U.S. patent application number 12/287040 was filed with the patent office on 2009-05-28 for pancreatic cancer targets and uses thereof.
This patent application is currently assigned to CELERA CORPORATION. Invention is credited to Bruno Domon, Ian McCaffery, Vaibhav Narayan, Scott Patterson.
Application Number | 20090138977 12/287040 |
Document ID | / |
Family ID | 40174938 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090138977 |
Kind Code |
A1 |
Domon; Bruno ; et
al. |
May 28, 2009 |
Pancreatic cancer targets and uses thereof
Abstract
The present invention provides a method for diagnosing and
detecting diseases associated with pancreas. The present invention
provides one or more proteins or fragments thereof, peptides or
nucleic acid molecules differentially expressed in pancreatic
diseases (PCAT) and antibodies binds to PCAT. The present invention
provides that PCAT is used as targets for screening agents that
modulates the PCAT activities. Further, the present invention
provides methods for treating diseases associated with
pancreas.
Inventors: |
Domon; Bruno; (Rockville,
MD) ; McCaffery; Ian; (Rockville, MD) ;
Narayan; Vaibhav; (Rockville, MD) ; Patterson;
Scott; (Newbury Park, CA) |
Correspondence
Address: |
CELERA CORPORATION
1401 HARBOR BAY PARKWAY
ALAMEDA
CA
94502
US
|
Assignee: |
CELERA CORPORATION
Alameda
CA
|
Family ID: |
40174938 |
Appl. No.: |
12/287040 |
Filed: |
October 3, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10912745 |
Aug 6, 2004 |
7473531 |
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12287040 |
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60493369 |
Aug 8, 2003 |
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60512690 |
Oct 21, 2003 |
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60519832 |
Nov 14, 2003 |
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60552390 |
Mar 12, 2004 |
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Current U.S.
Class: |
800/13 ;
424/133.1; 435/235.1; 435/326; 435/375; 436/501; 514/1.1; 530/350;
530/387.1; 530/387.3; 536/23.1; 536/24.5 |
Current CPC
Class: |
C07K 14/4705 20130101;
C12N 9/1044 20130101; C12Y 203/02013 20130101; C12Q 2600/106
20130101; C07K 16/2803 20130101; C07K 16/38 20130101; G01N 33/57438
20130101; C07K 16/2863 20130101; C07K 14/70596 20130101; C07K
14/705 20130101; C07K 14/8114 20130101; C07K 16/4266 20130101; C12N
15/113 20130101; G01N 2500/00 20130101; C07K 16/36 20130101; C12N
15/1138 20130101; C12Q 2600/158 20130101; C12N 2310/14 20130101;
C12Y 103/01071 20130101; C07K 2317/622 20130101; C07K 14/745
20130101; C07K 16/28 20130101; C07K 2317/24 20130101; C07K 16/2896
20130101; C12N 9/14 20130101; C12Q 2600/112 20130101; C12Q 1/6886
20130101; C07K 16/40 20130101; Y10T 436/25 20150115; C12N 9/001
20130101; C12Y 306/03009 20130101; C12Q 2600/136 20130101; C07K
14/71 20130101; C12Q 2600/118 20130101; C07K 14/70503 20130101;
A61P 35/00 20180101; C12N 15/1137 20130101; C07K 16/18
20130101 |
Class at
Publication: |
800/13 ; 530/350;
514/12; 536/23.1; 536/24.5; 530/387.1; 435/326; 435/235.1;
530/387.3; 424/133.1; 435/375; 436/501 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C07K 14/00 20060101 C07K014/00; A61K 38/16 20060101
A61K038/16; C07H 21/00 20060101 C07H021/00; C07H 21/02 20060101
C07H021/02; C12N 5/06 20060101 C12N005/06; A61P 35/00 20060101
A61P035/00; G01N 33/53 20060101 G01N033/53; A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C12N 5/16 20060101
C12N005/16; C12N 7/01 20060101 C12N007/01 |
Claims
1. An isolated protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS:1-288 and 703-873.
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:289-702; b) nucleotide sequences that encode a protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:1-288 and 703-873; 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 pancreatic cancer cells or stimulating
apoptosis of pancreatic 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
pancreatic 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 pancreatic 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:1-288 and 703-873; 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:289-702 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 pancreatic 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 pancreatic 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
[0001] This application is a divisional of U.S. application Ser.
No. 10/912,745, filed Aug. 6, 2004, which claims priority under 35
USC .sctn.119(e) to U.S. Provisional Application No. 60/493,369,
filed Aug. 8, 2003; U.S. Provisional Application No. 60/512,690,
filed Oct. 21, 2003; U.S. Provisional Application No. 60/519,832,
filed Nov. 14, 2003; and U.S. Provisional Application No.
60/552,390, filed Mar. 12, 2004.
FIELD OF THE INVENTION
[0002] 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 cancers.
BACKGROUND OF THE INVENTION
[0003] Cancer currently constitutes the second most common cause of
death in the United States. Carcinomas of the pancreas are the
eighth most prevalent form of cancer and fourth among the most
common causes of cancer deaths in this country.
[0004] The prognosis for pancreatic carcinoma is, at present, very
poor, it displays the lowest five-year survival rate among all
cancers. Such prognosis results primarily from delayed diagnosis,
due in part to the fact that the early symptoms are shared with
other more common abdominal ailments. Despite the advances in
diagnostic imaging methods like ultrasonography (US), endoscopic
ultrasonography (EUS), dualphase spiral computer tomography (CT),
magnetic resonance imaging (MRT), endoscopic retrograde
cholangiopancreatography (ERCP) and transcutaneous or EUS-guided
fine-needle aspiration (FNA), distinguishing pancreatic carcinoma
from benign pancreatic diseases, especially chronic pancreatitis,
is difficult because of the similarities in radiological and
imaging features and the lack of specific clinical symptoms for
pancreatic carcinoma.
[0005] Substantial efforts have been directed to developing tools
useful for early diagnosis of pancreatic carcinomas. Nonetheless, a
definitive diagnosis is often dependent on exploratory surgery
which is inevitably performed after the disease has advanced past
the point when early treatment may be effected.
[0006] One promising method for early diagnosis of various forms of
cancer is the identification of specific biochemical moieties,
termed targets, expressed differentially in the cancerous cells.
The targets are either cell surface proteins or cytosolic proteins.
Antibodies which will specifically recognize and bind to the
targets in the cancerous cells potentially provide powerful tools
for the diagnosis and treatment of the particular malignancy.
SUMMARY OF THE INVENTION
[0007] The present invention is based on the identification of
certain cell surface proteins or cytosolic proteins that are
differentially expressed in pancreatic disease. 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
pancreatic disease, as well as other cancers. 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 pancreatic cancer associated target, PCAT
proteins or PCAT nucleic acids or PCAT peptides, respectively.
[0008] The present invention provides peptides and protein
differentially expressed in pancreatic diseases (hereinafter PCAT),
particularly pancreatic cancer. Based on the site of protein
localization, e.g., surface or cytosolic, and protein
characterization, e.g. receptor or enzyme, specific uses of these
PCATs are provided. Some of the PCATs of the present invention
serve as targets for one or more classes of therapeutic agents,
while others may be suitable for antibody therapeutics.
[0009] Accordingly, the present invention provides a method for
diagnosing or detecting a pancreatic disease in a subject
comprising: determining the level of one or more PCAT proteins, or
any fragment(s) thereof, in a test sample from said subject,
wherein said PCAT protein comprises a sequence selected from a
group consisting of SEQ ID NOS: 1-288; wherein a differential level
of said PCAT 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 pancreatic disease.
[0010] The present invention also provides a method for detecting a
pancreatic disease in a subject comprising: determining the level
of one or more PCAT peptide(s) comprising a peptide sequence
selected from a group consisting of SEQ ID NOS: 703-873 in a test
sample from said subject, wherein a differential level of said PCAT
peptide(s) in said sample to the level of said PCAT peptide(s) in a
test sample from a healthy subject, or the level of said PCAT
peptide(s) established for a healthy subject, is indicative of the
pancreatic disease.
[0011] The present invention further provides a method for
detecting a pancreatic disease in a subject comprising: determining
the level of one or more PCAT nucleic acid(s), or any fragment(s)
thereof, in a test sample from said subject, wherein said PCAT
nucleic acid(s) encode a PCAT protein sequence selected from a
group consisting of SEQ ID NOS: 1-288; wherein a differential level
of said PCAT 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 the pancreatic disease.
[0012] The invention also provides methods for detecting the PCAT
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.
[0013] 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: 1-288.
[0014] The present invention further provides a composition
comprising an antibody that binds to a protein selected from a
group consisting of SEQ ID NOS: 1-288, and an acceptable
carrier.
[0015] The present invention further provides a method for treating
a pancreatic disease, comprising administering to a patient in need
of said treatment a therapeutically effective amount of one or more
antibody(ies) of this invention.
[0016] The present invention further provides a method for treating
a pancreatic disease comprising (i) identifying a subject having
pancreatic disease and (ii) administering to a said patient a
therapeutically effective amount of one or more antibody(ies) of
this invention.
[0017] The present invention further provides a method to screen
for agents that modulate PCAT protein activity, comprising the
steps of (i) contacting a test agent with a PCAT protein and (ii)
assaying for PCAT protein activity, wherein a change in said
activity in the presence of said agent relative to PCAT protein
activity in the absence of said agent indicates said agent
modulates said PCAT protein activity.
[0018] The present invention further provides a method to screen
for agents that bind to PCAT proteins, comprising the steps of (i)
contacting a test agent with a PCAT protein and (ii) measuring the
level of binding of agent to said PCAT protein.
[0019] The invention also provides diagnostic methods for human
disease, in particular for pancreatic cancer, its metastatic stage,
and therapeutic potential.
[0020] 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 cancers.
[0021] The invention also provides a method for monitoring the
disease progression and the treatment progress.
