate which can be measured using various
techniques. For example, the enzyme may catalyze a color change in
a substrate, which can be measured spectrophotometrically.
Alternatively, the enzyme may alter the fluorescence or
chemiluminescence of the substrate. Techniques for quantifying a
change in fluorescence are described above. The chemiluminescent
substrate becomes electronically excited by a chemical reaction and
may then emit light which can be measured (using a
chemiluminometer, for example) or donates energy to a fluorescent
acceptor. Examples of enzymatic labels include luciferases (e.g.,
firefly luciferase and bacterial luciferase; U.S. Pat. No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate
dehydrogenase, urease, peroxidase such as horseradish peroxidase
(HRPO), alkaline phosphatase, .beta.-galactosidase, glucoamylase,
lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose
oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic
oxidases (such as uricase and xanthine oxidase), lactoperoxidase,
microperoxidase, and the like. Techniques for conjugating enzymes
to antibodies are described in O'Sullivan et al., Methods for the
Preparation of Enzyme-Antibody Conjugates for Use in Enzyme
Immunoassay, in Methods in Enzyme. (Ed. J. Langone & H. Van
Vunakis), Academic press, New York, 73: 147-166 (1981).
[0272] Sometimes, the label is indirectly conjugated with the
antibody. The skilled artisan will be aware of various techniques
for achieving this. For example, the antibody can be conjugated
with biotin and any of the three broad categories of labels
mentioned above can be conjugated with avidin, or vice versa.
Biotin binds selectively to avidin and thus, the label can be
conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.
digloxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g. anti-digloxin
antibody). Thus, indirect conjugation of the label with the
antibody can be achieved.
[0273] In another embodiment of the invention, the antibody need
not be labeled, and the presence thereof can be detected using a
labeled antibody, which binds to the antibody.
[0274] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0275] The biological samples can then be tested directly for the
presence of CCAT by assays (e.g., ELISA or radioimmunoassay) and
format (e.g., microwells, dipstick (e.g., as described in
International Patent Publication WO 93/03367), etc). Alternatively,
proteins in the sample can be size separated (e.g., by
polyacrylamide gel electrophoresis (PAGE)), in the presence or
absence of sodium dodecyl sulfate (SDS), and the presence of CCAT
detected by immunoblotting (e.g., Western blotting). Immunoblotting
techniques are generally more effective with antibodies generated
against a peptide corresponding to an epitope of a protein, and
hence, are particularly suited to the present invention.
[0276] Antibody binding is detected by techniques known in the art
(e.g., radioimmunoas say, ELISA (enzyme-linked immunosorbant
assay), "sandwich" immunoassays, immunoradiometric assays, gel
diffusion precipitation reactions, immunodiffusion assays, in situ
immunoassays (e.g., using colloidal gold, enzyme or radioisotope
labels, for example), Western blots, precipitation reactions,
agglutination assays (e.g., gel agglutination assays,
hemagglutination assays, etc.), complement fixation assays,
immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
[0277] In one embodiment, antibody binding is detected by detecting
a label on the primary antibody. In another embodiment, the primary
antibody is detected by detecting binding of a secondary antibody
or reagent to the primary antibody. In a further embodiment, the
secondary antibody is labeled. Many means are known in the art for
detecting binding in an immunoassay and are within the scope of the
present invention. As is well known in the art, the immunogenic
peptide should be provided free of the carrier molecule used in any
immunization protocol. For example, if the peptide was conjugated
to KLH, it may be conjugated to BSA, or used directly, in a
screening assay. In some embodiments, an automated detection assay
is utilized. Methods for the automation of immunoassays are well
known in the art (See e.g., U.S. Pat. Nos. 5,885,530, 4,981,785,
6,159,750, and 5,358,691, each of which is herein incorporated by
reference). In some embodiments, the analysis and presentation of
results is also automated. For example, in some embodiments,
software that generates a prognosis based on the presence or
absence of a series of antigens is utilized.
[0278] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample for binding with a limited
amount of antibody. The amount of antigen in the test sample is
inversely proportional to the amount of standard that becomes bound
to the antibodies. To facilitate determining the amount of standard
that becomes bound, the antibodies generally are insolubilized
before or after the competition. As a result, the standard and test
sample that are bound to the antibodies may conveniently be
separated from the standard and test sample, which remain
unbound.
[0279] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
or the protein to be detected. In a sandwich assay, the test sample
to be analyzed is bound by a first antibody, which is immobilized
on a solid support, and thereafter a second antibody binds to the
test sample, thus forming an insoluble three-part complex. See
e.g., U.S. Pat. No. 4,376,110. The second antibody may itself be
labeled with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0280] The antibodies may also be used for in vivo diagnostic
assays. Generally, the antibody is labeled with a radionucleotide
(such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.3H,
.sup.32P or .sup.35S) so that the tumor can be localized using
immunoscintiography. In one embodiment, antibodies or fragments
thereof bind to the extracellular domains of two or more CCAT
targets and the affinity value (Kd) is less than 1.times.10.sup.8
M.
[0281] Antibodies for diagnostic use may be labeled with probes
suitable for detection by various imaging methods. Methods for
detection of probes include, but are not limited to, fluorescence,
light, confocal and electron microscopy; magnetic resonance imaging
and spectroscopy; fluoroscopy, computed tomography and positron
emission tomography. Suitable probes include, but are not limited
to, fluorescein, rhodamine, eosin and other fluorophores,
radioisotopes, gold, gadolinium and other lanthanides, paramagnetic
iron, fluorine-18 and other positron-emitting radionuclides.
Additionally, probes may be bi- or multi-functional and be
detectable by more than one of the methods listed. These antibodies
may be directly or indirectly labeled with said probes. Attachment
of probes to the antibodies includes covalent attachment of the
probe, incorporation of the probe into the antibody, and the
covalent attachment of a chelating compound for binding of probe,
amongst others well recognized in the art.
[0282] For immunohistochemistry, the disease tissue sample may be
fresh or frozen or may be embedded in paraffin and fixed with a
preservative such as formalin (see Example). The fixed or embedded
section contains the sample are contacted with a labeled primary
antibody and secondary antibody, wherein the antibody is used to
detect the CCAT protein express in situ. The detailed procedure is
shown in the Example.
Array:
[0283] Array technologies and quantitative PCR provide the means to
explore the expression profiles of a large number of related or
unrelated genes, and proteins. When an expression profile is
examined, arrays provide a platform for examining which genes or
proteins are tissue-specific, carrying out housekeeping functions,
parts of a signaling cascade, or specifically related to a
particular genetic predisposition, condition, disease, or disorder.
The potential application of gene or protein expression profiling
is particularly relevant to improving diagnosis, prognosis, and
treatment of disease. For example, both the sequences and the
amount of expression can be compared between tissues from subjects
with different types of colon diseases and cytologically normal
healthy tissue.
[0284] "Array" refers to an ordered arrangement of at least two
transcripts, proteins or peptides, or antibodies on a substrate. At
least one of the transcripts, proteins, or antibodies represents a
control or standard, and the other transcript, protein, or antibody
is of diagnostic or therapeutic interest. The arrangement of at
least two and up to about 40,000 transcripts, proteins, or
antibodies on the substrate assures that the size and signal
intensity of each labeled complex, formed between each transcript
and at least one nucleic acid, each protein and at least one ligand
or antibody, or each antibody and at least one protein to which the
antibody specifically binds, is individually distinguishable.
[0285] An "expression profile" is a representation of gene
expression in a sample. A nucleic acid expression profile is
produced using sequencing, hybridization, or amplification
technologies using transcripts from a sample. A protein expression
profile, although time delayed, minors the nucleic acid expression
profile and is produced using gel electrophoresis, mass
spectrometry, or an array and labeling moieties or antibodies which
specifically bind the protein. The nucleic acids, proteins, or
antibodies specifically binding the protein may be used in solution
or attached to a substrate, and their detection is based on methods
well known in the art.
[0286] A substrate includes but not limits to, paper, nylon or
other type of membrane, filter, chip, glass slide, or any other
suitable solid support.
[0287] The invention also provides an array with a cDNA or
transcript encoding CCAT proteins or peptides or fragments thereof,
antibodies which specifically bind CCAT proteins, peptides or
fragments thereof. Preferably, two or more of the nucleic acid
molecules (e.g., SEQ ID NOS:1234-2227), proteins (e.g., SEQ ID
NOS:1-1233) or peptides (e.g., SEQ ID NOS:2228-2638) are
immobilized on a substrate.
[0288] The present invention also provides an antibody array.
Antibody arrays have allowed the development of techniques for
high-throughput screening of recombinant antibodies. Such methods
use robots to pick and grid bacteria containing antibody genes, and
a filter-based ELISA to screen and identify clones that express
antibody fragments. Because liquid handling is eliminated and the
clones are arrayed from master stocks, the same antibodies can be
spotted multiple times and screened against multiple antigens
simultaneously. For more information, see de Wildt et al. (2000)
Nat Biotechnol 18:989-94.
[0289] The array is prepared and used according to the methods
described in U.S. Pat. No. 5,837,832, Chee et al., PCT application
WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat.
Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl.
Acad. Sci. 93: 10614-10619), U.S. Pat. No. 5,807,522, Brown et al.,
all of which are incorporated herein in their entirety by
reference.
[0290] In one embodiment, a nucleic acid array or a microarray,
preferably composed of a large number of unique, single-stranded
nucleic acid sequences, usually either synthetic antisense
oligonucleotides or fragments of cDNAs, fixed to a solid support.
The oligonucleotides are preferably about 6-60 nucleotides in
length, more preferably 15-30 nucleotides in length, and most
preferably about 20-25 nucleotides in length.
[0291] In order to produce oligonucleotides to a known sequence for
an array, the gene(s) of interest (or an ORF identified from the
contigs of the present invention) is typically examined using a
computer algorithm which starts at the 5' or at the 3' end of the
nucleotide sequence. Typical algorithms will then identify
oligomers of defined length that are unique to the gene, have a GC
content within a range suitable for hybridization, and lack
predicted secondary structure that may interfere with
hybridization. In certain situations it may be appropriate to use
pairs of oligonucleotides on an array. The "pairs" will be
identical, except for one nucleotide that preferably is located in
the center of the sequence. The second oligonucleotide in the pair
(mismatched by one) serves as a control. The number of
oligonucleotide pairs may range from two to one million. The
oligomers are synthesized at designated areas on a substrate using
a light-directed chemical process, wherein the substrate may be
paper, nylon or other type of membrane, filter, chip, glass slide
or any other suitable solid support as described above.
[0292] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference.
[0293] A gene expression profile comprises the expression of a
plurality of transcripts as measured by after hybridization with a
sample. The transcripts of the invention may be used as elements on
an array to produce a gene expression profile. In one embodiment,
the array is used to diagnose or monitor the progression of
disease. Researchers can assess and catalog the differences in gene
expression between healthy and diseased tissues or cells.
[0294] For example, the transcript or probe may be labeled by
standard methods and added to a biological sample from a patient
under conditions for the formation of hybridization complexes.
After an incubation period, the sample is washed and the amount of
label (or signal) associated with hybridization complexes, is
quantified and compared with a standard value. If complex formation
in the patient sample is significantly altered (higher or lower) in
comparison to either a normal or disease standard, then
differential expression indicates the presence of a disorder.
[0295] In order to provide standards for establishing differential
expression, normal and disease expression profiles are established.
This is accomplished by combining a sample taken from normal
subjects, either animal or human or nonmammal, with a transcript
under conditions for hybridization to occur. Standard hybridization
complexes may be quantified by comparing the values obtained using
normal subjects with values from an experiment in which a known
amount of a purified sequence is used. Standard values obtained in
this manner may be compared with values obtained from samples from
patients who were diagnosed with a particular condition, disease,
or disorder. Deviation from standard values toward those associated
with a particular disorder is used to diagnose that disorder.
[0296] By analyzing changes in patterns of gene expression, disease
can be diagnosed at earlier stages before the patient is
symptomatic. The invention can be used to formulate a prognosis and
to design a treatment regimen. The invention can also be used to
monitor the efficacy of treatment. For treatments with known side
effects, the array is employed to improve the treatment regimen. A
dosage is established that causes a change in genetic expression
patterns indicative of successful treatment. Expression patterns
associated with the onset of undesirable side effects are
avoided.
[0297] In another embodiment, animal models which mimic a human
disease can be used to characterize expression profiles associated
with a particular condition, disease, or disorder; or treatment of
the condition, disease, or disorder. Novel treatment regimens may
be tested in these animal models using arrays to establish and then
follow expression profiles over time. In addition, arrays may be
used with cell cultures or tissues removed from animal models to
rapidly screen large numbers of candidate drug molecules, looking
for ones that produce an expression profile similar to those of
known therapeutic drugs, with the expectation that molecules with
the same expression profile will likely have similar therapeutic
effects. Thus, the invention provides the means to rapidly
determine the molecular mode of action of a drug.
[0298] Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies or in
clinical trials or to monitor the treatment of an individual
patient. Once the presence of a condition is established and a
treatment protocol is initiated, diagnostic assays may be repeated
on a regular basis to determine if the level of expression in the
patient begins to approximate that which is observed in a normal
subject. The results obtained from successive assays may be used to
show the efficacy of treatment over a period ranging from several
days to years.
Treatment
[0299] The following terms, as used in the present specification
and claims, are intended to have the meaning as defined below,
unless indicated otherwise.
[0300] "Treat," "treating" or "treatment" of a disease
includes:
(1) inhibiting the disease, i.e., arresting or reducing the
development of the disease or its clinical symptoms, or (2)
relieving the disease, i.e., causing regression of the disease or
its clinical symptoms.
[0301] A "therapeutically effective amount" means the amount of an
agent that, when administered to a subject for treating a disease,
is sufficient to effect such treatment for the disease. The
"therapeutically effective amount" will vary depending on the
agent, the disease and its severity and the age, weight, etc., of
the subject to be treated.
[0302] A "Colon or colorectal disease" includes but not limited to
colon cancer, colon tumor, diverticulosis, diverticulitis, Crohn's
disease, ulcerative colitis, irritable bowel syndrome, inflammatory
bowel disease, hemorrhoids, and anal fissure.
[0303] A "cancer" is epithelial-cell related cancers include but
not limited to pancreatic, lung, colon, prostate, ovarian, breast,
bladder renal, hepatocellular, pharyngeal and gastric cancer.
[0304] The present invention provides an application of treatment
by using antibody, immunogenic peptides as well as other CCAT
agonists or antagonists.
[0305] CCATs are proteins differentially expressed in the colon
diseases cell lines or tissues. The proteins are either cell
surface proteins, cytosolic proteins, or secreted proteins (see the
list in Table 1). These proteins are associated with the diseases
especially colon diseases, particularly colon cancer; thus, they
serve as candidate targets for the treatment of the diseases.
[0306] In one embodiment, when decreased expression or activity of
the protein is desired, an inhibitor, antagonist, antibody and the
like or a pharmaceutical agent containing one or more of these
molecules may be delivered. Such delivery may be effected by
methods well known in the art and may include delivery by an
antibody specifically targeted to the protein. Neutralizing
antibodies, which inhibit dimer formation, are generally preferred
for therapeutic use.
[0307] In another embodiment, when increased expression or activity
of the protein is desired, the protein, an agonist, an enhancer and
the like or a pharmaceutical agent containing one or more of these
molecules may be delivered. Such delivery may be effected by
methods well known in the art and may include delivery of a
pharmaceutical agent by an antibody specifically targeted to the
protein.
[0308] Any of the transcripts, complementary molecules, or
fragments thereof, proteins or portions thereof, vectors delivering
these nucleic acid molecules or expressing the proteins, and their
ligands may be administered in combination with other therapeutic
agents. Selection of the agents for use in combination therapy may
be made by one of ordinary skill in the art according to
conventional pharmaceutical principles. A combination of
therapeutic agents may act synergistically to affect treatment of a
particular disorder at a lower dosage of each agent.
Antibody Therapy
[0309] The antibody of the present invention can be used for
therapeutic reason. It is contemplated that the antibody of the
present invention may be used to treat a mammal, preferably human
with colon diseases.
[0310] In general, the antibodies are also useful for inhibiting
protein function, for example, blocking the binding of the CCAT
protein or peptide to a binding partner such as a substrate. These
uses can also be applied in a therapeutic context in which
treatment involves inhibiting the protein's function. An antibody
can be used, for example, to block binding, thus modulating
(agonizing or antagonizing) the peptides activity. Antibodies can
be prepared against specific fragments containing sites required
for function or against intact protein that is associated within a
cell or cell membrane. The function blocking assays are provided in
detail in the Examples.
[0311] The antibodies of present invention can also be used as
means of enhancing the immune response. The antibodies can be
administered in amounts similar to those used for other therapeutic
administrations of antibody. For example, pooled gamma globulin is
administered at a range of about 1 mg to about 100 mg per patient.
Thus, antibodies reactive with the protein or peptides of CCAT can
be passively administered alone or in conjunction with other
anti-cancer therapies to a mammal afflicted--with colon diseases or
cancer. Examples of anti-cancer therapies include, but are not
limited to, chemotherapy, radiation therapy, adoptive immunotherapy
therapy with TIL (Tumor Infiltration Lymphocytes).
[0312] The selection of an antibody subclass for therapy will
depend upon the nature of the disease tumor antigen. For example,
an IgM may be preferred in situations where the antigen is highly
specific for the diseased target and rarely occurs on normal cells.
However, where the disease-associated antigen is also expressed in
normal tissues, although at much lower levels, the IgG subclass may
be preferred for the following reason: since the binding of at
least two IgG molecules in close proximity is required to activate
complement, less complement mediated damage may occur in the normal
tissues which express smaller amounts of the antigen and,
therefore, bind fewer IgG antibody molecules. Furthermore, IgG
molecules by being smaller may be more able than IgM molecules to
localize to the diseased tissue.
[0313] The mechanism for antibody therapy is that the therapeutic
antibody recognizes a cell surface protein, cytosolic protein, or a
secreted protein that is overexpressed in diseased cells. By NK
cell or complement activation, conjugation of the antibody with an
immunotoxin or radiolabel, the interaction can abrogate
ligand/receptor interaction or activation of apoptosis.
[0314] The potential mechanisms of antibody-mediated cytotoxicity
of diseased cells are phagocyte (antibody dependent cellular
cytotoxicity (ADCC)) (see Example), complement (Complement-mediated
cytotoxicity (CMC)) (see Example), naked antibody (receptor
cross-linking apoptosis and growth factor inhibition), or targeted
payload labeled with radionuclide or immunotoxins or
immunochemotherapeutics.
[0315] In one embodiment, the antibody is administered to a
nonhuman mammal for the purposes of obtaining preclinical data, for
example. Exemplary nonhuman mammals to be treated include nonhuman
primates, dogs, cats, rodents and other mammals in which
preclinical studies are performed. Such mammals may be established
animal models for a disease to be treated with the antibody or may
be used to study toxicity of the antibody of interest. In each of
these embodiments, dose escalation studies may be performed on the
mammal.
[0316] The antibody is administered by any suitable means,
including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
addition, the antibody variant is suitably administered by pulse
infusion, particularly with declining doses of the antibody
variant. Preferably the dosing is given by injections, most
preferably intravenous or subcutaneous injections, depending in
part on whether the administration is brief or chronic.
[0317] For the prevention or treatment of a disease, the
appropriate dosage of the antibody will depend on the type of
disease to be treated, the severity and the course of the disease,
whether the antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician.
[0318] Depending on the type and severity of the disease, about 1
.mu.g/kg to 150 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. A typical daily dosage might range from
about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment is sustained
until a desired suppression of disease symptoms occurs. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0319] The antibody composition will be formulated, dosed and
administered in a manner consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners.
[0320] The therapeutically effective amount of the antibody to be
administered will be governed by such considerations, and is the
minimum amount necessary to prevent, ameliorate, or treat a disease
or disorder. The antibody need not be, but is optionally formulated
with one or more agents currently used to prevent or treat the
disorder in question.
[0321] Antibodies of the present invention may also be used as
therapeutic reagents, to diminish or eliminate cancer or tumors.
For example, the antibodies may be used on their own (for instance,
to inhibit metastases) or coupled to one or more therapeutic
agents. Suitable agents in this regard include radionuclides,
differentiation inducers, drugs, toxins, and derivatives thereof.
Preferred radionuclides include .sup.90Y, .sup.123I, .sup.125I,
.sup.131I, .sup.186Re, .sup.188Re, .sup.211At, and .sup.212Bi.
Preferred drugs include methotrexate, and pyrimidine and purine
analogs. Preferred differentiation inducers include phorbol esters
and butyric acid. Preferred toxins include ricin, abrin, diptheria
toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella
toxin, and pokeweed antiviral protein
[0322] A therapeutic agent may be coupled (e.g., covalently bonded)
to a suitable monoclonal antibody either directly or indirectly
(e.g., via a linker group). A direct reaction between an agent and
an antibody is possible when each possesses a substituent capable
of reacting with the other. For example, a nucleophilic group, such
as an amino or sulfhydryl group, on one may be capable of reacting
with a carbonyl-containing group, such as an anhydride or an acid
halide, or with an alkyl group containing a good leaving group
(e.g., a halide) on the other.
[0323] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0324] It will be evident to those skilled in the art that a
variety of bifunctional or polyfunctional reagents, both homo- and
hetero-functional (such as those described in the catalog of the
Pierce Chemical Co., Rockford, Ill.), may be employed as the linker
group. Coupling may be affected, for example, through amino groups,
carboxyl groups, sulfhydryl groups or oxidized carbohydrate
residues. There are numerous references describing such
methodology, e.g. U.S. Pat. No. 4,671,958, to Rodwell et al.
[0325] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,
U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,
to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,
U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).
[0326] It may be desirable to couple more than one agent to an
antibody. In one embodiment, multiple molecules of an agent are
coupled to one antibody molecule. In another embodiment, more than
one type of agent may be coupled to one antibody. Regardless of the
particular embodiment, immunoconjugates with more than one agent
may be prepared in a variety of ways as described above.
[0327] While it is possible for the immunogen to be administered in
a pure or substantially pure form, it is preferable to present it
as a pharmaceutical composition, formulation or preparation with a
carrier.
[0328] The formulations of the present invention, both for
veterinary and for human use, comprise an immunogen as described
above, together with one or more pharmaceutically acceptable
carriers and, optionally, other therapeutic ingredients. The
carrier(s) must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not deleterious
to the recipient thereof. The formulations may conveniently be
presented in unit dosage form and may be prepared by any method
well-known in the pharmaceutical art.
[0329] Suitable pharmaceutical carriers include proteins such as
albumins (e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides
and polysaccharides such as aminodextran (e.g., U.S. Pat. No.
4,699,784, to Shih et al.), or water. A carrier may also bear an
agent by noncovalent bonding or by encapsulation, such as within a
liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088).
Carriers specific for radionuclide agents include radiohalogenated
small molecules and chelating compounds. For example, U.S. Pat. No.
4,735,792 discloses representative radiohalogenated small molecules
and their synthesis. A radionuclide chelate may be formed from
chelating compounds that include those containing nitrogen and
sulfur atoms as the donor atoms for binding the metal, metal oxide,
radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et
al. discloses representative chelating compounds and their
synthesis.
[0330] All methods include the step of bringing into association
the active ingredient with the carrier, which constitutes one or
more accessory ingredients. In general, the formulations are
prepared by uniformly and intimately bringing into association the
active ingredient with liquid carriers or finely divided solid
carriers or both, and then, if necessary, shaping the product into
the desired formulation.
[0331] Formulations suitable for intravenous intramuscular,
subcutaneous, or intraperitoneal administration conveniently
comprise sterile aqueous solutions of the active ingredient with
solutions, which are preferably isotonic with the blood of the
recipient. Such formulations may be conveniently prepared by
dissolving solid active ingredient in water containing
physiologically compatible substances such as sodium chloride (e.g.
0.1-2.0M), glycine, and the like, and having a buffered pH
compatible with physiological conditions to produce an aqueous
solution, and rendering said solution sterile. These may be present
in unit or multi-dose containers, for example, sealed ampoules or
vials.