[0022] The invention further provide a method of diagnosis by an
array, wherein the array is immobilized with two or more PCAT
proteins, peptides or nucleic acid molecules. The proteins,
peptides or nucleic acid molecules include but are not limited to
the SEQ ID NOS: 1-873.
[0023] The invention also provides monoclonal or polyclonal
antibodies and composition thereof reactive with antigenic portion
of PCAT protein, peptides or fragments thereof in a form for use in
pancreatic diseases diagnosis.
[0024] The invention further provides an immunogenic antibody for
treating pancreatic disease or diseases associated with pancreatic
diseases, preferably pancreatic cancer.
[0025] The present invention provides a method for screening agents
that modulate PCAT activity, comprising the steps of (a) contacting
a sample comprising a PCAT with an agent; and (b) assaying for PCAT
activity, wherein a change in said PCAT activity in the presence of
said agent relative to PCAT activity in the absence of said
compound indicates said agent modulates PCAT. The agents include
but are not limited to protein, peptide, antibody, nucleic acid
such as antisense RNA, RNAi fragments, small molecules.
[0026] The present invention further provides a method for treating
pancreatic diseases, comprising: administering to a patient one or
more agents in a therapeutically effective amount to treat
pancreatic diseases.
[0027] The present invention provides a method for treating
pancreatic diseases, comprising: identifying a subject having
pancreatic diseases; and administering to a patient to one or more
antibodies in a therapeutically effective amount to treat
pancreatic diseases.
[0028] The present invention further provide method for diagnosis
and treatment for pancreatic cancer.
[0029] 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 cancers.
Description of the Files Contained on the CD-R Named
CL001538CDR
[0030] The CD-R named CL001538CDR contains the following three text
(ASCII) files:
1) File SEQLIST.sub.--1538DIV.txt provides the Sequence Listing.
The Sequence Listing provides the protein sequences (SEQ ID NOS:
1-288); transcript sequences (SEQ ID NOS: 289-702) and peptide
sequences (SEQ ID NOS: 703-873) as shown in Table 2. File
SEQLIST.sub.--1538DIV.txt is 3,231 KB in size. 2) File
Table1.sub.--1538.txt is 185 KB in size. 3) File
Table2.sub.--1538.txt is 316 KB in size.
[0031] The material contained on the CD-R labeled CL001538 CDR is
hereby incorporated by reference pursuant to 37 CFR 1.77(b)(4).
Description of Table 1 and Table 2
[0032] Table 1 (provided on the CD-R) discloses the peptides which
correspond to the protein, the pancreatic cancer cell lines, the
expression information, the ratio compare to the control sample.
The expression is based on measuring the level of the peptides.
Numerical representation of overexpression is indicated by more
than two, whereas numerical representation of underexpression is
indicated by less than 0.5. Overexpressed singleton indicates that
the peptide peak in diseased sample was detected and there was no
peak detected in control samples. Underexpressed singleton
indicates that the peptide peak was detected in the control sample
and there was no peak in the diseased sample.
[0033] Table 2 (provided on the CD-R) discloses the PCAT proteins,
transcripts, and peptides sequences that correlated to the Table
1.
[0034] The transcript/protein information includes:
[0035] a protein number (1 through 288)
[0036] a Celera protein internal identification number for the
protein encoded by the Celera transcript (hCP and/or UID)
[0037] a public protein accession number (Genbank e.g., RefSeq NP
number, Swiss-prot, or Derwent) for the protein
[0038] an art-known gene/protein name
[0039] a Celera transcript internal identification number (hCT
and/or UID)
[0040] a public transcript accession number (Genbank e.g., RefSeq
NM number, or Derwent)
[0041] a Celera hCG and UID internal identification numbers for the
gene
[0042] an art-known gene symbol
[0043] Celera genomic axis position (indicating start nucleotide
position-stop nucleotide position)
[0044] the chromosome number of the chromosome on which the gene is
located
[0045] 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
[0046] alternative gene/protein name(s) and/or symbol(s) in the
OMIM entry
DESCRIPTION OF FIGURES
[0047] FIG. 1. Tissue Factor expression in various tumor types by
immunohistochemistry.
[0048] FIG. 2. Kunitz-type 1 protease inhibitor expression in
various tumor types by immunohistochemistry.
[0049] FIG. 3. Mucin 4 expression in various tumor types by
immunohistochemistry.
[0050] FIG. 4. Kunitz-type 1 protease inhibitor mRNA expression in
pancreatic cell lines. Kunitz-type 1 protease inhibitor mRNA
expression was measured across the pancreatic cell line panel and
calculated as fold difference from the reference cell line Hs766t.
mRNA expression was found to be over-expressed in several of the
cell lines relative to Hs766t, including the SU.86.86 cell line
consistent ICAT data collected by mass spectrometry.
[0051] FIG. 5. Caspase 3/7 Analysis of Mucin 4 (PA-116) knockdown.
Mucin siRNA induced apoptosis of ASPC-1 cells 3 days after
transfection, and level of apoptosis was dose-dependent on muscin
siRNA (n=3). The level of apoptosis on day 3 was similar to that
given by TRAIL+actinomycin D positive control.
[0052] FIG. 6. Knockdown effects on MTS proliferation. Mucin 4
siRNA inhibited the proliferation of ASPC-1 cells (n=3). The level
of inhibition was shown to be dose-dependent on the concentration
of Mucin 4 siRNA (n=3)
[0053] FIG. 7. Viability test on Mucin 4 (PA-116) siRNA after
transfection. The result shows that Mucin siRNA can inhibit about
50% of viable ASPC-1 cells 3 days after transfection (n=2)
[0054] FIG. 8. Analysis of individual Mucin 4 (PA-116) siRNA vs.
SMARTPool. The result shows that both SMARTPool Mucin 4 siRNA and
Mucin 4 duplex 1 siRNA can induce an inhibition of proliferation
and an increase in apoptosis 3 days after transfection
[0055] FIG. 9. Morphology of ASPC-1 Pancreatic Cancer Cells with
Mucin 4 SiRNA
[0056] FIG. 10. Target protein expression was measured in whole
cell extracts prepared from the pancreatic cell line panel.
Polyclonal antibodies raised in rabbits (Green Mountain Antibodies)
were used to detect Gs3786 or Retinol Dehydrogenase Type II
homolog. Targets were found to be over-expressed in several cell
lines relative to the reference cell line Hs766t.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
General Description
[0057] 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 a pancreatic disease by determining the level of one or
more PCAT protein(s) or any fragment(s) thereof is wherein the
level of PCAT protein(s) are determined by contacting one or more
antibody(ies) that specifically bind to the antigenic regions of
the PCAT 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.
[0058] A preferred method for detecting a pancreatic disease by
determining the level of one or more PCAT peptide(s) is wherein the
level of PCAT peptides(s) are determined by contacting one or more
antibody(ies) that specifically bind to the antigenic regions of
the PCAT 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.
[0059] A preferred method for detecting a pancreatic disease by
determining the level of one or more PCAT nucleic acid(s) is
wherein the level of said PCAT 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.
[0060] The methods for detecting a pancreatic disease provided by
the present invention may be used for diagnosing the presence of
disease in a patent, monitoring the presence of pancreatic disease
in patients undergoing treatment and testing for the reoccurrence
of pancreatic disease in patients that were successfully treated
for the pancreatic disease; preferably wherein the pancreatic
disease is pancreatic cancer. The test sample may be, but is not
limited to, a biological sample such as tissue, blood, serum or
biological fluid.
[0061] The present invention is based on the discovery of
protein(s) and peptide(s) that are differentially expressed in
pancreatic disease samples, for example, cancer sample versus
normal pancreatic samples. These proteins and peptide, and the
encoding nucleic acid molecules are associated with pancreatic
diseases, hereinafter the PCAT proteins, peptides or nucleic
acids.
[0062] 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 pancreatic disease tissues or cell lines
in an effort to discover and identify novel molecules associated
with the disease.
[0063] Based on these discoveries, the present invention provides
proteins, peptides, nucleic acids that are differential in
pancreatic 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 pancreatic diseases. The present invention provides a
detection reagent, markers for pancreatic diseases at various
stages, comprises PCAT sequences isolated from human pancreatic
disease tissue, sera, cell lines, blood or biological fluids.
[0064] The present invention provides a method for treating
pancreatic diseases targeting at PCAT. The treatment includes
administration of a therapeutically effective amount of composition
comprise, but not limit to, an antibody, an immunogentic 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 PCAT. A "Pancreatic disease" can include
pancreatic cancer, pancreatic tumor (exocrine or endocrine),
pancreatic cysts, acute pancreatitis, chronic pancreatitis,
diabetes (type I and II) as well as pancreatic trauma, preferably
pancreatic cancer.
[0065] 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.
[0066] The present invention further provides the target for
screening an agent for PCAT, wherein the agent is compounds of
small molecules, proteins, peptides, nucleic acids, antibodies or
other agonists or antigonists.
[0067] Specifically, the following targets are selected for
targeting purpose: Tissue Factor, GS3786, Na/K ATPase Beta-3,
Kunitz Type inhibitor-2 (HAI-2), CD46, CD166, Neutral Amino Acid
Transporter (ASCT2), Transglutaminase-2 (TGM2), Kunitz Inhibitor-1,
ErbB3, Mucin 4, Decay Accelerating Factor (DAF). These factors are
associated with pancreatic cancer and are the potential target for
treating pancreatic cancer.
[0068] Tissue factor is known for its function for activation of
fvII resulting in generation of tumor growth promoting factors.
fVIIa is known to bind TF and may stimulate mitogenic signaling and
inhibit apoptosis of tumor cells.