[0332] The formulations of the present invention may incorporate a
stabilizer. Illustrative stabilizers are polyethylene glycol,
proteins, saccharides, amino acids, inorganic acids, and organic
acids, which may be used either on their own or as admixtures.
These stabilizers are preferably incorporated in an amount of
0.11-10,000 parts by weight per part by weight of immunogen. If two
or more stabilizers are to be used, their total amount is
preferably within the range specified above. These stabilizers are
used in aqueous solutions at the appropriate concentration and pH.
The specific osmotic pressure of such aqueous solutions is
generally in the range of 0.1-3.0 osmoles, preferably in the range
of 0.8-1.2. The pH of the aqueous solution is adjusted to be within
the range of 5.0-9.0, preferably within the range of 6-8. In
formulating the antibody of the present invention, anti-adsorption
agent may be used.
[0333] Additional pharmaceutical methods may be employed to control
the duration of action. Controlled release preparations may be
achieved through the use of polymer to complex or absorb the
proteins or their derivatives. The controlled delivery may be
exercised by selecting appropriate macromolecules (for example
polyester, polyamino acids, polyvinyl, pyrrolidone,
ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or
protamine sulfate) and the concentration of macromolecules as well
as the methods of incorporation in order to control release.
Another possible method to control the duration of action by
controlled-release preparations is to incorporate the CCAT antibody
into particles of a polymeric material such as polyesters,
polyamino acids, hydrogels, poly(lactic acid) or ethylene
vinylacetate copolymers. Alternatively, instead of incorporating
these agents into polymeric particles, it is possible to entrap
these materials in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly(methylmethacylate) microcapsules, respectively, or in
colloidal drug delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and nanocapsules or in
macroemulsions.
[0334] When oral preparations are desired, the compositions may be
combined with typical carriers, such as lactose, sucrose, starch,
talc magnesium stearate, crystalline cellulose, methyl cellulose,
carboxymethyl cellulose, glycerin, sodium alginate or gum arabic
among others.
[0335] The therapeutic antibody may be supplied in the form of a
kit, alone, or in the form of a pharmaceutical composition as
described above.
Other Immunotherapy
[0336] The CCAT proteins or peptides or fragments thereof of this
invention are also intended for use in producing antiserum designed
for pre- or post-disease prophylaxis. Here the protein, peptides or
fragment thereof, is formulated with a suitable adjuvant and
administered by injection to human volunteers, according to known
methods for producing human antisera. Antibody response to the
injected proteins is monitored, during a several-week period
following immunization, by periodic serum sampling to detect the
presence of antiserum antibodies, using an immunoassay as described
herein.
[0337] The antiserum from immunized individuals may be administered
as a prophylactic measure for individuals who are at risk of
developing colon diseases or cancer. The antiserum is also useful
in treating an individual afflicted with colon diseases or cancer
for post-disease prophylaxis.
[0338] Alternatively, peptides derived form the CCAT protein
sequence may be modified to increase their immunogenicity by
enhancing binding of the peptide to the MHC molecules in which the
peptide is presented. The peptide or modified peptide may be
conjugated to a carrier molecule to enhance the antigenicity of the
peptide. Examples of carrier molecules, include, but are not
limited to, human albumin, bovine albumin, lipoprotein and keyhole
limpet hemo-cyanin ("Basic and Clinical Immunology" (1991) Stites,
D. P. and Ten A. I. (eds) Appleton and Lange, Norwalk Conn., San
Mateo, Calif.).
[0339] An "immunogenic peptide" is a peptide, which comprises an
allele-specific motif such that the peptide will bind the MHC
allele (HLA in human) and be capable of inducing a CTL (cytoxic
T-lymphocytes) response. Thus, immunogenic peptides are capable of
binding to an appropriate class I or II MHC molecule and inducing a
cytotoxic T cell or T helper cell response against the antigen from
which the immunogenic peptide is derived.
[0340] Alternatively, amino acid sequence variants of the peptide
can be prepared by mutations in the DNA, which encodes the peptide,
or by peptide synthesis.
[0341] At the genetic level, these variants ordinarily are prepared
by site-directed mutagenesis of nucleotides in the DNA encoding the
peptide molecule, thereby producing DNA encoding the variant, and
thereafter expressing the DNA in recombinant cell culture. The
variants typically exhibit the same qualitative biological activity
as the nonvariant peptide.
[0342] T-lymphocytes recognize antigen in association with Class I
or Class II MHC molecules in the form of a peptide fragment bound
to an MHC molecule. The degree of peptide binding to a given MHC
allele is based on amino acids at particular positions within the
peptide (Parker et al. (1992) Journal of Immunology 149:3580; Kubo,
et al. (1994) Journal of Immunology 52:3913-3924; Ruppert J. et al.
(1993) Cell 74:929-937; Falk et al. (1991) Nature 351:290-296). The
peptides of the present invention are useful as an epitope for
immunogenic response (see more detailed description below).
[0343] In human, MHC is called HLA, wherein class I molecules are
encoded by the HLA-A, B, and C loci. HLA-A and B antigens are
expressed at the cell surface at approximately equal densities,
whereas the expression of HLA-C is significantly lower (about
10-fold lower). Each of these loci has a number of alleles. MHC
class II molecules are encoded by three pairs of MHC II alpha- and
beta-chain genes, called HLA DR, -DP, and -DQ in human. In many
haplotypes the HLA-DR cluster contains an extra beta-chain gene
whose product can pair with the DR alpha chain. Each MHC class I
and II molecule binds a different rage of peptides. The present of
several loci means that any one individual is equipped to present a
much broader ranger of different peptides than if only one MHC
protein of each class were expressed at the cell surface. The
peptide binding motifs of the present invention are designed to be
specific for each allelic subtype.
[0344] The peptides of the present invention are used for treatment
of the colon diseases. Treatment involves administration of the
protective composition after the appearance of the disease.
[0345] The present invention is also applied to prevent and
suppress the disease. It is not always possible to distinguish
between "preventing" and "suppressing" since the ultimate inductive
event or events may be unknown, latent, or the patient is not
ascertained until well after the occurrence of the event or events.
Therefore, it is common to use the term "prophylaxis" as distinct
from "treatment" to encompass both "preventing" and "suppressing"
as defined herein. The term "protection," as used herein, is meant
to include "prophylaxis."
[0346] The peptides are used for treating T cell-mediated
pathology. The term "T cell-mediated pathology" refers to any
condition in which an inappropriate T cell response is a component
of the pathology. The term is intended to encompass both T cell
mediated colon diseases and diseases resulting from unregulated
clonal T cell replication.
[0347] Therefore, the present invention relates to peptides or
modified peptides derived from the protein sequences of the CCAT
proteins that differentially expressed in the colon diseases. By
way of example, modification may include substitution, deletion or
addition of an amino acid in the given immunogenic peptide sequence
or mutation of existing amino acids within the given immunogenic
peptide sequence, or derivatization of existing amino acids within
the given immunogenic peptide sequence. Any amino acid comprising
the immunogenic peptide sequence may be modified in accordance with
this invention. In one aspect, at least one amino acid is
substituted or replaced within the given immunogenic peptide
sequence. Any amino acid may be used to substitute or replace a
given amino acid within the immunogenic peptide sequence. Modified
peptides are intended to include any immunogenic peptide obtained
from differentially expressed proteins, which has been modified and
exhibits enhanced binding to the MHC molecule with which it
associates when presented to the T-cell. These modified peptides
may be synthetically or recombinantly produced by conventional
methods.
[0348] In another embodiment, the peptides of the present invention
comprise, or consisting sequences of about 5-8, 8-10, 10-15 or
15-30 amino acids which are immunogenic, that is, capable of
inducing an immune response when injected into a subject.
[0349] The recombinant or natural protein, peptides, or fragment
thereof of CCAT, or modified peptides, may be used as a vaccine
either prophylactically or therapeutically. When provided
prophylactically the vaccine is provided in advance of any evidence
of colon diseases, particularly, cancer. The prophylactic
administration of the colon diseases vaccine should serve to
prevent or attenuate colon diseases, preferably cancer, in a
mammal.
[0350] Preparation of vaccine is using recombinant protein or
peptide expression vectors comprising all or part of nucleic acid
sequence of CCAT proteins encoding peptides. Examples of vectors
that may be used in the aforementioned vaccines include, but are
not limited to, defective retroviral vectors, adenoviral vectors
vaccinia viral vectors, fowl pox viral vectors, or other viral
vectors (Mulligan, R. C., (1993) Science 260:926-932). The viral
vectors carrying all or part of nucleic sequence of SEQ ID
NOS:1234-2227 can be introduced into a mammal either prior to any
evidence of colon diseases or to mediate regression of the disease
in a mammal afflicted with colon diseases. Examples of methods for
administering the viral vector into the mammals include, but are
not limited to, exposure of cells to the virus ex vivo, or
injection of the retrovirus or a producer cell line of the virus
into the affected tissue or intravenous administration of the
virus. Alternatively the viral vector carrying all or part of the
CCAT nucleic acid sequence that encode peptides may be administered
locally by direct injection into the cancer lesion or topical
application in a pharmaceutically acceptable carrier. The quantity
of viral vector, carrying all or part of the CCAT nucleic acid
sequence, to be administered is based on the titer of virus
particles. A preferred range of the immunogen to be administered
may be about 106 to about 1011 virus particles per mammal,
preferably a human. After immunization the efficacy of the vaccine
can be assessed by production of antibodies or immune cells that
recognize the antigen, as assessed by specific lytic activity or
specific cytokine production or by tumor regression. One skilled in
the art would know the conventional methods to assess the
aforementioned parameters. If the mammal to be immunized is already
afflicted with cancer, the vaccine can be administered in
conjunction with other therapeutic treatments. Examples of other
therapeutic treatments includes, but are not limited to, adoptive T
cell immunotherapy, coadministration of cytokines or other
therapeutic drugs for cancer.
[0351] Alternatively all or parts thereof of a substantially or
partially purified the CCAT protein or their peptides may be
administered as a vaccine in a pharmaceutically acceptable carrier.
Ranges of the protein that may be administered are about 0.001 to
about 100 mg per patient, preferred doses are about 0.01 to about
100 mg per patient. In a preferred embodiment, the peptides or
modified peptides thereof is administered therapeutically or
prophylactically to a mammal in need of such treatment. The peptide
may be synthetically or recombinantly produced. Immunization is
repeated as necessary, until a sufficient titer of anti-immunogen
antibody or immune cells has been obtained.
[0352] In yet another alternative embodiment a viral vector, such
as a retroviral vector, can be introduced into mammalian cells.
Examples of mammalian cells into which the retroviral vector can be
introduced include, but are not limited to, primary mammalian
cultures or continuous mammalian cultures, COS cells, NIH3T3, or
293 cells (ATTC #CRL 1573), dendritic cells. The means by which the
vector carrying the gene may be introduced into a cell includes,
but is not limited to, microinjection, electroporation,
transfection or transfection using DEAE dextran, lipofection,
calcium phosphate or other procedures known to one skilled in the
art (Sambrook et al. (EDS) (2001) in "Molecular Cloning. A
laboratory manual", Cold Spring Harbor Press Plainview, N.Y.).
[0353] The vaccine formulation of the present invention comprises
an immunogen that induces an immune response directed against the
cancer associated antigens such as the CCATs, and in nonhuman
primates and finally in humans. The safety of the immunization
procedures is determined by looking for the effect of immunization
on the general health of the immunized animal (weight change,
fever, appetite behavior etc.) and looking for pathological changes
on autopsies. After initial testing in animals, cancer patients can
be tested. Conventional methods would be used to evaluate the
immune response of the patient to determine the efficiency of the
vaccine.
[0354] Measurement of candidate disease tumor antigen or vaccine
expression in patients is the first step of the present invention.
Subsequent steps will focus on measuring immune responses to these
candidate antigens or vaccine. Sera from disease patients,
particularly cancer patients, and healthy donors will be screened
for antibodies to the candidate antigens as well as for levels of
circulating tumor derived antigens. antigen. The vaccine
formulations may be evaluated first in animal models, initially
rodents
[0355] In one embodiment mammals, preferably human, at high risk
for colon diseases, particularly cancer, are prophylactically
treated with the vaccines of this invention. Examples of such
mammals include, but are not limited to, humans with a family
history of colon diseases, humans with a history of colon diseases,
particular cancer, or humans afflicted with colon cancer previously
resected and therefore at risk for reoccurrence. When provided
therapeutically, the vaccine is provided to enhance the patient's
own immune response to the diseased antigen present on the colon
diseases or advanced stage of colon diseases. The vaccine, which
acts as an immunogen, may be a cell, cell lysate from cells
transfected with a recombinant expression vector, cell lysates from
cells transfected with a recombinant expression vector, or a
culture supernatant containing the expressed protein.
Alternatively, the immunogen is a partially or substantially
purified recombinant protein, peptide or analog thereof or modified
peptides or analogs thereof. The proteins or peptides may be
conjugated with lipoprotein or administered in liposomal form or
with adjuvant.
[0356] While it is possible for the immunogen to be administered in
a pure or substantially pure form, it is preferable to present it
as a pharmaceutical composition, formulation or preparation. The
formulations of the present invention are described in the previous
section.
[0357] Vaccination can be conducted by conventional methods. For
example, the immunogen can be used in a suitable diluent such as
saline or water, or complete or incomplete adjuvants. Further, the
immunogen may or may not be bound to a carrier to make the protein
immunogenic. Examples of such carrier molecules include but are not
limited to bovine serum albumin (BSA), keyhole limpet hemocyanin
(KLH), tetanus toxoid, and the like. The immunogen also may be
coupled with lipoproteins or administered in liposomal form or with
adjuvants. The immunogen can be administered by any
route-appropriate for antibody production such as intravenous,
intraperitoneal, intramuscular, subcutaneous, and the like. The
immunogen may be administered once or at periodic intervals until a
significant titer of anti-CCAT immune cells or anti-CCAT antibody
is produced. The presence of anti-CCAT immune cells may be assessed
by measuring the frequency of precursor CTL (cytoxic T-lymphocytes)
against CCAT antigen prior to and after immunization by a CTL
precursor analysis assay (Coulie, P. et al., (1992) International
Journal Of Cancer 50:289-297). The antibody may be detected in the
serum using the immunoassay described above.
[0358] The safety of the immunization procedures is determined by
looking for the effect of immunization on the general health of the
immunized animal (weight change, fever, appetite behavior etc.) and
looking for pathological changes on autopsies. After initial
testing in animals, colon diseases patients can be tested.
Conventional methods would be used to evaluate the immune response
of the patient to determine the efficiency of the vaccine.
[0359] In yet another embodiment of this invention all, part, or
parts of the CCAT proteins or peptides or fragments thereof, or
modified peptides, may be exposed to dendritic cells cultured in
vitro. The cultured dendritic cells provide a means of producing
T-cell dependent antigens comprised of dendritic cell modified
antigen or dendritic cells pulsed with antigen, in which the
antigen is processed and expressed on the antigen activated
dendritic cell. The CCAT antigen activated dendritic cells or
processed dendritic cell antigens may be used as immunogens for
vaccines or for the treatment of colon diseases, particularly colon
cancer. The dendritic cells should be exposed to antigen for
sufficient time to allow the antigens to be internalized and
presented on the dendritic cells surface. The resulting dendritic
cells or the dendritic cell process antigens can than be
administered to an individual in need of therapy. Such methods are
described in Steinman et al. (WO93/208185) and in Banchereau et al.
(EPO Application 0563485A1).
[0360] In yet another aspect of this invention T-cells isolated
from individuals can be exposed to the CCAT proteins, peptides or
fragment thereof, or modified peptides in vitro and then
administered to a patient in need of such treatment in a
therapeutically effective amount. Examples of where T-lymphocytes
can be isolated include but are not limited to, peripheral blood
cells lymphocytes (PBL), lymph nodes, or tumor infiltrating
lymphocytes (TIL). Such lymphocytes can be isolated from the
individual to be treated or from a donor by methods known in the
art and cultured in vitro (Kawakami, Y. et al. (1989) J. Immunol.
142: 2453-3461). Lymphocytes are cultured in media such as RPMI or
RPMI 1640 or AIM V for 1-10 weeks. Viability is assessed by trypan
blue dye exclusion assay. Examples of how these sensitized T-cells
can be administered to the mammal include but are not limited to,
intravenously, intraperitoneally or intralesionally. Parameters
that may be assessed to determine the efficacy of these sensitized
T-lymphocytes include, but are not limited to, production of immune
cells in the mammal being treated or tumor regression. Conventional
methods are used to assess these parameters. Such treatment can be
given in conjunction with cytokines or gene modified cells
(Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 75-90;
Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 57-73).
[0361] The present invention is further described by the following
example. The example is provided solely to illustrate the invention
by reference to specific embodiments. This exemplification, while
illustrating certain aspects of the invention, does not offer the
limitations or circumscribe the scope of the disclosed
invention.
[0362] All examples outlined here were carried out using standard
techniques, which are well known and routine to those of skill in
the art. Routine molecular biology techniques of the following
example can be carried out as described in standard laboratory
manuals, such as Sambrook et al., Molecular Cloning: A laboratory
Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (2001).
WORKING EXAMPLES
1. Colon Tissues and Cell Lines
Colon Tissues
Tissue Processing
[0363] All tissues were procured as fresh specimens. Tissues were
collected as remnant tissues following surgical resection of
colorectal tissues. Remnant tissues were supplied following
processing for pathological diagnosis according to proper standards
of patient care. Procurement of all tissues was performed in an
anonymised manner in strict compliance with Federal mandated
ethical and legal guidelines (HIPAA) and in accordance with
clinical institution ethical review board as well as the internal
institutional review board. Tissues were transported on ice in
ice-cold transport buffer by courier for processing.
[0364] i) Enrichment of Epithelial Cells from Normal Colorectal
Mucosa
[0365] Normal colorectal tissue was transferred from the transport
vessel to a sterile dish containing 25 ml of ice-cold transport
buffer. The tissue was measured, weighed and photographed. The
tissue was dissected to isolate colorectal mucosa which was
transferred to a fresh dish containing 25 ml ice-cold Hanks
buffered saline solution. The tissue section was washed by vigorous
shaking and the HBSS replaced. This was repeated 2 further times or
until all visible mucus was removed. Mucosa was measured, weighed
and diced into 1 mm2 sections. The tissues sections were
transferred to a 50 ml polypropylene centrifuge tube containing 50
ml of A52 media (Biosource) supplemented with 2 mM L-glutamine and
1.5 mg/ml dispase (Roche Biochemicals). The digest was incubated
for 1 h at 37.degree. C. with frequent agitation. Following the
incubation, the suspension was poured through a 40-mesh cell sieve
situated in the base of a 15 cm culture dish. The filtrate was
diluted to 50 ml using A52 media supplemented with 2 mM L-glutamine
and passed through a 200-mesh cell sieve. The filtrate was
collected into a 50 ml polypropylene centrifuge tube and the
suspension was triturated several times followed by vortexing for 2
min at setting 6. The density and viability of nucleated cells was
determined by flow cytometry using propidium iodide as a negative
stain for viability (Guava system). Erythrocytes were lysed using a
standard ammonium chloride lysis protocol with incubation at room
temperature for 10 s. Cells were harvested by centrifugation at 500
g for 5 min at 4.degree. C. The cell pellet was resuspended in 50
ml of ice-cold HBSS and recentrifuged. The final cell pellet was
resuspended in 3 ml of ice-cold HBSS supplemented with 0.1% BSA and
0.25M EDTA. Cell density and viability were estimated using the
Guava system and the density adjusted to 1.times.10.sup.7 cells per
ml. Epithelial cells were stained with a FITC-labeled anti-EpCAM
murine monoclonal antibody and enriched by cell sorting using flow
cytometry.
[0366] ii) Enrichment of Tumor Cells from Colorectal Tumor
Tissue
[0367] Colorectal tumor tissue was transferred from the transport
vessel to a sterile dish containing 25 ml of ice-cold transport
buffer. The tissue was measured, weighed and photographed. The
tissue was dissected to remove necrotic and fibrotic tissue plaques
and the tumour tissue transferred to a fresh dish containing 25 ml
ice-cold Hanks buffered saline solution. The tissue section was
washed by vigorous shaking and the HBSS replaced. This was repeated
2 further times or until all visible mucus was removed. Tumour
tissue was measured, weighed and extensively diced. The tissues
slurry was transferred to a 50 ml polypropylene centrifuge tube
containing 50 ml of A52 media (Biosource) supplemented with 2 mM
L-glutamine and 1.5 mg/ml dispase (Roche Biochemicals). The digest
was incubated for 1 h at 37.degree. C. with frequent agitation.
Following the incubation, the suspension was poured through a
40-mesh cell sieve situated in the base of a 15 cm culture dish.
The filtrate was diluted to 50 ml using A52 media supplemented with
2 mM L-glutamine and passed through a 200-mesh cell sieve. The
filtrate was collected into a 50 ml polypropylene centrifuge tube
and the suspension was triturated several times followed by
vortexing for 2 min at setting 6. The density and viability of
nucleated cells was determined by flow cytometry using propidium
iodide as a negative stain for viability (Guava system).
Erythrocytes were lysed using a standard ammonium chloride lysis
protocol with incubation at room temperature for 10 s. Cells were
harvested by centrifugation at 500 g for 5 min at 4.degree. C. The
cell pellet was resuspended in 50 ml of ice-cold HBSS and
recentrifuged. The final cell pellet was resuspended in 3 ml of
ice-cold HBSS supplemented with 0.1% BSA and 0.25M EDTA. Cell
density and viability were estimated using the Guava system and the
density adjusted to 1.times.10.sup.7 cells per ml. Epithelial cells
were stained with a FITC-labeled anti-EpCAM murine monoclonal
antibody and enriched by cell sorting using flow cytometry.
[0368] iii) Enrichment of Cell Surface Proteins from Sorted
Epithelial and Tumor Cells
[0369] Sorted cells were centrifuged at 500 g at 4.degree. C. for 5
min and resuspended in 50 ml of ice-cold DPBS. The cell suspension
was washed by 2 further cycles of centrifugation 500 g at 4.degree.
C. for 5 min and resuspension of the cell pellet in 50 ml of
ice-cold DPBS. Finally, the cell pellet was resuspended in 9.5 ml
of ice-cold DPBS and sodium metaperiodate added to a final
concentration of 1 mM. The cell suspension was incubated on ice for
10 min with frequent agitation in the dark. Cells were centrifuged
at 500 g at 4.degree. C. for 5 min and resuspended in 50 ml of
ice-cold DPBS. The cell suspension was washed by 2 further cycles
of centrifugation 500 g at 4.degree. C. for 5 min and resuspension
of the cell pellet in 50 ml of ice-cold DPBS. Finally, the cell
pellet was resuspended in lysis buffer (1% SDS [w/v]; 0.1M HEPES;
10 mM MgCl.sub.2; 0.1% Non ionic detergent P40; 100 ml protease
inhibitor cocktail [P8340, Sigma]) and homogenisation performed by
passage of lysate through a 18 G syringe needle 10 times. Protein
concentrations were assayed relative to a Bovine serum albumin
standard by a modified Lowry assay (DC assay, BioRAD) and 1 mg of
total cellular protein transferred to a fresh tube and diluted to 1
mg/ml in acetate buffer (0.1M, pH 5.0).
Colon Cancer Cell Line Model System:
[0370] The model system employed here involves the use of Caco2 as
a "normal" reference (i.e., control) to which cell surface
expression in tumor derived cell lines is compared. These
differentials or candidates are then validated in tissues, colon
cancer and normal colon, to confirm that they are differentially
expressed between these tissues as well as within the cell line
model system.
Colon Cancer Cell Line Culture
[0371] Cell lines were grown in a culturing medium that is
supplemented as necessary with growth factors and serum, in
accordance with the American Type Culture Collection (ATCC)
(Mannassas, Va.) guidelines for each particular cell line (the ATCC
catalog number for each cell line is indicated in Table 1).