[0069] Mucin 4 is a transmembrane mucin composed of two subunits
(alpha and beta) generated from proteolytic cleavage (beta is
transmembrane, alpha is extracellular). Mucin 4 and erbB2 is
localized to the apical membrane and stimulates differentiation of
polarized epithelium. Expression of neuregulin[NRG] by stromal
elements enables erbB2/erbB3 complex formation which stimulates
proliferation. Loss of polarization of epithelial cell allows Mucin
4/erbB2 and erbB3 complex formation, which potentiates
proliferative signaling. ErbB3 is unique among the ErbB receptor
family in that it possesses little or no intrinsic tyrosine kinase
activity. When ErbB3 is co-expressed with ErbB2, an active
signaling complex is formed and antidies directed against ErbB2 are
capable of disrupting this complex. Monoclonal antibody against
ErbB3 has an agonistic effect on the anchorage-independent growth
of cell lines expressing this receptor (Rajkumar et al., British J.
Cancer, 70(3): 459-465 (1994)).
[0070] Kunitz Type Protease Inhibitor-1, also called HAI-1, Serine
protease inhibitors or serpins. HAI is associate with hepatocyte
growth factor HGF/scatter factor/SF which is thought to play an
important role in the regeneration of injured gastrointestinal
mucosa by promoting the proliferation and migration of epithelial
cells. HGF is activated by HGFA which is activated locally by
thrombin. HAI-1 is upregulated and inhibits and sequesters HGFA at
the site of injury, thereby ensuring pericellular activation of HGF
through upregulation of injury associated proteases. HGFA is
released from HAI-1 upon cleavage with cell surface
metalloprotease.
[0071] Neutral Amino Acid Transporter (ASCT2) is a member of ASC/B0
amino acid transporter and it functions as Na+ dependent glutamine
transport.
[0072] Decay accelerating factor (CD55)'s primary function is to
inactivate the C3 convertases of the complement cascade by
dissociating them into their constituent proteins. It is expressed
on all serum exposed cells and provides protection from complement
activated proteases.
PCAT Peptide/Proteins and Peptides
[0073] The present invention provides isolated PCAT peptide and
protein molecules that consisting of, consisting essentially of, or
comprising the amino acid sequences of the PCAT peptides and
proteins disclosed in the Tables 1 and 2, (encoded by the nucleic
acid molecule shown in Table 2), 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.
[0074] In one embodiment PCAT peptides include, but are not limited
to, the amino acid sequence of SEQ ID NOS: 703-873 and variants
thereof. A PCAT protein includes, but is not limited to, the amino
acid sequence of SEQ ID NOS: 1-288 and variants thereof. PACT
proteins may be differentially expressed in pancreatic cell line,
blood, tissue, serum or body fluids.
[0075] 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.
[0076] The PCAT 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).
[0077] As used herein, a "peptide" is defined as amino acid
sequences between 5-20 amino acids derived from PCAT proteins such
as SEQ ID NOS: 1-288 or variants thereof. The peptide
differentially expressed in either pancreatic 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: 703-873, or variants thereof.
[0078] As used herein, a "protein" is full-length protein
differentially expressed in pancreatic 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: 1-288.
[0079] 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).
[0080] 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.
[0081] 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 PCAT 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.
[0082] The isolated PCAT 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 humans pancreatic cell lines. For
example, a nucleic acid molecule encoding the PCAT 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.
[0083] A PCAT 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.
[0084] 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 PCAT 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.
[0085] A chimeric or fusion PCAT 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 PCAT-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the PCAT protein or peptide.
[0086] As mentioned above, the PCAT peptide or the PCAT protein has
obvious variants of the amino acid sequence, such as naturally
occurring mature forms of the PCAT, allelic/sequence variants of
the PCAT, non-naturally occurring recombinantly derived variants of
the PCAT, and orthologs and paralogs of the PCAT proteins or
peptides. Such variants can readily be generated using art-known
techniques in the fields of recombinant nucleic acid technology and
protein biochemistry.
[0087] It is understood, however, that PCAT and variants exclude
any amino acid sequences disclosed prior to the invention.
[0088] 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 PCAT 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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 PCAT peptides of the present invention as
well as being encoded by the same genetic locus as the PCAT peptide
provided herein (See Table 2).
[0093] Allelic variants of a PCAT 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 PCAT
peptide as well as being encoded by the same genetic locus as the
PCAT peptide provided herein. Genetic locus can readily be
determined based on the genomic information provided in Table 2,
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 PCAT peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0094] Paralogs of a PCAT peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the PCAT 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 PCAT peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0095] Orthologs of a PCAT peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the PCAT 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 PCAT 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.
[0096] Non-naturally occurring variants of the PCAT 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 PCAT peptide. For example, one class of substitutions is
conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a PCAT 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).
[0097] Variant PCAT 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.
[0098] 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.
[0099] 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 PCAT 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)).
[0100] The present invention further provides fragments of the
PCATs, in addition to proteins and peptides that comprise and
consist of such fragments, particularly those comprising the
residues identified in Tables 1 and 2. As used herein, a fragment
comprises at least 8, 10, 12, 14, 16, 18, 20 or more contiguous
amino acid residues from a PCAT. Such fragments can be chosen based
on the ability to retain one or more of the biological activities
of the PCAT 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 PCAT, 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).
[0101] 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 PCATs are described in basic
texts, detailed monographs, and the research literature, and they
are well known to those of skill in the art.
[0102] 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.
[0103] 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)).
[0104] Accordingly, the PCATs 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 PCAT is fused
with another compound, such as a compound to increase the half-life
of the PCAT (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature PCAT, such as a
leader or secretory sequence or a sequence for purification of the
mature PCAT or a pro-protein sequence.
Protein/Peptide Uses
[0105] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in the Tables; 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 PCAT-effector
protein interaction or PCAT-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.
[0106] 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", 3rd ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 2001, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0107] 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, PCATs 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 PCAT.
Experimental data as provided in Table 1 indicate that the PCATs of
the present invention are expressed at differential level in
various pancreatic cell lines, for example SEQ ID NOS: 1-119 are
overexpressed in all tested cell lines, whereas other proteins or
peptides are underexpressed in selected cell lines. In one example,
protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) is
overexpressed in one cell line (HPAC), yet underexpressed in
another cell line (Su.86.86). A large percentage of pharmaceutical
agents are being developed that modulate the activity of PCAT
proteins, particularly members of the PCAT 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 humans pancreatic cell lines. Such uses can
readily be determined using the information provided herein, that
which is known in the art, and routine experimentation.
[0108] 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 PCATs that
are related to members of the PCAT subfamily. Such assays involve
any of the known PCAT functions or activities or properties useful
for diagnosis and treatment of PCAT-related conditions that are
specific for the subfamily of PCATs that the one of the present
invention belongs to, particularly in cells and tissues that
express the PCAT. Experimental data as provided in Table 1 indicate
that the PCATs of the present invention are expressed at
differential level in various pancreatic cell lines, for example
SEQ ID NOS: 1-119 are overexpressed in all tested cell lines,
whereas other proteins or peptides are underexpressed in selected
cell lines. In one example, protein (SEQ ID NO: 120) or peptide
(SEQ ID NO: 761) is overexpressed in one cell line (HPAC), yet
underexpressed in another cell line (Su.86.86).
[0109] 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 PCAT, as a biopsy or expanded in cell culture. Experimental
data as provided in Table 1 indicates expression in human
pancreatic cell lines. In an alternate embodiment, cell-based
assays involve recombinant host cells expressing the PCAT
protein.
[0110] The polypeptides can be used to identify compounds or agents
that modulate PCAT activity of the protein in its natural state or
an altered form that causes a specific disease or pathology
associated with the PCAT. Both the PCATs 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 PCAT. These compounds can be further
screened against a functional PCAT to determine the effect of the
compound on the PCAT 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 PCAT to a desired
degree.
[0111] 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 PCAT protein and a molecule that
normally interacts with the PCAT protein, e.g. a substrate or or an
extracellular binding ligand or a component of the signal pathway
that the PCAT protein normally interacts (for example, a cytosolic
signal protein or another PCAT). Such assays typically include the
steps of combining the PCAT protein with a candidate compound under
conditions that allow the PCAT 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 PCAT 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.
[0112] 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).
[0113] One candidate compound or agent is a soluble fragment of the
PCAT that competes for substrate binding. Other candidate compounds
include mutant PCATs or appropriate fragments containing mutations
that affect PCAT 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.
[0114] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) PCAT
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate PCAT 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 PCAT protein dependent signal cascade can be
assayed.
[0115] Any of the biological or biochemical functions mediated by
the PCAT 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 the Tables, particularly Table 1.
Specifically, a biological function of a cell or tissues that
expresses the PCAT can be assayed. Experimental data as provided in
Table 1 indicate that the PCATs of the present invention are
expressed at differential level in various pancreatic cell lines,
for example SEQ ID NOS: 1-119 are overexpressed in all tested cell
lines, whereas other proteins or peptides are underexpressed in
selected cell lines. In one example, protein (SEQ ID NO: 120) or
peptide (SEQ ID NO: 761) is overexpressed in one cell line (HPAC),
yet underexpressed in another cell line (Su.86.86).
[0116] Binding and/or activating compounds can also be screened by
using chimeric PCAT 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
PCAT. 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 PCAT is derived.
[0117] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the PCAT (e.g. binding partners and/or
ligands). Thus, a compound is exposed to a PCAT polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble PCAT polypeptide is also added to the
mixture. If the test compound interacts with the soluble PCAT
polypeptide, it decreases the amount of complex formed or activity
from the PCAT. This type of assay is particularly useful in cases
in which compounds are sought that interact with specific regions
of the PCAT. Thus, the soluble polypeptide that competes with the
target PCAT region is designed to contain peptide sequences
corresponding to the region of interest.
[0118] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the PCAT 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.
[0119] 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 PCAT-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 PCAT-binding
protein and a candidate compound are incubated in the PCAT
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 PCAT protein target molecule, or which are
reactive with PCAT protein and compete with the target molecule, as
well as PCAT-linked assays which rely on detecting an enzymatic
activity associated with the target molecule.
[0120] Agents that modulate one of the PCATs 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.