Cultures were established from frozen stocks in which the cells
were suspended in a freezing medium (cell culture medium with 10%
DMSO [v/v]) and flash frozen in liquid nitrogen. Frozen stocks
prepared in this way were stored in the liquid nitrogen vapour.
Cell cultures were established by rapidly thawing frozen stocks at
37.degree. C. Thawed stock cultures were slowly transferred to a
culture vessel containing a large volume of culture medium that was
supplemented. For maintenance of culture, cells were seeded at
1.times.10.sup.5 cells/per ml in medium and incubated at 37.degree.
C. until confluence of cells in the culture vessel exceeds 50% by
area. At this time, cells were harvested from the culture vessel
using enzymes or EDTA where necessary. The density of harvested,
viable cells was estimated by hemocytometry and the culture
reseeded as above. A passage of this nature was repeated no more
than 25 times at which point the culture was destroyed and
reestablished from frozen stocks as described above.
[0372] For the analyses of cell surface protein expression in
cultured cell lines, cells were grown as described above. At a
period 24 h prior to the experiment, the cell line was passaged as
described above. This yielded cell densities that were <50%
confluent and growing exponentially. Typically, triplicate analyses
of differential expression were performed for each line relative to
Caco2 for the purpose of identifying statistically significant
reproducible differentially expressed proteins.
2. Cloning and Expression of Target Proteins
[0373] cDNA Retrieval
[0374] Peptide sequences were searched by BlastP against the Celera
Discovery System (CDS) and public database to identify the
corresponding full-length open reading frames (ORFs). Each ORF
sequence was then searched by BlastN against the Celera in-house
human cDNA clone collection. For each sequence of interest, up to
three clones are pulled and streaked onto LB/Ampicillin (100 ug/ml)
plates. Plasmid DNA is isolated using Qiagen spin mini-prep kit and
verified by restriction digest. Subsequently, the isolated plasmid
DNA is sequence verified against the ORF reference sequence.
Sequencing reactions are carried out using Applied Biosystems
BigDye Terminator kit followed by ethanol precipitation. Sequence
data is collected using the Applied Biosystems 3100 Genetic
Analyzer and analyzed by alignment to the reference sequence using
the Clone Manager alignment tool.
PCR
[0375] PCR primers are designed to amplify the full-length ORF as
well as any regions of the ORF that are interest for expression
(antigenic or hydrophilic regions as determined by the Clone
Manager sequence analysis tool). Primers also contain 5' and 3'
overhangs to facilitate cloning (see below). PCR reactions contain
2.5 units Platinum Taq DNA Polymerase High Fidelity (Invitrogen),
50 ng cDNA plasmid template, 1 uM forward and reverse primers, 800
uM dNTP cocktail (Applied Biosystems) and 2 mM MgSO4. After 20-30
cycles (94.degree. C. for 30 seconds, 55.degree. C. for 1 minutes
and 73.degree. C. for 2 minutes), product is verified and
quantitated by agarose gel electrophoresis.
Construction of Entry Clones
[0376] PCR products are cloned into an entry vector for use with
the Gateway recombination based cloning system (Invitrogen). These
vectors include pDonr221, pDonr201, pEntr/D-TOPO or pEntr/SD/D-TOPO
and are used as described in the cloning methods below.
TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO
[0377] For cloning using this method, the forward PCR primer
contained a 5' overhang containing the sequence "CACC". PCR
products are generated as described above and cloned into the entry
vector using the Invitrogen TOPO cloning kit. Reactions are
typically carried out at room temperature for 10 minutes and
subsequently transformed into TOP10 chemically competent cells
(Invitrogen, CA). Candidate clones are picked, plasmid DNA is
prepared using Qiagen spin mini-prep kit and screened using
restriction digest. Inserts are subsequently sequence verified as
described above.
Gateway Cloning into pDonr201 or pDonr221
[0378] For cloning using this method, PCR primers contained the
following overhangs:
TABLE-US-00001 Forward 5' overhang:
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3' Reverse 5' overhang:
5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'
[0379] PCR products are generated as described above. ORFs are
recombined into the entry vector using the Invitrogen Gateway BP
Clonase enzyme mix. Reactions are typically carried out at
25.degree. C. for 1 hour, treated with Proteinase K at 37.degree.
C. for 10 minutes and transformed into Library Efficiency
DH5.alpha. chemically competent cells (Invitrogen, CA). Candidate
clones are picked, plasmid DNA is prepared using Qiagen spin
mini-prep kit and screened using restriction digest. Inserts are
subsequently sequence verified as described above.
Construction of Expression Clones
[0380] ORFs are transferred from the entry construct into a series
of expression vectors using the Gateway LR Clonase enzyme mix.
Reactions are typically carried out for 1 hour at 25.degree. C.,
treated with Proteinase K at 37.degree. C. for 10 minutes and
subsequently transformed into Library Efficiency DH5a chemically
competent cells (Invitrogen). Candidate clones are picked, plasmid
DNA is prepared using Qiagen spin mini-prep kit and screened using
restriction digest. Expression vectors include but are not limited
to pDest14, pDest15, pDest17, pDest8, pDest10 and pDest20. These
vectors allow expression in systems such as E. coli and recombinant
baculovirus. Other vectors not listed here allow expression in
yeast, mammalian cells, or in vitro.
Expression of Recombinant Proteins in E. coli
[0381] Constructs are transformed into one or more of the following
host strains: BL21 SI, BL21 AI, (Invitrogen); Origami B (DE3),
Origami B (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS,
Rosetta-Gami (DE3), Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B
(DE3) pLysS (Novagen). The transformants are grown in LB with or
without NaCl and with appropriate antibiotics, at temperatures in
the range of 20-37.degree. C., with aeration. Expression is induced
with the addition of IPTG (0.03-0.3 mM) or NaCl (75-300 mM) when
the cells are in mid-log growth. Growth is continued for one to 24
hours post-induction. Cells are harvested by centrifugation in a
Sorvall RC-3C centrifuge in a H6000A rotor for 10 minutes at 3000
rpm, at 4.degree. C. Cell pellets are stored at -80.degree. C.
Expression of Recombinant Proteins Using Baculovirus
[0382] Recombinant proteins are expressed using baculovirus in Sf21
fall army worm ovarian cells. Recombinant baculoviruses are
prepared using the Bac-to-Bac system (Invitrogen) per the
manufacturer's instructions. Proteins are expressed on the large
scale in Sf900 .mu.l serum-free medium (Invitrogen) in a 10 L
bioreactor tank (27.degree. C., 130 rpm, 50% dissolved oxygen for
48 hours).
3. Recombinant Protein Purification
[0383] Recombinant proteins are purified from E. coli and/or insect
cells using a variety of standard chromatography methods. Briefly,
cells are lysed using sonication or detergents. The insoluble
material is pelleted by centrifugation at 10,000.times.g for 15
minutes. The supernatant is applied to an appropriate affinity
column, e.g. His-tagged proteins are separated using a pre-packed
chelating sepharose column (Pharmacia) or GST-tagged proteins are
separated using a glutathione sepharose column (Pharmacia). After
using the affinity column, proteins are further separated using
various techniques, such as ion exchange chromatography (columns
from Pharmacia) to separate on the basis of electrical charge or
size exclusion chromatography (columns from Tosohaas) to separate
on the basis of molecular weight, size and shape.
[0384] Expression and purification of the protein are also achieved
using either a mammalian cell expression system or an insect cell
expression system. The pUB6/V5-His vector system (Invitrogen, CA)
is used to express GSCC in CHO cells. The vector contains the
selectable bsd gene, multiple cloning sites, the promoter/enhancer
sequence from the human ubiquitin C gene, a C-terminal V5 epitope
for antibody detection with anti-V5 antibodies, and a C-terminal
polyhistidine (6.times.His) sequence for rapid purification on
PROBOND resin (Invitrogen, CA). Transformed cells are selected on
media containing blasticidin.
[0385] Spodoptera frugiperda (Sf9) insect cells are infected with
recombinant Autographica californica nuclear polyhedrosis virus
(baculovirus). The polyhedrin gene is replaced with the cDNA by
homologous recombination and the polyhedrin promoter drives cDNA
transcription. The protein is synthesized as a fusion protein with
6.times.his which enables purification as described above. Purified
protein is used in the following activity and to make
antibodies
4. Chemical Synthesis of Peptides
[0386] Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in a
batchwise or continuous flow process which sequentially adds
.alpha.-amino- and side chain-protected amino acid residues to an
insoluble polymeric support via a linker group. A linker group such
as methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-a-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin. Trifluoroacetic acid
or piperidine are used to remove the protecting group in the case
of Boc or Fmoc, respectively. Each additional amino acid is added
to the anchored residue using a coupling agent or pre-activated
amino acid derivative, and the resin is washed. The full length
peptide is synthesized by sequential deprotection, coupling of
derivitized amino acids, and washing with dichloromethane and/or
N,N-dimethylformamide. The peptide is cleaved between the peptide
carboxy terminus and the linker group to yield a peptide acid or
amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook,
San Diego Calif. pp. S1-S20). Automated synthesis may also be
carried out on machines such as the 431A peptide synthesizer (ABI).
A protein or portion thereof may be purified by preparative high
performance liquid chromatography and its composition confirmed by
amino acid analysis or by sequencing (Creighton (1984) Proteins,
Structures and Molecular Properties, W H Freeman, New York
N.Y.).
5. Antibody Development
Polyclonal Antibody Preparations:
[0387] Polyclonal antibodies against recombinant proteins are
raised in rabbits (Green Mountain Antibodies, Burlington, Vt.).
Briefly, two New Zealand rabbits are immunized with 0.1 mg of
antigen in complete Freund's adjuvant. Subsequent immunizations are
carried out using 0.05 mg of antigen in incomplete Freund's
adjuvant at days 14, 21 and 49. Bleeds are collected and screened
for recognition of the antigen by solid phase ELISA and western
blot analysis. The IgG fraction is separated by centrifugation at
20,000.times.g for 20 minutes followed by a 50% ammonium sulfate
cut. The pelleted protein is resuspended in 5 mM Tris and separated
by ion exchange chromatography. Fractions are pooled based on IgG
content. Antigen-specific antibody is affinity purified using
Pierce AminoLink resin coupled to the appropriate antigen.
Isolation of Antibody Fragments Directed Against CCATs from A
Library of scFvs
[0388] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against CCAT to which the donor may or may not have
been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein
by reference in its entirety).
[0389] Rescue of the Library: A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times.TY containing 1% glucose and 100
.mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of
0.8 with shaking. Five ml of this culture is used to innoculate 50
ml of 2.times.TY-AMP-GLU, 2.times.108 TU of delta gene 3 helper
(M13 delta gene III, see PCT publication WO 92/01047) are added and
the culture incubated at 37.degree. C. for 45 minutes without
shaking and then at 37.degree. C. for 45 minutes with shaking. The
culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100 .mu.g/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are
prepared as described in PCT publication WO 92/01047.
[0390] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phagre particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 2001), resuspended in 2 ml PBS
and passed through a 0.45 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[0391] Panning of the Library: Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phages are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 .mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[0392] Characterization of Binders: Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 .mu.g/ml of the polypeptide of the present invention in
50 mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., PCT publication WO
92/01047) and then by sequencing.
Monoclonal Antibody Generation
[0393] i) Materials:
[0394] 1) Complete Media No Sera (CMNS) for washing of the myeloma
and spleen cells; Hybridoma medium CM-HAT {Cell Mab (BD), 10% FBS
(or HS); 5% Origen HCF (hybridoma cloning factor) containing 4 mM
L-glutamine and antibiotics} to be used for plating hybridomas
after the fusion.
[0395] 2) Hybridoma medium CM-HT (NO AMINOPTERIN) (Cell Mab (BD),
10% FBS 5% Origen HCF containing 4 mM L-glutamine and antibiotics)
to be used for fusion maintenance are stored in the refrigerator at
4-6.degree. C. The fusions are fed on days 4, 8, and 12, and
subsequent passages. Inactivated and pre-filtered commercial Fetal
Bovine serum (FBS) or Horse Serum (HS) are thawed and stored in the
refrigerator at 4.degree. C. and must be pretested for myeloma
growth from single cells.
[0396] 3) The L-glntamine (200 mM, 100.times. solution), which is
stored at -20.degree. C. freezer, is thawed and warmed until
completely in solution. The L-glntamin is dispensed into media to
supplement growth. L-glntamin is added to 2 mM for myelomas, and 4
mM for hybridoma media. Further the Penicillin, Streptomycin,
Amphotericin (antibacterial-antifungal stored at -20.degree. C.) is
thawed and added to Cell Mab Media to 1%.
[0397] 4) Myeloma growth media is Cell Mab Media (Cell Mab Media,
Quantum Yield from BD is stored in the refrigerator at 4.degree. C.
in the dark) which are added L-glntamine to 2 mM and
antibiotic/antimycotic solution to 1% and is called CMNS.
[0398] 5) 1 bottle of PEG 1500 in Hepes (Roche, N.J.)
[0399] 6) 8-Azaguanine is stored as the dried powder supplied by
SIGMA at -700.degree. C. until needed. Reconstitute 1 vial/500 ml
of media and add entire contents to 500 ml media (eg. 2
vials/litre).
[0400] 7) Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza
(1.times.) stored in the refrigerator at 4.degree. C.
[0401] 8) Clonal cell medium D (Stemcell, Vancouver) contains HAT
and methyl cellulose for semi-solid direct cloning from the fusion.
This comes in 90 ml bottles with a CoA and must be "melted at
37.degree. C. in a waterbath in the morning of the day of the
fusion. Loosen the cap and leave in CO2 incubator to sufficiently
gas the medium D and bring the pH down.
[0402] 9) Hybridoma supplements HT [hypoxanthine, thymidine] are to
be used in medium for the section of hybridomas and maintenance of
hybridomas through the cloning stages respectively.
[0403] 10) Origen HCF can be obtained directly from Igen and is a
cell supernatant produced from a macrophage-like cell-line. It can
be thawed and aliquoted to 15 ml tubes at 5 ml per tube and stored
frozen at -20.degree. C. Positive Hybridomas are fed HCF through
the first subcloning and are gradually weaned. It is not necessary
to continue to supplement unless you have a particularly difficult
hybridoma clone. This and other additives have been shown to be
more effective in promoting new hybridoma growth than conventional
feeder layers.
[0404] ii) Procedure
[0405] To generate monoclonal antibodies, mice are immunized with
5-50 ug of antigen either intra-peritoneally (i.p.) or by
intravenous injection in the tail vein (i.v.). Typically, the
antigen used is a recombinant protein that is generated as
described above. The primary immunization takes place 2 months
prior to the harvesting of splenocytes from the mouse and the
immunization is typically boosted by i.v. injection of 5-50 ug of
antigen every two weeks. At least one week prior to expected fusion
date, a fresh vial of myeloma cells is thawed and cultured. Several
flasks at different densities are maintained in order that a
culture at the optimum density is ensured at the time of fusion.
The optimum density is determined to be 3-6.times.10.sup.5
cells/ml. Two to five days before the scheduled fusion, a final
immunization is administered of .about.5 ug of antigen in PBS i.p.
or i.v.
[0406] Myeloma cells are washed with 30 ml serum free media by
centrifugation at 500 g at 4.degree. C. for 5 minutes. Viable cell
density is determined in resuspended cells using hemocytometry and
vital stains. Cells resuspended in complete growth medium are
stored at 37.degree. C. during the preparation of splenocytes.
Meanwhile, to test aminopterin sensitivity, 1.times.10.sup.6
myeloma cells are transferred to a 15 ml conical tube and
centrifuged at 500 g at 4.degree. C. for 5 minutes. The resulting
pellet is resuspended in 15 ml of HAT media and cells plated at 2
drops/well on a 96 well plate.
[0407] To prepare splenocytes from immunized mice, the animals are
euthanised and submerged in 70% ETOH. Under sterile conditions, the
spleen is surgically removed and placed in 10 ml of RPMI medium
supplemented with 20% fetal calf serum in a Petri dish. Cells are
extricated from the spleen by infusing the organ with medium>50
times using a 21 g syringe.
[0408] Cells are harvested and washed by centrifugation (at 500 g
at 4.degree. C. for 5 minutes) with 30 ml of medium. Cells are
resuspended in 10 ml of medium and the density of viable cells
determined by hemocytometry using vital stains. The splenocytes are
mixed with myeloma cells at a ratio of 5:1 (spleen cells: myeloma
cells). Both the myeloma and spleen cells are washed 2 more times
with 30 ml of RPMI-CMNS. Spin at 800 rpm for 12 minutes.
[0409] Supernatant is removed and cells are resuspended in 5 ml of
RPMI-CMNS and are pooled to fill volume to 30 ml and spin down as
before. Then, the pellet is broken up by gently tapping on the flow
hood surface and resuspended in 1 ml of BMB REG1500 (prewarmed to
37.degree. C.) dropwise with 1 cc needle over 1 minute.
[0410] RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly
dilute out the PEG. Cells are centrifuged and diluted in 5 ml of
Complete media and 95 ml of Clonacell Medium D (HAT) media (with 5
ml of HCF). The cells are plated out 10 ml per small petri
plate.
[0411] Myeloma/HAT control. P is prepared as follows: dilute about
1000 P3X63 Ag8.653 myeloma cells into 1 ml of medium D and transfer
into a single well of a 24 well plate. Plates are placed in
incubator, with two plates inside of a large petri plate, with an
additional petri plate full of distilled water, for 10-18 days
under 5% CO2 overlay at 37.degree. C. Clones are picked from
semisolid agarose into 96 well plates containing 150-200 ul of
CM-HT. Supernatants are screened 4 days later in ELISA, and
positive clones are moved up to 24 well plates. Heavy growth will
require changing of the media at day 8 (+/-150 ml). One should
further decrease the HCF to 0.5% (gradually-2%, then 1%, then 0.5%)
in the cloning plates.
[0412] For further references see Kohler G, and C. Milstein
Continuous cultures of fused cells secreting antibody of predefined
specificity. 1975. Nature 256: 495-497; Lane, R. D. A short
duration polyethylene glycol fusion technique for increasing
production of monoclonal antibody-secreting hybridomas. 1985. J.
Immunol. Meth. 81:223-228;
[0413] Harlow, E. and D. Lane. Antibodies: A laboratory manual.
Cold Spring Harbour Laboratory Press. 1988; Kubitz, D. The Scripps
Research Institute. La Jolla. Personal Communication; Zhong, G.,
Berry, J. D., and Choukri, S. (1996) Mapping epitopes of Chlamydia
trachomatis neutralizing monoclonal antibodies using phage random
peptide libraries. J. Indust. Microbiol. Biotech. 19, 71-76; Berry,
J. D., Licea, A., Popkov, M., Cortez, X., Fuller, R., Elia, M.,
Kerwin, L., and C. F. Barbas III. (2003) Rapid monoclonal antibody
generation via dendritic cell targeting in vivo. Hybridoma and
Hybridomics 22 (1), 23-31.
6. mRNA Expression
Validation in Tissues by Taqman
[0414] Expression of mRNA is quantitated by RT-PCR using
TaqMan.RTM. technology. The Taqman system couples a 5' fluorogenic
nuclease assay with PCR for real time quantitation. A probe is used
to monitor the formation of the amplification product.
[0415] Total RNA is isolated from cancer model cell lines using the
RNEasy Kit.RTM. (Qiagen) per manufacturer's instructions and
included DNase treatment. Normal human tissue RNAs are acquired
from commercial vendors (Ambion, Austin, Tex.; Stratagene, La
Jolla, Calif., BioChain Institute, Newington, N.H.) as were RNAs
from matched disease/normal tissues.
[0416] Target transcript sequences are identified for the
differentially expressed peptides by searching the BlastP database.
TaqMan assays (PCR primer/probe set) specific for those transcripts
are identified by searching the Celera Discovery System.TM. (CDS)
database. The assays are designed to span exon-exon borders and do
not amplify genomic DNA.
[0417] The TaqMan primers and probe sequences are as designed by
Applied Biosystems (AB) as part of the Assays on Demand.TM. product
line or by custom design through the AB Assays by Design.sup.SM
service.
[0418] RT-PCR is accomplished using AmpliTaqGold and MultiScribe
reverse transcriptase in the One Step RT-PCR Master Mix reagent kit
(AB) according to the manufacturer's instructions. Probe and primer
concentrations are 250 nM and 900 nM, respectively, in a 15 .mu.l
reaction. For each experiment, a master mix of the above components
is made and aliquoted into each optical reaction well. Eight
nanograms of total RNA is the template. Each sample is assayed in
triplicate. Quantitative RT-PCR is performed using the ABI
Prism.RTM. 7900HT Sequence Detection System (SDS). Cycling
parameters follow: 48.degree. C. for 30 min. for one cycle;
95.degree. C. for 10 min for one cycle; 95.degree. C. for 15 sec,
60.degree. C. for 1 min. for 40 cycles.
[0419] The SDS software calculates the threshold cycle (C.sub.T)
for each reaction, and C.sub.T values are used to quantitate the
relative amount of starting template in the reaction. The C.sub.T
values for each set of three reactions are averaged for all
subsequent calculations
[0420] Data are analyzed for fold difference in expression using an
endogenous control for normalization and is expressed relative to a
normal tissue or normal cell line reference. The choice of
endogenous control is determined empirically by testing various
candidates against the cell line and tissue RNA panels and
selecting the one with the least variation in expression. Relative
changes in expression are quantitated using the
2.sup.-.DELTA..DELTA.CT Method. Livak, K. J. and Schmittgen, T. D.
(2001) Methods 25: 402-408; User bulletin #2: ABI Prism 7700
Sequence Detection System.
Validation by Tissue Flow Cytometry Analysis
[0421] Post tissue processing, cells are sorted by flow cytometry
known in the art to enrich for epithelial cells. Alternatively,
cells isolated from lung tissue are stained directly with EpCAM
(for epithelial cells) and the specific antibody to CCAT. Cell
numbers and viability are determined by PI exclusion (GUAVA) for
cells isolated from both normal and tumor lung tissue. A minimum of
0.5.times.10.sup.6 cells are used for each analysis. Cells are
washed once with Flow Staining Buffer (0.5% BSA, 0.05% NaN3 in
D-PBS). To the cells, 20 ul of each antibody for CCAT are added. An
additional 5 ul of EpCAM antibody conjugated to APC were added when
unsorted cells are used in the experiment. Cells are incubated with
antibodies for 30 minutes at 4.degree. C. Cells are washed once
with Flow Staining Buffer and either analyzed immediately on the
LSR flow cytometry apparatus or fixed in 1% formaldehyde and store
at 4.degree. C. until LSR analysis. The antibodies used to detect
CCAT targets are all purchased by BD Biosciences and PE-conjugated.
The isotype control antibody used for these experiments is
PE-conjugated mouse IgG1k.
7. Detection and Diagnosis of CCAT by Liquid Chromatography and
Mass Spectrometry (LC/MS)
[0422] The proteins from cells can be prepared by reduction,
alkylation and cysteine-containing peptide enrichment of
concentrated conditioned media.
[0423] The differential expression of proteins in disease and
healthy samples are quantitated using Liquid Chromatography Mass
Spectrometry. The LC/MS spectra from disease and healthy (control)
samples are collected and processed using the following steps:
[0424] The raw scans from the LC/MS instrument are subjected to
peak detection and noise reduction software. Filtered peak lists
are then used to detect `features` corresponding to specific
peptides from the original sample(s). Features are characterized by
their mass/charge, charge, retention time, isotope pattern and
intensity.
[0425] Similar experiments are repeated in order to increase the
confidence in detection of a peptide. These multiple acquisitions
are computationally aggregated into one experiment. The intensity
of a peptide present in both healthy and disease samples is used to
calculate the differential expression, or relative abundance, of
the peptide. The intensity of a peptide found exclusively in one
sample is used to calculate a theoretical expression ratio for that
peptide (singleton). Expression ratios are calculated for each
peptide of each replicate of the experiment
[0426] Statistical tests are performed to assess the robustness of
the data and select statistically significant differentials. To
assess general quality of the data, a) ensured that similar
features are detected in all replicates of the experiment; b)
number of matched ions between replicates; c) calculated the
overall pair wise intensity correlations between LC/MS maps of
process replicates to ensure that the expression ratios for
peptides are reproducible across the multiple replicates; d)
aggregated multiple experiments in order to compare the expression
ratio of a peptide in multiple diseases or disease samples.