[0121] Modulators of PCAT protein activity identified according to
these drug screening assays can be used to treat a subject with a
disorder mediated by the PCAT pathway, by treating cells or tissues
that express the PCAT. Experimental data as provided in Table 1
indicates expression in human pancreatic cell lines. These methods
of treatment include the steps of administering a modulator of PCAT
activity in a pharmaceutical composition to a subject in need of
such treatment, the modulator being identified as described
herein.
[0122] In yet another aspect of the invention, the PCAT 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) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with the PCAT
and are involved in PCAT activity. Such PCAT-binding proteins are
also likely to be involved in the propagation of signals by the
PCAT proteins or PCAT targets as, for example, downstream elements
of a PCAT-mediated signaling pathway. Alternatively, such
PCAT-binding proteins are likely to be PCAT inhibitors.
[0123] 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 PCAT
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 PCAT-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 PCAT protein.
[0124] 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 PCAT-modulating
agent, an antisense PCAT nucleic acid molecule, an PCAT-RNAi
fragment, a PCAT-specific antibody, or a PCAT-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.
[0125] The PCAT 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 pancreatic cell lines. The method involves contacting a
biological sample with a compound capable of interacting with the
PCAT 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.
[0126] 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.
[0127] 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 PCAT 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.
[0128] 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.
[0129] 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
PCAT protein in which one or more of the PCAT 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 PCAT 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.
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 pancreatic cell lines. Accordingly, methods for
treatment include the use of the PCAT protein or fragments.
Antibodies
[0130] The present invention provides antibodies specifically bind
to PCAT proteins or fragments thereof, peptides, or antigenic
portion thereof.
[0131] 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.
[0132] The antibody of present invention selectively binds a target
PCAT 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.
[0133] 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.
[0134] 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).
[0135] 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.
[0136] 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.
[0137] "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).
[0138] 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.
[0139] 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.
[0140] 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(ab').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.
[0141] 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.
[0142] 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.
[0143] The present invention further provides monoclonal
antibodies, polyclonal antibodies 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 PCAT protein can be used.
Particularly important fragments are those covering functional
domains, some but not all the examples of the domains are
identified in Tables. 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).
[0144] 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
procedures for making a monoclonal antibody, see the Example
below.
[0145] "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).
[0146] 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 PCAT
proteins, 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.
[0147] 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.
[0148] The protein used herein 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 PCAT
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: 703-873 and fragments of SEQ ID NOS:
1-288. 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
elecrophoresis, 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.
[0154] 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
[0155] 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 PCATs of
the present invention are expressed at differential level in
various pancreatic cell lines, for example SEQ ID NOS: 1-119 are
overexpressed in all tested cell lines, whereas other proteins or
peptides are underexpressed in selected cell lines. In one example,
protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) is
overexpressed in one cell line (HPAC), yet underexpressed in
another cell line (Su.86.86). 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.
[0156] 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 pancreatic cell lines. 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.
[0157] 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 pancreatic cell lines. 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 diagnostic methods are described in detail below.
[0158] 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.
[0159] The antibodies are also useful for tissue typing.
Experimental data as provided in Table 1 indicates expression in
human pancreatic cell lines. 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.
[0160] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the PCAT peptide to
a binding partner such as a substrate or another antibody binding
to the PCAT. 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.
[0161] 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
[0162] The present invention further provides isolated nucleic acid
molecules that encode a PCAT 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 PCAT 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in Table 2,
(SEQ ID NOS: 289-702), or any nucleic acid molecule that encodes
the protein provided in Table 2, (SEQ ID NOS: 1-288). 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.
[0167] In Table 2, 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.
[0168] 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.
[0169] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the PCAT
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.
[0170] 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).
[0171] 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 PCAT
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.
[0172] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in Tables 1 and 2. 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.
[0173] 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 the gene.
[0174] 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.
[0175] 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 the Table 2 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.
[0176] 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 is 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
[0177] 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 Tables 1 and 2 and to isolate cDNA and genomic
clones that correspond to variants (alleles, orthologs, etc.)
producing the same or related peptides shown in Tables 1 and 2.
[0178] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Tables.
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.
[0179] 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.
[0180] 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.
[0181] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0182] 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.
[0183] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0184] 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.
[0185] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0186] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0187] 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 PCATs of the present invention are expressed at
differential level in various pancreatic cell lines, for example
SEQ ID NOS: 1-119 are overexpressed in all tested cell lines,
whereas other proteins or peptides are underexpressed in selected
cell lines. In one example, protein (SEQ ID NO: 120) or peptide
(SEQ ID NO: 761) is overexpressed in one cell line (HPAC), yet
underexpressed in another cell line (Su.86.86). 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 PCAT
protein expression relative to normal results.
[0188] 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.
[0189] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a PCAT protein, such as
by measuring a level of a PCAT-encoding nucleic acid in a sample of
cells from a subject e.g., mRNA or genomic DNA, or determining if a
PCAT gene has been mutated. Experimental data as provided in Table
1 indicate that the PCATs of the present invention are expressed at
differential level in various pancreatic cell lines, for example
SEQ ID NOS: 1-119 are overexpressed in all tested cell lines,
whereas other proteins or peptides are underexpressed in selected
cell lines. In one example, protein (SEQ ID NO: 120) or peptide
(SEQ ID NO: 761) is overexpressed in one cell line (HPAC), yet
underexpressed in another cell line (Su.86.86). More detection and
diagnostic methods are described in detail below.
[0190] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate PCAT nucleic acid
expression.
[0191] 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 PCAT gene, particularly biological
and pathological processes that are mediated by the PCAT in cells
and tissues that express it. Experimental data as provided in Table
1 indicates expression in human pancreatic cell lines. The method
typically includes assaying the ability of the compound to modulate
the expression of the PCAT nucleic acid and thus identifying a
compound that can be used to treat a disorder characterized by
undesired PCAT nucleic acid expression. The assays can be performed
in cell-based and cell-free systems. Cell-based assays include
cells naturally expressing the PCAT nucleic acid or recombinant
cells genetically engineered to express specific nucleic acid
sequences.
[0192] The assay for PCAT 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 PCAT 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.
[0193] Thus, modulators of PCAT 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 PCAT mRNA in the presence of the candidate compound
or agent is compared to the level of expression of PCAT 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.
[0194] 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
PCAT nucleic acid expression in cells and tissues that express the
PCAT. Experimental data as provided in Table 1 indicate that the
PCATs of the present invention are expressed at differential level
in various pancreatic cell lines, for example SEQ ID NOS: 1-119 are
overexpressed in all tested cell lines, whereas other proteins or
peptides are underexpressed in selected cell lines. In one example,
protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) is
overexpressed in one cell line (HPAC), yet underexpressed in
another cell line (Su.86.86). Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0195] 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
PCAT nucleic acid expression in the cells and tissues that express
the protein. Experimental data as provided in Table 1 indicates
expression in human pancreatic cell lines.
[0196] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds or agents on the
expression or activity of the PCAT 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.
[0197] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in PCAT nucleic acid expression, and
particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in PCAT
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 PCAT 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 PCAT 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 PCAT protein.
[0198] Individuals carrying mutations in the PCAT 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.
[0199] Alternatively, mutations in a PCAT gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0200] 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.
[0201] 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 PCAT 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)).
[0202] 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.
[0203] 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 PCAT gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment.
[0204] 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.
[0205] The nucleic acid molecules are thus useful as antisense
constructs to control PCAT 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 PCAT protein. An
antisense RNA or DNA nucleic acid molecule would hybridize to the
mRNA and thus block translation of mRNA into PCAT protein.
[0206] 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.
[0207] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of PCAT nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired PCAT 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 PCAT protein, such as substrate
binding.
[0208] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in PCAT 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 PCAT protein to treat the individual.
[0209] The invention also encompasses kits for detecting the
presence of a PCAT nucleic acid in a biological sample.
Experimental data as provided in Table 1 indicate that the PCATs of
the present invention are expressed at differential level in
various pancreatic cell lines, for example SEQ ID NOS: 1-119 are
overexpressed in all tested cell lines, whereas other proteins or
peptides are underexpressed in selected cell lines. In one example,
protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) is
overexpressed in one cell line (HPAC), yet underexpressed in
another cell line (Su.86.86). For example, the kit can comprise
reagents such as a labeled or labelable nucleic acid or agent
capable of detecting PCAT nucleic acid in a biological sample;
means for determining the amount of PCAT nucleic acid in the
sample; and means for comparing the amount of PCAT 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 PCAT protein mRNA or DNA.
Vectors/Host Cells
[0210] 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.
[0211] 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.
[0212] 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).
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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).
[0217] 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).
[0218] 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.
[0219] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodologies. 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.
[0220] 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.
[0221] 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)).
[0222] 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.).
[0223] 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)).
[0224] 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)).
[0225] 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, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 2001.
[0226] 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).
[0227] 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.
[0228] 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, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 2001).
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] Where secretion of the peptide is desired, which may be
difficult to achieve with multi-transmembrane domain containing
proteins such as PCATs, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0234] Where the peptide is not secreted into the medium, 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.
[0235] 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
[0236] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a PCAT protein or peptide that can be further purified to
produce desired amounts of PCAT protein or fragments. Thus, host
cells containing expression vectors are useful for peptide
production.
[0237] Host cells are also useful for conducting cell-based assays
involving the PCAT protein or PCAT protein fragments, such as those
described above as well as other formats known in the art. Thus, a
recombinant host cell expressing a native PCAT protein is useful
for assaying compounds that stimulate or inhibit PCAT protein
function.
[0238] Host cells are also useful for identifying PCAT 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 PCAT protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native PCAT protein.
[0239] 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 PCAT protein and identifying
and evaluating modulators of PCAT protein activity. Other examples
of transgenic animals include non-human primates, sheep, dogs,
cows, goats, chickens, and amphibians.
[0240] 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 PCAT
protein nucleotide sequences can be introduced as a transgene into
the genome of a non-human animal, such as a mouse.
[0241] 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 PCAT
protein to particular cells.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] 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, PCAT 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 PCAT protein function,
including substrate interaction, the effect of specific mutant PCAT
proteins on PCAT protein function and substrate interaction, and
the effect of chimeric PCAT proteins. It is also possible to assess
the effect of null mutations, that is, mutations that substantially
or completely eliminate one or more PCAT protein functions.
Detection and Diagnosis
[0246] The present invention provides a method for detecting PCAT
nucleic acids, proteins, peptides and fragments thereof that are
differentially expressed in pancreatic diseases in a test sample,
preferably in a biological sample.
[0247] The present invention further provides a method for
diagnosing the pancreatic diseases, by detecting the nucleic acids,
proteins, peptides and fragments thereof. The further embodiment
includes but is not limited to, monitoring the disease prognosis
(recurrence), diagnosing disease stage, preventing the disease and
treating the disease.
[0248] 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).
[0249] As used herein, a "differential level" is defined as the
level of PCAT 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 from a control cell line,
a normal tissue or body fluids, or combination thereof, or from a
healthy subject. While the protein is overexpressed, the expression
of PCAT is preferably greater than about 20%, or preferably greater
than about 30%, and most preferably greater than about 50% or more
of pancreatic disease sample, at a level that is at least two fold,
and preferably at least five fold, greater than the level of
expression in control samples, as determined using a representative
assay provided herein. While the protein is underexpressed, the
expression of PCAT is preferably less than about 20%, or preferably
less than 30%, and most preferably less than about 50% or more of
the pancreatic disease sample, at a level that is at least 0.5
fold, and preferably at least 0.2 fold less than the level of the
expression in control samples, as determined using a representative
assay provided herein.
[0250] 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 pancreatic
diseases or pancreatic associated diseases by using conventional
diagnostic methods.
[0251] As used herein, the "diseases" include pancreatic diseases
and pancreatic associated diseases. Preferably, the pancreatic
disease is pancreatic cancer.
[0252] 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
[0253] The present invention is not limited to the detection
methods described above. Any suitable detection method that allows
for the specific detection of pancreatic disease cells, tissues or
organs may be utilized. For example, in some embodiments, the
expression of RNA corresponding to a PCAT 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.).
[0254] In another embodiment, the present invention provides a
method for diagnosing or detecting pancreatic diseases in a subject
comprising: determining the level of one or more PCAT nucleic acid
molecules or any fragment(s) thereof in a test sample from said
subject, wherein said PCAT nucleic acid molecule(s) comprises a
sequence selected from a group consisting of SEQ ID NOS: 289-702
and a combination thereof; wherein a differential level of said
PCAT 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
pancreatic diseases.
[0255] 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: 289-702 and combination
thereof.
[0256] In further embodiments of the present invention, the
presence of particular sequences in the genome of a subject is
detected. Such sequences include PCAT sequences associated with
abnormal expression of PCAT (e.g., overexpression or expression at
a physiological inappropriate time). These sequences include
polymorphisms, including polymorphisms in the transcribed sequence
(e.g., that effect PCAT 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
[0257] The present invention provides methods for diagnosing or
detecting the differential presence of PCAT proteins. In some
embodiments (e.g., where PCATs are overexpressed in diseased
cells), PCAT proteins are detected directly. In other embodiments
(e.g., where the presence of a PCATs are underexpressed), PCATs to
the disease antigens are detected non-existence.
[0258] 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 PCAT may be indicative of
a specific stage or sub-type of the same cancer or disease.
[0259] The information obtained is also used to determine prognosis
and appropriate course of treatment. For example, it is
contemplated that individuals with a specific PCAT expression or
stage of pancreatic diseases may respond differently to a given
treatment that individuals lacking the PCAT expression. The
information obtained from the diagnostic methods of the present
invention thus provides for the personalization of diagnosis and
treatment.
[0260] In one embodiment, the present invention provides a method
for monitoring pancreatic diseases treatment in a subject
comprising: determining the level of one or more PCAT proteins or
any fragment(s) or peptide(s) thereof in a test sample from said
subject, wherein said PCAT protein(s) comprises a sequence selected
from a group consisting of SEQ ID NOS: 1-288, SEQ ID NOS: 703-873
and a combination thereof; wherein an level of said PCAT 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.
[0261] In another embodiment, the present invention provides a
method for diagnosing recurrence of pancreatic diseases following
successful treatment in a subject comprising: determining the level
of one or more PCAT proteins or any fragment(s) or peptide(s)
thereof in a test sample from said subject, wherein said PCAT
protein(s) comprises a sequence selected from a group consisting of
SEQ ID NOS: 1-288, SEQ ID NOS: 703-873 and a combination thereof;
wherein a changed level of said PCAT 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 pancreatic diseases.
[0262] In yet another embodiment, the present invention provides a
method for diagnosing or detecting pancreatic diseases in a subject
comprising: determining the level of one or more PCAT proteins or
any fragment(s) or peptides thereof in a test sample from said
subject, wherein said PCAT protein(s) comprises a sequence selected
from a group consisting of SEQ ID NOS: 1-288, SEQ ID NOS: 703-873
and a combination thereof; wherein a differential level of said
PCAT 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 pancreatic diseases.
[0263] 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: 1-288 and combination thereof.
[0264] Further, the detecting or diagnosing method comprises
determining level of differential expression of 5, 10, 15, 20, 40,
60, 80, 100 or more PCAT peptides, preferably the peptides are
selected from the group consisting of SEQ ID NOS: 703-873 and
combination thereof.
[0265] 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.
[0266] In yet another embodiment, the present invention provides a
detection or diagnostic method of PCATs by using LC/MS. The
proteins from cells are prepared by methods known in the art (for
example, 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.
[0267] 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 PCAT protein or peptide are similar to the expression
pattern in Table 1 in a test subject indicates the likelihood of
having pancreatic diseases or diseases associated with
pancreas.
[0268] 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 the
sections below.
Antibody Detections
[0269] 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
proteins among various tissues in an organism and over the course
of normal development.
[0270] 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.
[0271] 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 PCAT
protein, peptide or fragment thereof, in specific cells, tissues,
blood, serum or body fluids.
[0272] 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:
[0273] (a) Radioisotopes, such as .sup.36S, .sup.14C, .sup.125I,
.sup.3H, 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.
[0274] (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.
[0275] (c) Various enzyme-substrate labels are available and U.S.
Pat. Nos. 4,275,149, and 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).
[0276] 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.
[0277] In another embodiment of the invention, the antibody needs
not be labeled, and the presence thereof can be detected using a
labeled antibody, which binds to the antibody.
[0278] 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).
[0279] The biological samples can then be tested directly for the
presence of PCAT by assays (e.g., ELISA or radioimmunoassay) and
format (e.g., microwells, dipstick as described in International
Patent Publication WO 93/03367). 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 PCAT 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.
[0280] Antibody binding is detected by techniques known in the art
(e.g., radioimmunoassay, 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.
[0281] 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.
[0282] 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.
[0283] 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.
[0284] 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 PCAT
targets and the affinity value(Kd) is less than 1.times.10.sup.8
M.
[0285] 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.
[0286] 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 Examples). 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 PCAT protein express in situ. The detailed procedure is
shown in the Example.
Array:
[0287] "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.
[0288] 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, mirrors 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.
[0289] A substrate includes but not limits to, paper, nylon or
other type of membrane, filter, chip, glass slide, or any other
suitable solid support. The invention also provides an array with a
cDNA or transcript encoding PCAT proteins or peptides or fragments
thereof, antibodies that specifically bind PCAT proteins, peptides
or fragments thereof. Preferably, two or more of the nucleic acid
molecules (e.g., SEQ ID NOS: 289-702), proteins (e.g., SEQ ID NOS:
1-288) or peptides (e.g., SEQ ID NOS: 703-873) are immobilized on a
substrate. Specifically, the following targets are selected for
targeting purpose:Tissue Factor, GS3786, Na/K ATPase Beta-3, Kunitz
Type inhibitor-2 (HAI-2), CD46, CD166, Neutral Amino Acid
Transporter (ASCT2), Transglutaminase-2 (TGM2), Kunitz Inhibitor-1,
ErbB3, Mucin 4, Decay Accelerating Factor (DAF).
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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.
[0295] A gene expression profile comprises the expression of a
plurality of transcripts as measured by 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.
[0296] 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.
[0297] 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.
[0298] By analyzing changes in patterns of gene/protein 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.
[0299] 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.
[0300] 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.
[0301] In one embodiment, the detected targets comprise, consist
essentially of or consist of combinations of PCAT proteins or
nucleic acids encoding such proteins. The combinations are either
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80,
90, 100, 150, 200, 250 proteins (SEQ ID NOS: 1-288), nucleic acids
(SEQ ID NOS: 289-702) encoding such proteins, or peptides (SEQ ID
NOS: 703-873).
[0302] In one embodiment, the combinations of the protein or
nucleic acid targets for detection of a pancreatic cancer comprise
targets selected from a group consisting of Tissue Factor, GS3786,
Na/K ATPase Beta-3, Kunitz Type inhibitor-1 (HAI-1), Kunitz Type
inhibitor-2 (HAI-2), CD46, CD 166, Neutral Amino Acid Transporter
(ASCT2), Transglutaminase-2 (TGM2), Kunitz Inhibitor-1, ErbB3,
Mucin 4, Decay Accelerating Factor (DAF), wherein their
corresponding sequences are listed in Table 2.
[0303] In another embodiment, the detection targets are combination
of all the targets of Tissue Factor, GS3786, Na/K ATPase Beta-3,
Kunitz Type inhibitor-1 (HAI-1), Kunitz Type inhibitor-2 (HAI-2),
CD46, CD166, Neutral Amino Acid Transporter (ASCT2),
Transglutaminase-2 (TGM2), Kunitz Inhibitor-1, ErbB3, Mucin 4,
Decay Accelerating Factor (DAF).