8. Expression Validation by IHC in Tissue Sections
Tissue Sections
[0427] Paraffin embedded, fixed tissue sections are obtained from a
panel of normal tissues (Adrenal, Bladder, Lymphocytes, Bone
Marrow, Breast, Cerebellum, Cerebral cortex, Colon, Endothelium,
Eye, Fallopian tube, Small Intestine, Heart, Kidney [glomerulus,
tubule], Liver, Lung, Testes and Thyroid) as well as 30 tumor
samples with matched normal adjacent tissues from pancreas, lung,
colon, prostate, ovarian and breast. In addition, other tissues are
selected for testing such as bladder renal, hepatocellular,
pharyngeal and gastric tumor tissues. Replicate sections are also
obtained from numerous tumor types (Bladder Cancer, Lung Cancer,
Breast Cancer, Melanoma, Colon Cancer, Non-Hodgkins Lymphoma,
Endometrial Cancer, Ovarian Cancer, Head and Neck Cancer, Prostate
Cancer, Leukemia [ALL and CML] and Rectal Cancer). Sections are
stained with hemotoxylin and eosin and histologically examined to
ensure adequate representation of cell types in each tissue
section.
[0428] An identical set of tissues will be obtained from frozen
sections and are used in those instances where it is not possible
to generate antibodies that are suitable for fixed sections. Frozen
tissues do not require an antigen retrieval step.
Paraffin Fixed Tissue Sections
[0429] Hemotoxylin and Eosin staining of paraffin embedded, fixed
tissue sections.
[0430] Sections are deparaffinized in 3 changes of xylene or xylene
substitute for 2-5 minutes each. Sections are rinsed in 2 changes
of absolute alcohol for 1-2 minutes each, in 95% alcohol for 1
minute, followed by 80% alcohol for 1 minute. Slides are washed
well in running water and stained in Gill solution 3 hemotoxylin
for 3 to 5 minutes. Following a vigorous wash in running water for
1 minute, sections are stained in Scott's solution for 2 minutes.
Sections are washed for 1 min in running water then counterstained
in Eosin solution for 2-3 minutes depending upon development of
desired staining intensity. Following a brief wash in 95% alcohol,
sections are dehydrated in three changes of absolute alcohol for 1
minute each and three changes of xylene or xylene substitute for
1-2 minutes each. Slides are coverslipped and stored for
analysis.
Optimisation of Antibody Staining
[0431] or each antibody, a positive and negative control sample are
generated using data from the ICAT analysis of the colon cancer
cell lines/tissues. Cells are selected that are known to express
low levels of a particular target as determined from the ICAT data.
This cell line is the reference normal control. Similarly, a colon
tumor line is selected that is determined to overexpress the target
is selected.
Antigen Retrieval
[0432] Sections are deparaffinized and rehydrated by washing 3
times for 5 minutes in xylene; two times for 5 minutes in 100%
ethanol; two times for 5 minutes in 95% ethanol; and once for 5
minutes in 80% ethanol. Sections are then placed in endogenous
blocking solution (methanol+2% hydrogen peroxide) and incubated for
20 minutes at room temperature. Sections are rinsed twice for 5
minutes each in deionized water and twice for 5 minutes in
phosphate buffered saline (PBS), pH 7.4. Alternatively, where
necessary sections are deparrafinized by High Energy Antigen
Retrieval as follows: sections are washed three times for 5 minutes
in xylene; two times for 5 minutes in 100% ethanol; two times for 5
minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.
Sections are placed in a Coplin jar with dilute antigen retrieval
solution (10 mM citrate acid, pH 6). The Coplin jar containing
slides is placed in a vessel filled with water and microwaved on
high for 2-3 minutes (700 watt oven). Following cooling for 2-3
minutes, steps 3 and 4 are repeated four times (depending on
tissue), followed by cooling for 20 minutes at room temperature.
Sections are then rinsed in deionized water, two times for 5
minutes, placed in modified endogenous oxidation blocking solution
(PBS+2% hydrogen peroxide) and rinsed for 5 minutes in PBS.
Blocking and Staining
[0433] Sections are blocked with PBS/1% bovine serum albumin (PBA)
for 1 hour at room temperature followed by incubation in normal
serum diluted in PBA (2%) for 30 minutes at room temperature to
reduce non-specific binding of antibody. Incubations are performed
in a sealed humidity chamber to prevent air-drying of the tissue
sections. (The choice of blocking serum is the same as the species
of the biotinylated secondary antibody). Excess antibody is gently
removed by shaking and sections covered with primary antibody
diluted in PBA and incubated either at room temperature for 1 hour
or overnight at 4.degree. C. (Care is taken that the sections do
not touch during incubation). Sections are rinsed twice for 5
minutes in PBS, shaking gently. Excess PBS is removed by gently
shaking. The sections are covered with diluted biotinylated
secondary antibody in PBA and incubated for 30 minutes to 1 hour at
room temperature in the humidity chamber. If using a monoclonal
primary antibody, addition of 2% rat serum is used to decrease the
background on rat tissue sections. Following incubation, sections
are rinsed twice for 5 minutes in PBS, shaking gently. Excess PBS
is removed and sections incubated for 1 hour at room temperature in
Vectastain ABC reagent (as per kit instructions). The lid of the
humidity chamber is secured during all incubations to ensure a
moist environment. Sections are rinsed twice for 5 minutes in PBS,
shaking gently.
Develop and Counterstain
[0434] Sections are incubated for 2 minutes in peroxidase substrate
solution that is made up immediately prior to use as follows:
[0435] 10 mg diaminobenzidine (DAB) dissolved in 10 ml 50 mM sodium
phosphate buffer, pH 7.4. [0436] 12.5 microliters 3% CoCl2/NiCl2 in
deionized water [0437] 1.25 microliters hydrogen peroxide
[0438] Slides are rinsed well three times for 10 min in deionized
water and counterstained with 0.01% Light Green acidified with
0.01% acetic acid for 1-2 minutes depending on intensity of
counterstain desired.
[0439] Slides are rinsed three times for 5 minutes with deionized
water and dehydrated two times for 2 minutes in 95% ethanol; two
times for 2 minutes in 100% ethanol; and two times for 2 minutes in
xylene. Stained slides are mounted for visualization by
microscopy.
9. IHC Staining of Frozen Tissue Sections
[0440] Fresh tissues are embedded carefully in OCT in plastic mold,
without trapping air bubbles surrounding the tissue. Tissues are
frozen by setting the mold on top of liquid nitrogen until 70-80%
of the block turns white at which point the mold is placed on dry
ice. The frozen blocks were stored at -80.degree. C. Blocks are
sectioned with a cryostat with care taken to avoid warming to
greater than -10.degree. C. Initially, the block is equilibrated in
the cryostat for about 5 minutes and 6-10 mm sections are cut
sequentially. Sections are allowed to dry for at least 30 minutes
at room temperature. Following drying, tissues are stored at
4.degree. C. for short term and -80.degree. C. for long term
storage.)
[0441] Sections are fixed by immersing in acetone jar for 1-2
minutes at room temperature, followed by drying at room temp.
Primary antibody is added (diluted in 0.05 M Tris-saline [0.05 M
Tris, 0.15 M NaCl, pH 7.4], 2.5% serum) directly to the sections by
covering the section dropwise to cover the tissue entirely. Binding
is carried out by incubation a chamber for 1 hour at room
temperature. Without letting the sections dry out, the secondary
antibody (diluted in Tris-saline/2.5% serum) is added in a similar
manner to the primary and incubated as before (at least 45
minutes).
[0442] Following incubation, the sections are washed gently in
Tris-saline for 3-5 minutes and then in Tris-saline/2.5% serum for
another 3-5 minutes. If a biotinylated primary antibody is used, in
place of the secondary antibody incubation, slides are covered with
100 ul of diluted alkaline phosphatase conjugated streptavidin,
incubated for 30 minutes at room temperature and washed as above.
Sections are incubated with alkaline phosphates substrate (1 mg/ml
Fast Violet; 0.2 mg/ml Napthol AS-MX phosphate in Tris-Saline pH
8.5) for 10-20 minutes until the desired positive staining is
achieved at which point the reaction is stopped by washing twice
with Tris-saline. Slides are counter-stained with Mayer's
hematoxylin for 30 seconds and washed with tap water for 2-5
minutes. Sections are mounted with Mount coverslips and mounting
media.
10. Assay for Antibody Dependent Cellular Cytotoxicity
[0443] Cultured tumor cells are labeled with 100 .mu.Ci 51Cr for 1
hour; Livingston, P. O., Zhang, S., Adluri, S., Yao, T.-J.,
Graeber, L., Ragupathi, G., Helling, F., & Fleischer, M.
(1997). Cancer Immunol. Immunother. 43, 324-330. After being washed
three times with culture medium, cells are resuspended at
10.sup.5/ml, and 100 .mu.l/well are plated onto 96-well
round-bottom plates. A range of antibody concentrations are applied
to the wells, including an isotype control together with donor
peripheral blood mononuclear cells that are plated at a 100:1 and
50:1 ratio. After an 18-h incubation at 37.degree. C., supernatant
(30 .mu.l/well) is harvested and transferred onto Lumaplate 96
(Packard), dried, and read in a Packard Top-Count NXT .gamma.
counter. Each measurement is carried out in triplicate. Spontaneous
release is determined by cpm of tumor cells incubated with medium
and maximum release by cpm of tumor cells plus 1% Triton X-100
(Sigma). Specific lysis is defined as: % specific
lysis=[(experimental release-spontaneous release)/(maximum
release-spontaneous release)].times.100. The percent ADCC is
expressed as peak specific lysis postimmune subtracted by preimmune
percent specific lysis. A doubling of the ADCC to >20% is
considered significant.
11. Assay for Complement Dependent Cytotoxicity
[0444] Chromium release assays to assess complement-mediated
cytotoxicity are performed for each patient at various time points;
Dickler, M. N., Ragupathi, G., Liu, N. X., Musselli, C., Martino,
D. J., Miller, V. A., Kris, M. G., Brezicka, F. T., Livingston, P.
O. & Grant, S. C. (1999) Clin. Cancer Res. 5, 2773-2779.
Cultured tumor cells are washed in FCS-free media two times,
resuspended in 500 .mu.l of media, and incubated with 100 .mu.Ci
.sup.51Cr per 10 million cells for 2 h at 37.degree. C. The cells
are then shaken every 15 min for 2 h, washed 3 times in media to
achieve a concentration of approximately 20,000 cells/well, and
then plated in round-bottom plates. The plates contain either 50
.mu.l cells plus 50 .mu.l monoclonal antibody, 50 .mu.l cells plus
serum (pre- and posttherapy), or 50 .mu.l cells plus mouse serum as
a control. The plates are incubated in a cold room on a shaker for
45 min. Human complement of a 1:5 dilution (resuspended in 1 ml of
ice-cold water and diluted with 3% human serum albumin) is added to
each well at a volume of 100 .mu.l. Control wells include those for
maximum release of isotope in 10% Triton X-100 (Sigma) and for
spontaneous release in the absence of complement with medium alone.
The plates are incubated for 2 h at 37.degree. C., centrifuged for
3 min, and then 100 .mu.l of supernatant is removed for
radioactivity counting. The percentage of specific lysis is
calculated as follows: % cytotoxicity=[(experimental
release-spontaneous release)/(maximum release-spontaneous
release)].times.100.A doubling of the CDC to >20% is considered
significant.
12. In Vitro Assays in Cell Lines; RNAi
[0445] Lipofectamine is purchased from Invitrogen (Carlsbad,
Calif.) and GeneSilencer from Gene Therapy Systems (San Diego,
Calif.). Synthetic siRNA oligonucleotides are from Dharmacon
(Lafayette, Colo.), Qiagen (Valencia, Calif.) or Ambion (Austin.