[0304] In yet another embodiment, the invention provides a
composition comprising a plurality of PCAT nucleic acid sequences
for use in detecting the differential expression of genes in a
disease state, wherein said plurality of nucleic acid sequences
comprise two or more sequences of SEQ ID NOS: 289-702, or all the
sequences of SEQ ID NOS: 289-702, or the complete complements
thereof. Further, the nucleic acid sequences are immobilized on a
substrate, and the said nucleic acid sequences are hybridizable
elements on a microarray.
[0305] In yet another embodiment, the invention provides a
composition comprising a plurality of PCAT proteins or peptides for
use in detecting the differential expression of proteins in a
disease state wherein said plurality of protein sequences comprise
two or more sequences of SEQ ID NOS: 1-288 or all sequence of SEQ
ID NOS: 1-288; and wherein said plurality of peptide sequences
comprise two or more sequences of SEQ ID NOS: 703-873 or all
sequence of SEQ ID NOS: 703-873.
[0306] In yet another embodiment, the compositions can be used in
diagnosing or monitoring the treatment of pancreatic cancer
Treatment
[0307] The following terms, as used in the present specification
and claims, are intended to have the meaning as defined below,
unless indicated otherwise.
[0308] "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.
[0309] 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.
[0310] A "pancreatic disease" includes pancreatic cancer,
pancreatic tumor (exocrine or endocrine), pancreatic cysts, acute
pancreatitis, chronic pancreatitis, diabetes (type I and II) as
well as pancreatic trauma, preferably pancreatic cancer.
[0311] A "cancer" is epithelial-cell related cancers include but
not limited to pancreatic, lung, colon, prostate, ovarian, breast,
bladder renal, hepatocellular, pharyngeal and gastric cancers.
[0312] The present invention provides an application of treatment
by using antibody, immunogenic peptides as well as other PCAT
agonists or antagonists.
[0313] PCATs are proteins differentially expressed in the
pancreatic diseases cell lines or tissues. The proteins are either
cell surface proteins or cytosolic proteins (see the list in Table
2). These proteins are associated with the diseases especially
pancreatic diseases, particularly pancreatic cancer; thus, they
serve as candidate targets for the treatment of the diseases.
[0314] 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.
[0315] 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.
[0316] 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
[0317] The antibody of the present invention can be used for
therapeutic reasons. It is contemplated that the antibody of the
present invention may be used to treat a mammal, preferably a human
with pancreatic diseases.
[0318] In general, the antibodies are also useful for inhibiting
protein function, for example, blocking the binding of the PCAT
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. Other evidence is provided in U.S. Pat. No.
6,207,152, and U.S. Pat. No. 6,387,371.
[0319] 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 PCAT can
be passively administered alone or in conjunction with other
anti-cancer therapies to a mammal afflicted-with pancreatic
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).
[0320] 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.
[0321] The mechanism for antibody therapy is that the therapeutic
antibody recognizes a cell surface protein or a cytosolic 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.
[0322] 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.
[0323] 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.
[0324] 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.
[0325] 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.
[0326] 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.
[0327] 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.
[0328] 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.
[0329] 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
[0330] 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.
[0331] 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.
[0332] 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.
[0333] 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.).
[0334] 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.
[0335] 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.
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 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 PCAT 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.
[0342] 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.
[0343] 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
[0344] The PCAT proteins, 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.
[0345] The antiserum from immunized individuals may be administered
as a prophylactic measure for individuals who are at risk of
developing pancreatic diseases or cancer. The antiserum is also
useful in treating an individual afflicted with pancreatic diseases
or cancer for post-disease prophylaxis.
[0346] Alternatively, peptides derived form the PCAT 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 Terr A. I. (eds) Appleton and Lange, Norwalk Conn., San
Mateo, Calif.).
[0347] 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.
[0348] Alternatively, amino acid sequence variants of the peptide
can be prepared by mutations in the DNA, which encodes the peptide,
or by peptide synthesis.
[0349] 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.
[0350] 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).
[0351] 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.
[0352] The peptides of the present invention are used for treatment
of the pancreatic diseases. Treatment involves administration of
the protective composition after the appearance of the disease.
[0353] 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."
[0354] 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 pancreatic diseases and diseases resulting from
unregulated clonal T cell replication.
[0355] Therefore, the present invention relates to peptides or
modified peptides derived from the protein sequences of the PCAT
proteins that differentially expressed in the pancreatic 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.
[0356] 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.
[0357] The recombinant or natural protein, peptides, or fragment
thereof of PCAT, 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 pancreatic diseases, particularly, cancer. The prophylactic
administration of the pancreatic disease vaccine should serve to
prevent or attenuate pancreatic diseases, preferably cancer, in a
mammal.
[0358] Preparation of Vaccine is Using Recombinant Protein or
Peptide Expression vectors comprising all or part of nucleic acid
sequence of PCAT 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:
289-702 can be introduced into a mammal either prior to any
evidence of pancreatic diseases or to mediate regression of the
disease in a mammal afflicted with pancreatic 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
PCAT 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 PCAT 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.
[0359] Alternatively all or parts thereof of a substantially or
partially purified the PCAT proteins 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.
[0360] 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.).
[0361] The vaccine formulation of the present invention comprises
an immunogen that induces an immune response directed against the
cancer associated antigens such as the PCATs, 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.
[0362] 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
[0363] In one embodiment mammals, preferably human, at high risk
for pancreatic 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 pancreatic diseases, humans with a history of pancreatic
diseases, particular cancer, or humans afflicted with pancreatic
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 pancreatic diseases or advanced stage of pancreatic
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.
[0364] 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.
[0365] 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-PCAT immune cells or anti-PCAT antibody
is produced. The presence of anti-PCAT immune cells may be assessed
by measuring the frequency of precursor CTL (cytoxic T-lymphocytes)
against PCAT 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.
[0366] 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, pancreatic diseases in patients can be tested.
Conventional methods would be used to evaluate the immune response
of the patient to determine the efficiency of the vaccine.
[0367] In yet another embodiment of this invention all, part, or
parts of the PCAT 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 PCAT antigen activated dendritic cells or
processed dendritic cell antigens may be used as immunogens for
vaccines or for the treatment of pancreatic diseases, particularly
pancreatic 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).
[0368] In yet another aspect of this invention T-cells isolated
from individuals can be exposed to the PCAT proteins, peptides or
fragments 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).
[0369] 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.
[0370] 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, Cold Spring Harbor,
N.Y. (2001).
WORKING EXAMPLES
1. Pancreatic Cell Line Model System
[0371] Analysis of gene expression in various pancreatic cancer
cell lines as well as pancreatic duct epithelial tissue has shown
that the cell line Hs766T correlates well with normal tissue. For
this reason, this cell line is reported in the literature as being
a good surrogate for normal tissue in analyses of differential
expression between pancreatic adenomcarcinoma (and derived tumor
lines) and normal tissue (or surrogate, Hs766T). The model system
employed here involves the use of Hs766T as a "normal" reference to
which cell surface expression in tumor derived cell lines is
compared. These differentials or candidates are then validated in
tissues, pancreatic cancer and normal pancreas, to confirm that
they are differentially expressed between these tissues as well as
within the cell line model system. Details of the pancreatic tumor
lines that were used for this study, as well as the pancreatic line
Hs766T are provided below.
TABLE-US-00001 TABLE 3 Cell Lines and Media Fetal ATCC Base
Nonessential Sodium Sodium Bovine Cell line Reference medium
Glutamine amino acids Carbonate Pyruvate Hepes Serum Panc-1
CRL-1469 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) Hs766t
HTB-134 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) SU.86.86
CRL-1837 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) AsPC1
CRL-1682 RPMI 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10 mM 20% (v/v) HPAF II
CRL-1997 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) HPAC CRL-2119
DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) Mia-Paca-2 CRL-1420
DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) Mpanc-96 CRL-2380 RPMI
2 mM 1% (w/v) 0.1% (w/v) 1 mM 10 mM 10% (v/v) BxPC-3 CRL-1687 RPMI
2 mM 1% (w/v) 0.1% (w/v) 1 mM 10 mM 10% (v/v) Capan-2 HTB-80 DMEM 2
mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v)
Pancreatic Cancer Cell Line Culture
[0372] Cell lines were grown in a culturing medium that is
supplemented as necessary with growth factors and serum (as
described in Table 3). 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.
[0373] 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
Hs766T for the purpose of identifying statistically significant
reproducible differentially expressed proteins.
2. Cloning and Expression of Target Proteins
[0374] cDNA Retrieval
[0375] 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 were pulled and streaked onto LB/Ampicillin (100
ug/ml) plates. Plasmid DNA was isolated using Qiagen spin mini-prep
kit and verified by restriction digest. Subsequently, the isolated
plasmid DNA was sequence verified against the ORF reference
sequence. Sequencing reactions were carried out using Applied
Biosystems BigDye Terminator kit followed by ethanol precipitation.
Sequence data was collected using the Applied Biosystems 3100
Genetic Analyzer and analyzed by alignment to the reference
sequence using the Clone Manager alignment tool.
PCR
[0376] PCR primers were designed to amplify the full-length ORF as
well as any regions of the ORF that were interest for expression
(antigenic or hydrophilic regions as determined by the Clone
Manager sequence analysis tool). Primers also contained 5' and 3'
overhangs to facilitate cloning (see below). PCR reactions
contained 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 was verified
and quantitated by agarose gel electrophoresis.
Construction of Entry Clones
[0377] PCR products were cloned into an entry vector for use with
the Gateway recombination based cloning system (Invitrogen). These
vectors included pDonr221, pDonr201, pEntr/D-TOPO or
pEntr/SD/D-TOPO and were used as described in the cloning methods
below.
TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO
[0378] For cloning using this method, the forward PCR primer
contained a 5' overhang containing the sequence "CACC". PCR
products were generated as described above and cloned into the
entry vector using the Invitrogen TOPO cloning kit. Reactions were
typically carried out at room temperature for 10 minutes and
subsequently transformed into TOP10 chemically competent cells
(Invitrogen, CA). Candidate clones were picked, plasmid DNA was
prepared using Qiagen spin mini-prep kit and screened using
restriction digest. Inserts were subsequently sequence verified as
described above.
Gateway Cloning into pDonr201 or pDonr221
[0379] For cloning using this method, PCR primers contained the
following overhangs:
TABLE-US-00002 Forward 5' overhang:
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3' Reverse 5' overhang:
5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'
[0380] PCR products were generated as described above. ORFs were
recombined into the entry vector using the Invitrogen Gateway BP
Clonase enzyme mix. Reactions were 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 were picked, plasmid DNA was prepared using Qiagen spin
mini-prep kit and screened using restriction digest. Inserts were
subsequently sequence verified as described above.
Construction of Expression Clones
[0381] ORFs were transferred from the entry construct into a series
of expression vectors using the Gateway LR Clonase enzyme mix.
Reactions were 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 DH5.alpha.
chemically competent cells (Invitrogen). Candidate clones were
picked, plasmid DNA was prepared using Qiagen spin mini-prep kit
and screened using restriction digest. Expression vectors included
but were 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
[0382] Constructs were transformed into one or more of the
following host strains: BL21 S1, 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 were grown in LB with or
without NaCl and with appropriate antibiotics, at temperatures in
the range of 20-37.degree. C., with aeration. Expression was
induced with the addition of IPTG (0.03-0.3 mM) or NaCl (75-300 mM)
when the cells were in mid-log growth. Growth was continued for one
to 24 hours post-induction. Cells were 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 were stored at -80.degree.
C.
Expression of Recombinant Proteins Using Baculovirus
[0383] Recombinant proteins were expressed using baculovirus in
Sf21 fall army worm ovarian cells. Recombinant baculoviruses were
prepared using the Bac-to-Bac system (Invitrogen) per the
manufacturer's instructions. Proteins were expressed on the large
scale in Sf900II 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
[0384] Recombinant proteins were purified from E. coli and/or
insect cells using a variety of standard chromatography methods.
Briefly, cells were lysed using sonication or detergents. The
insoluble material was pelleted by centrifugation at 10,000.times.g
for 15 minutes. The supernatant was applied to an appropriate
affinity column, e.g. His-tagged proteins were separated using a
pre-packed chelating sepharose column (Pharmacia) or GST-tagged
proteins were separated using a glutathione sepharose column
(Pharmacia). After using the affinity column, proteins were 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.
[0385] 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.
[0386] 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
[0387] 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
derivatized 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:
[0388] Polyclonal antibodies against recombinant proteins were
raised in rabbits (Green Mountain Antibodies, Burlington, Vt.).
Briefly, two New Zealand rabbits were immunized with 0.1 mg of
antigen in complete Freund's adjuvant. Subsequent immunizations
were carried out using 0.05 mg of antigen in incomplete Freund's
adjuvant at days 14, 21 and 49. Bleeds were collected and screened
for recognition of the antigen by solid phase ELISA and western
blot analysis. The IgG fraction was separated by centrifugation at
20,000.times.g for 20 minutes followed by a 50% ammonium sulfate
cut. The pelleted protein was resuspended in 5 mM Tris and
separated by ion exchange chromatography. Fractions were pooled
based on IgG content. Antigen-specific antibody was affinity
purified using Pierce AminoLink resin coupled to the appropriate
antigen.
Isolation of Antibody Fragments Directed Against PCATs from a
Library of scFvs
[0389] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against PCAT 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).
[0390] 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.
[0391] 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).
[0392] 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.
[0393] 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
[0394] i) Materials:
[0395] 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.
[0396] 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.
[0397] 3) The L-glutamine (200 mM, 100.times. solution), which is
stored at -20.degree. C. freezer, is thawed and warmed until
completely in solution. The L-glutamin is dispensed into media to
supplement growth. L-glutamin 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%.
[0398] 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-glutamine to 2 mM and
antibiotic/antimycotic solution to 1% and is called CMNS.
[0399] 5) 1 bottle of PEG 1500 in Hepes (Roche, N.J.)
[0400] 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 (e.g. 2
vials/litre).
[0401] 7) Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza
(1.times.) stored in the refrigerator at 4.degree. C.
[0402] 8) Clonal cell medium D (Stemcell, Vancouver) contains HAT
and methyl cellulose for semi-solid direct cloning from the
fusion.
[0403] 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.
[0404] 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 aliqouted 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.
[0405] ii) Procedure
[0406] 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.
[0407] Myeloma cells are washed with 30 ml serum free media by
centrifugation at 500.times.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.
[0408] 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.
[0409] Cells are harvested and washed by centrifugation (at
500.times.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.
[0410] Supernatant is removed and cells are resuspended in 5 ml of
RPMI-CMNS and are pooled to bring the volume to 30 ml and spun down
as before. The cell pellet is broken up by gentle tapping and
resuspended in 1 ml of BMB PEG1500 (prewarmed to 37.degree. C.)
added dropwise with 1 cc needle over 1 minute.
[0411] RPMI-CMNS is added to the PEG cells to slowly to 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 at 10 ml per small petri plate.
[0412] Myeloma/HAT control 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.
[0413] 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;
[0414] 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. Expression Validation
[0415] mRNA Expression Validation by TAQMAN
[0416] Expression of mRNA was 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 was
used to monitor the formation of the amplification product.
[0417] Total RNA was isolated from cancer model cell lines using
the RNEasy Kit.RTM. (Qiagen) per manufacturer's instructions and
included DNase treatment. Normal human tissue RNAs were acquired
from commercial vendors (Ambion, Austin, Tex.; Stratagene, La
Jolla, Calif., BioChain Institute, Newington, N.H.) as were RNAs
from matched disease/normal tissues.
[0418] Target transcript sequences were identified for the
differentially expressed peptides by searching the BlastP database.
TaqMan assays (PCR primer/probe set) specific for those transcripts
were identified by searching the Celera Discovery System.TM. (CDS)
database. The assays were designed to span exon-exon borders and do
not amplify genomic DNA.
[0419] The TaqMan primers and probe sequences were 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.
[0420] RT-PCR was 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
was made and aliquoted into each optical reaction well. Eight
nanograms of total RNA was the template. Each sample was 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.
[0421] The SDS software calculates the threshold cycle (C.sub.T)
for each reaction, and C.sub.T values were used to quantitate the
relative amount of starting template in the reaction. The C.sub.T
values for each set of three reactions were averaged for all
subsequent calculations
[0422] Data were analyzed for fold differences in expression using
an endogenous control for normalization, and measuring expression
expressed relative to a normal tissue or normal cell line
reference. The choice of endogenous control was 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 were 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.
Protein Expression Validation by Western
[0423] Western blot analysis of target proteins was carried out
using whole cell extracts prepared from each of the pancreatic cell
lines. To make cell extracts, the cells were resuspended in Lysis
buffer (125 mM Tris, pH 7.5, 150 mM NaCl, 2% SDS, 5 mM EDTA, 0.5%
NP-40) and passed through a 20-gauge needle. Lysates were
centrifuged at 5,000.times.g for 5 minutes at 4.degree. C. The
supernatants were collected and a protease inhibitor cocktail
(Sigma) was added. The Pierce BCA assay was used to quantitate
total protein. Samples were separated by SDS-PAGE and transferred
to either a nitrocellulose or PVDF membrane. The Western Breeze kit
from Invitrogen was used for western blot analysis. Primary
antibodies were either purchased from commercially available
sources or prepared using one of the methods described in Section
5. For this application, antibodies were typically diluted 1:500 to
1:10,000 in diluent buffer. Blots were developed using Pierce
NBT.
Results
[0424] Both mRNA expression and protein expression were performed
and many targets were found to have a positive correlation between
mRNA and protein expression relative to Hs766t. The expression of
each of the targets was first analyzed in the pancreas cell line
panel to show that in fact mRNA expression is also upregulated and
that it correlates with the protein data obtained from the LC/MS.
The exemplary targets are Muc4, Kunitz-1 protease inhibitor,
Kunitz-2 protease inhibitor, Neutral amino acid transporter B(0),
Transmembrane 4 superfamily member 3, KIAA 1363, Gs3786, HSPC as
well as Tissue Factor.
[0425] In addition to Hs766t, mRNAs from normal pancreas tissue or
other normal tissues were obtained for control. The targets listed
above showed high mRNA expression in pancreatic cancer cell line.
Moreover, mRNAs from pancreatic tumors were analysed and were shown
to be overexpressed in target such as TM4SF3.
[0426] FIG. 4 shows the Kunitz-type 1 protease inhibitor mRNA
expression in pancreatic cell lines. By using Hs766t as a control,
the result shows that Kunitz-type 1 protease inhibitor is
overexpressed in AsPc-1, BxPc-3, Capan-2, HPAC, HPAF-II, MPanc-96,
Panc1 as well as SU 86.86. Moreover, the data is also consistent
with the result from detection of the target by using LC/MS with
ICAT labeling (see the method below).
[0427] FIG. 10 shows that both Gs3786 and Retinol Dehydrogenase
Type II homolog were found to be over-expressed in several cell
lines relative to the reference cell line Hs766t.
Tissue Flow Cytometry Analysis
[0428] Post tissue processing, cells were sorted by flow cytometry
known in the art to enrich for epithelial cells. Alternatively,
cells isolated from pancreatic tissue were stained directly with
EpCAM (for epithelial cells) and the specific antibody to PCAT.
Cell numbers and viability were determined by PI exclusion (GUAVA)
for cells isolated from both normal and tumor pancreatic tissue. A
minimum of 0.5.times.10.sup.6 cells were used for each analysis.