TX) RNeasy 96 Kit is purchased from Qiagen (Valencia, Calif.).
Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous One
Solution Cell Proliferation Assay are both purchased from Promega
(Madison, Wis.). Alamar Blue proliferation assay can be purchased
from Biosource (Camarillo, Calif.). Function blocking antibodies
are purchased from Chemicon (Temecula, Calif.), Biotrend (Cologne,
Germany) or Alexis Corporation (San Diego, Calif.). Cell invasion
assay kits from purchased from Chemicon (Temecula, Calif.).
RiboGreen RNA Quantitation Kit is purchased from Molecular probes
(Eugene, Oreg.).
RNAi
[0446] RNAi is performed by using Smartpools (Dharmacon), 4--for
Silencing siRNA duplexes (Qiagen) or scrambled negative control
siRNA (Ambion). Transient transfections are carried out in
triplicate by using either Lipofectamine 2000 from Invitrogen
(Carlsbad, Calif.) or by using GeneSilencer from Gene Therapy
Systems (San Diego, Calif.) in methods described below. 1 to 4 days
after transfections, total RNA is isolated by using the RNeasy 96
Kit (Qiagen) according to manufacturer's instructions and
expression of mRNA is quantitated by using TaqMan technology.
Protein expression levels are examined by flow cytometry and
apoptosis and proliferation assays are performed daily using
Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous One
Solution Cell Proliferation Assay (see protocols below).
[0447] i) RNAi Transfections--Lipofectamine 2000
[0448] Transient transfections are carried out on sub-confluent
colon cancer cell lines as previously described. Elbashir, S. M. et
al. (2001) Nature 411: 494-498; Caplen, N. J. et al. (2001) Proc
Natl Acad Sci USA 98: 9742-9747; Sharp, P. A. (2001) Genes and
Development 15: 485-490. Synthetic RNA to gene of interest or
scrambled negative control siRNA is transfected using lipofectamine
according to manufacturer's instructions. Cells are plated in 96
well plates in antibiotics free medium. The next day, the
transfection reagent and siRNA are prepared for transfections as
follows: Each 0.1-1 ul of lipofectamine 2000 and 10-150 mM siRNA
are resuspended 25 ul serum-free media and incubated at room
temperature for 5 minutes. After incubation, the diluted siRNA and
the lipofectamine 2000 are combined and incubated for 20 minutes at
room temperature. The cells are then washed and the combined
siRNA-Lipofectamine 2000 reagent added. After further 4 hours
incubation, 50 ul serum containing medium is added to each well. 1
and 4 days after transfection, expression of mRNA is quantitated by
RT-PCR using TaqMan technology and protein expression levels are
examined by flow cytometry. Apoptosis and proliferation assays are
performed daily using Apop-one homogeneous caspase-3/7 kit and
CellTiter 96 AQueous One Solution Cell Proliferation Assay (see
protocols below).
[0449] ii) RNAi Transfections--GeneSilencer
[0450] Transient transfections are carried out on sub-confluent
colon cancer cell lines as previously described. Elbashir, S. M. et
al. (2001) Nature 411: 494-498; Caplen, N. J. et al. (2001) Proc
Natl Acad Sci USA 98: 9742-9747; Sharp, P. A. (2001) Genes and
Development 15: 485-490. Synthetic RNA to gene of interest or
scrambled negative control siRNA is transfected using GeneSilencer
according to manufacturer's instructions. Cells are plated in 96
well plates in antibiotics free medium. The next day, the
transfection reagent and the synthetic siRNA are prepared for
transfections as follows: predetermined amount of Gene Silencer is
diluted in serum-free media to a final volume of 20 ul per well.
After resuspending 10-150 mM siRNA in 20 ul serum-free media, the
reagents are combined and incubated at room temperature for 5-20
minutes. After incubation, the siRNA-Gene Silencer reagent is added
to each well and incubated in a 37.degree. C. incubator for 4 hours
before an equal volume of serum containing media is added back to
the cultured cells. The cells are then incubated for 1 to 4 days
before mRNA, protein expression and effects on apoptosis and
proliferation are examined.
Testing of Function Blocking Antibodies
[0451] Sub-confluent colon cancer cell lines are serum-staved
overnight. The next day, serum-containing media is added back to
the cells in the presence of 5-50 ng/ml of function blocking
antibodies. After 2 or 5 days incubation at 37.degree. C. 5%
CO.sub.2, antibody binding is examined by flow cytometry and
apoptosis and proliferation are examined by using protocols
described below.
Apoptosis
[0452] Apoptosis assay is performed by using the Apop-one
homogeneous caspase-3/7 kit from Promega. Briefly, the caspase-3/7
substrate is thawed to room temperature and diluted 1:100 with
buffer. The diluted substrate is then added 1:1 to cells, control
or blank. The plates are then placed on a plate shaker for 30
minutes to 18 hours at 300-500 rpm. The fluorescence of each well
is then measured at using an excitation wavelength of 485+/-20 nm
and an emission wavelength of 530+/-25 nm.
Proliferation--MTS
[0453] Proliferation assay is performed by using the CellTiter 96
AQueous One Solution Cell Proliferation Assay kit from Promega. 20
ul of CellTiter 96 AQueous One Solution is added to 100 ul of
culture medium. The plates are then incubated for 1-4 hours at
37.degree. C. in a humidified 5% CO.sub.2 incubator. After
incubation, the change in absorbance is read at 490 nm.
Proliferation--Alamar Blue
[0454] Proliferation assay is performed by using the Alamar Blue
assay from Biosource. 10 ul of Alamar Blue reagent is added to 100
ul of cells in culture medium. The plates are then incubated for
1-4 hours at 37.degree. C. in a humidified 5% CO.sub.2 incubator.
After incubation, the change in fluorescence is measured at using
an excitation wavelength of 530 nm and an emission wavelength of
595 nm.
Cell Invasion
[0455] Cell invasion assay is performed by using the 96 well cell
invasion assay kit available from Chemicon. After the cell invasion
chamber plates are adjusted to room temperature, 100 ul serum-free
media is added to the interior of the inserts. 1-2 hours later,
cell suspensions of 1.times.10.sup.6 cells/ml are prepared. Media
is then carefully removed from the inserts and 100 ul of prepared
cells are added into the insert +/-0 to 50 ng function blocking
antibodies. The cells are pre-incubated for 15 minutes at
37.degree. C. before 150 ul of media containing 10% FBS is added to
the lower chamber. The cells are then incubated for 48 hours at
37.degree. C. After incubation, the cells from the top side of the
insert are discarded and the invasion chamber plates are then
placed on a new 96-well feeder tray containing 150 ul of pre-warmed
cell detachment solution in the wells. The plates are incubated for
30 minutes at 37.degree. C. and are periodically shaken. Lysis
buffer/dye solution (4 ul CyQuant Dye/300 ul 4.times. lysis buffer)
is prepared and added to each well of dissociation buffer/cells on
feeder tray. The plates are incubated for 15 minutes at room
temperature before 150 ul is transferred to a new 96-well plate.
Fluorescence of invading cells is then read at 480 excitation and
520 emission.
Receptor Internalization
[0456] For quantification of receptor internalization, ELISA assays
are performed essentially as described by Daunt et al. Daunt, D.
A., Hurtz, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K.
(1997) Mol. Pharmacol. 51, 711-720. The cell lines are plated at
6.times.10.sup.5 cells per in a 24-well tissue culture dishes that
have previously been coated with 0.1 mg/ml poly-L-lysine. The next
day, the cells are washed once with PBS and incubated in DMEM at
37.degree. C. for several minutes. Agonist to the cell surface
target of interest is then added at a pre-determined concentration
in prewarmed DMEM to the wells. The cells are then incubated for
various times at 37.degree. C. and reactions are stopped by
removing the media and fixing the cells in 3.7% formaldehyde/TBS
for 5 min at room temperature. The cells are then washed three
times with TBS and nonspecific binding blocked with TBS containing
1% BSA for 45 min at room temperature. The first antibody is added
at a pre-determined dilution in TBS/BSA for 1 h at room
temperature. Three washes with TBS followed, and cells are briefly
reblocked for 15 min at room temperature. Incubation with goat
anti-mouse conjugated alkaline phosphatase (Bio-Rad) diluted 1:1000
in TBS/BSA is carried out for 1 h at room temperature. The cells
are washed three times with TBS and a colorimetric alkaline
phosphatase substrate is added. When the adequate color change is
reached, 100-0 samples are taken for colorimetric readings.
mRNA Expression
[0457] Expression of mRNA is quantitated by RT-PCR using
TaqMan.RTM. technology. Total RNA is isolated from cancer model
cell lines using the RNEasy 96 kit (Qiagen) per manufacturer's
instructions and included DNase treatment. Target transcript
sequences are identified for the differentially expressed peptides
by searching the BlastP database. TaqMan assays (PCR primer/probe
set) specific for those transcripts are identified by searching the
Celera Discovery System.TM. (CDS) database. The assays are designed
to span exon-exon borders and do not amplify genomic DNA. The
TaqMan primers and probe sequences are as designed by Applied
Biosystems (AB) as part of the Assays on Demand.TM. product line or
by custom design through the AB Assays by Design.sup.SM service.
RT-PCR is accomplished using AmpliTaqGold and MultiScribe reverse
transcriptase in the One Step RT-PCR Master Mix reagent kit (AB)
according to the manufacturer's instructions. Probe and primer
concentrations are 900 nM and 250 nM, respectively, in a 250
reaction. For each experiment, a master mix of the above components
is made and aliquoted into each optical reaction well. 5 ul of
total RNA is the template. Each sample is assayed in triplicate.
Quantitative RT-PCR is performed using the ABI Prism.RTM. 7900HT
Sequence Detection System (SDS). Cycling parameters follow:
48.degree. C. for 30 min. for one cycle; 95.degree. C. for 10 min
for one cycle; 95.degree. C. for 15 sec, 60.degree. C. for 1 min.
for 40 cycles.
[0458] The SDS software calculates the threshold cycle (C.sub.T)
for each reaction, and C.sub.T values are used to quantitate the
relative amount of starting template in the reaction. The C.sub.T
values for each set of three reactions are averaged for all
subsequent calculations.
[0459] Total RNA is quantitated by using RiboGreen RNA Quantitation
Kit according to manufacturer's instructions and the % mRNA
expression is calculated using total RNA for normalization. %
knockdown is then calculated relative to the no addition
control.
13. In Vivo Studies by Using Antibodies
[0460] Treatment of Colon Cancer Cells with Monoclonal
Antibodies.
[0461] colon cancer cells are seeded at a density of
4.times.10.sup.4 cells per well in 96-well microtiter plates and
allowed to adhere for 2 hours. The cells are then treated with
different concentrations of anti-CCAT monoclonal antibody (Mab) or
irrelevant isotype matched (anti-rHuIFN-. gamma. Mab) at 0.05, 0.5
or 5.0 mug/ml. After a 72 hour incubation, the cell monolayers are
stained with crystal violet dye for determination of relative
percent viability (RPV) compared to control (untreated) cells. Each
treatment group consists of replicates. Cell growth inhibition is
monitored.
Treatment of NIH 3T3 Cells Overexpression CCAT Protein with
Monoclonal Antibodies.
[0462] NIH 3T3 expressing CCAT protein are treated with different
concentrations of anti-CCAT MAbs. Cell growth inhibition is
monitored.
In Vivo Treatment of NIH 3T3 Cells Overexpressing CCAT with
Anti-CCAT Monoclonal Antibodies.
[0463] NIH 3T3 cells transfected with either a CCAT expression
plasmid or the neo-DHFR vector are injected into nu/nu (athymic)
mice subcutaneously at a dose of 10.sup.6 cells in 0.1 ml of
phosphate-buffered saline. On days 0, 1, 5 and every 4 days
thereafter, 100 mug (0.1 ml in PBS) of either an irrelevant or
anti-CCAT monoclonal antibody of the IG2A subclass is injected
intraperitoneally. Tumor occurrence and size are monitored for 1
month period of treatment.
[0464] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention, which are obvious to those skilled in the field of
molecular biology or related fields, are intended to be within the
scope of the following claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110271357A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110271357A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References