Cells were washed once with Flow Staining Buffer (0.5% BSA, 0.05%
NaN3 in D-PBS). To the cells, 20 ul of each antibody for PCAT were
added. An additional 5 ul of EpCAM antibody conjugated to APC were
added when unsorted cells were used in the experiment. Cells were
incubated with antibodies for 30 minutes at 4.degree. C. Cells were
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 PCAT targets were all purchased by BD
Biosciences and PE-conjugated. The isotype control antibody used
for these experiments was PE-conjugated mouse IgGlk.
[0429] Tumors tissue from pancreatic cancer and from Xenograft
mouse pancreatic tissues were obtained and detected via Flow
Cytometry analysis. Tissue factor was shown to be overexpressed
among the pancreatic tumor tissues (data not shown). Xenograft
mouse was obtained by using cultured human BXPC pancreatic cells
(ATCC) that were injected subcutaneously in BALB nude mouse. The
pancreatic tumors were obtained from the xenograft mouse for
further FACS study.
7. Detection and Diagnosis of PCAT by Liquid Chromatography and
Mass Spectrometry (LC/MS)
[0430] 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 that are pooled together for experimental purposes
or two acquisitions of the same sample for classification of true
sample peptides from LC/MS noise artifacts. The LC/MS spectra are
collected for the labeled samples and processed using the following
steps:
[0431] 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.
[0432] Similar experiments can be repeated in order to increase the
confidence in detection of a peptide. These multiple acquisitions
are computationally aggregated into one experiment. Experiments
involving healthy and disease samples use the known effects of the
ICAT label to classify the peptides as originating from a
particular sample or from both samples. 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 (for example, Table 1).
[0433] Statistical tests are performed to assess the robustness of
the data and select statistically significant differentials. To
assess general quality of the data, one: a) ensure that similar
features are detected in all replicates of the experiment; b)
assess the distribution of the log ratios of all peptides (a
Gaussian was expected); c) calculate the overall pair wise
correlations between ICAT LC/MS maps to ensure that the expression
ratios for peptides are reproducible across the multiple
replicates; d) aggregate 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
[0434] Paraffin embedded, fixed tissue sections were 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.
[0435] Esophageal replicate sections were 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 were stained with
hemotoxylin and eosin and histologically examined to ensure
adequate representation of cell types in each tissue section.
[0436] 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
[0437] Hemotoxylin and Eosin staining of paraffin embedded, fixed
tissue sections. Sections were deparaffinized in 3 changes of
xylene or xylene substitute for 2-5 minutes each. Sections were
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 were 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 were stained in
Scott's solution for 2 minutes. Sections were 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 were 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 were
coverslipped and stored for analysis.
Optimisation of Antibody Staining
[0438] For each antibody, a positive and negative control sample
was generated using data from the ICAT analysis of the pancreatic
cancer cell lines. Cell lines were selected that are known to
express low levels of a particular target as determined from the
ICAT data. This cell line was the reference normal control
"Hs766T". Similarly, a pancreatic tumour line was selected that was
determined to overexpress the target was selected.
Antigen Retrieval
[0439] Sections were 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 were then placed in endogenous
blocking solution (methanol+2% hydrogen peroxide) and incubated for
20 minutes at room temperature. Sections were 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 were deparrafinized by High Energy Antigen
Retrieval as follows: sections were 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 were placed in a Coplin jar with dilute antigen
retrieval solution (10 mM citrate acid, pH 6). The Coplin jar
containing slides was 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 were repeated four times
(depending on tissue), followed by cooling for 20 minutes at room
temperature. Sections were 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
[0440] Sections were 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 were performed
in a sealed humidity chamber to prevent air-drying of the tissue
sections. (The choice of blocking serum was 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 was taken that the sections do
not touch during incubation). Sections were rinsed twice for 5
minutes in PBS, shaking gently. Excess PBS was removed by gently
shaking. The sections were 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 was used to decrease the
background on rat tissue sections. Following incubation, sections
were rinsed twice for 5 minutes in PBS, shaking gently. Excess PBS
was removed and sections incubated for 1 hour at room temperature
in Vectastain ABC reagent (as per kit instructions). The lid of the
humidity chamber was secured during all incunations to ensure a
moist environment. Sections were rinsed twice for 5 minutes in PBS,
shaking gently.
Develop and Counterstain
[0441] Sections were incubated for 2 minutes in peroxidase
substrate solution that was made up immediately prior to use as
follows: 10 mg diaminobenzidine (DAB) dissolved in 10 ml 50 mM
sodium phosphate buffer, pH 7.4; 12.5 microliters 3%
CoCl.sub.2/NiCl.sub.2 in deionized water; 1.25 microliters hydrogen
peroxide
[0442] Slides were 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.
[0443] Slides were 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 were mounted for visualization by
microscopy.
Results
[0444] Tissue Factor: The protein was overexpressed in fourteen out
of twenty two pancreatic tumor and two out seven pancreatic tumor
metastases. In normal tissues, antibody to tissue factor or CD 142
showed minimal staining. Occasional cytoplasmic staining was
present in subsets of inflammatory cells, specifically lymphocytes
and mast cells. Gastric chief cells showed cytoplasmic and nuclear
staining. Many cell types and tissues, including urothelium, breast
epithelium, respiratory epithelium, adrenal cortex, ovarian stroma,
endometrial stroma, pancreatic acinar epithelium, and placental
trophoblasts showed focal nuclear staining. However, in contrast to
normal tissues, a number of malignancies showed significant
membranous and cytoplasmic staining in malignant cells among the
tumor tissues. The most prominent positivity was identified in
pancreatic carcinoma, followed by prostatic carcinoma. Individual
samples of colon, lung, breast and ovarian carcinoma showed
positive staining. The color scale or number scale indicates the
color intensity of the staining. Each number represents 100% of
staining of all the tumor cells at this staining scale (see FIG.
1). In addition, CD142 (tissue factor) is overexpressed by 2
pathology grades relative to normal comparative cell type in
esophageal 10%, pharyngeal 20%, gastric 10%.
[0445] Decay Accelerating Factor (DAF): The protein was
overexpressed in seven out of twenty-three pancreatic tumors. The
target also overexpressed in colon cancer. DAF was overexpressed by
2 pathology grades in esophageal 20%, liver 30%, gastric 30%.
[0446] Mucin 4: Mucin 4 is a transmembrane mucin composed of two
subunits (a and b) generated from proteolytic cleavage, wherein a
is extracellular and b is transmembrane. The b subunit contains two
EGF-like domains and interacts with ErbB2 and the ErbB2/ErbB3
heterodimer with the presence of neuregulin. Interaction with ErbB2
results phosphorylation of ErbB2 and upregulation of
cyclin-dependent kinase inhibitor p27kip. The interaction causes
cell cycle arrest and epithelial cell differentiation. Interaction
of Mucin 4 with ErbB2/B3 results in hyperphosphorylation of the
ErbB2/ErbB3/NRG heterotrimer and activation of MAPK, PI3K pathways
with downregulation of p27kip. This results in the stimulation of
epithelial cell proliferation. Apoptosis is repressed by expression
of Mucin 4 and tumor cell growth therefore stimulated.
[0447] In normal tissues, antibody to Mucin 4 showed the most
prominent positive staining in colonic epithelium and scattered
individual cells within the prostatic epithelium. Additional
positive cell types included pancreatic islets, urothelium,
astrocytes, gastric chief cells, and subsets of renal tubular
epithelial cells. Focal positivity was present in the adrenal
cortex, endometrium, and testis. Rarely, inflammatory cells and
single cells (neuroendocrine cells) in the intestinal epithelium
were positive. Among malignancies tumor tissues, FIG. 3 showed that
Mucin 4 protein is overexpressed in breast, non-small lung,
prostate, ovarian and pancreatic cancers.
[0448] The Mucin 4 was overexpressed in fourteen out of twenty four
pancreatic tumors and six out of eight metastatic tissues. The
protein was expressed in several normal epithelia. However, both
Mucin 4 and ErbB3 were overexpressed in pancreatic tumors. Mucin 4
was also overexpressed in breast cancer. In addition, Muc4 is
overexpressed by 2 pathology grades in esophageal 80% (relative to
gastric epithelium), renal cell carcinoma 90%, hepatocellular 20%,
pharyngeal 70% (relative to gastric epithelium), gastric 20%.
[0449] ErbB3: The protein was specifically expressed in pancreatic
tumor BxPC3 cell line and Breast cancer MCF7 cell line by Western
analysis. Further IHC data showed that ErbB3 is overexpressed in
50% of pancreatic cancer, 30% ovarian cancer and 10% non-small cell
lung cancer. Overlap of expression of Mucin 4 and ErbB3 was
predominantly tumor specific where both are overexpressed.
[0450] Kunitz Inhibitor-1: the protein was overexpressed in
thirteen out of twenty three pancreatic tumors and three out seven
metastatic pancreatic cancer tissues. In normal tissues, antibody
to Kunitz Inhibitor-1 or HAI-1 showed the most prominent positivity
in placental trophoblasts. Colonic epithelium was also positive.
Less intense positivity was present in urothelium and endometrial
glands. Focal positivity was present in breast epithelium,
respiratory epithelium, centroacinar cells in the pancreas, subsets
of renal tubular epithelial cells, tonsillar epithelium, and
hepatic bile ducts. However, in tumor tissues, a majority of
samples of all subtypes tested showed positive staining of
malignant cells. The staining was often membranous and diffused
within the samples. A few samples of breast and lung cancer
contained adjacent benign epithelium, which showed significantly
less intense staining than the malignant cells. In pancreatic
carcinoma, the level of staining in malignant cells was frequently
similar to that in benign centroacinar cells, but in a few cases,
the staining was less intense in malignant cells than in the
adjacent benign epithelium. FIG. 2 concludes that Kunitz
inhibitor-1 was overexpressed in breast, non-small cell lung,
prostate, ovarian as well as pancreatic cancers. In addition,
Kunitz-1 is overexpressed by 2 pathology grades in: esophageal 40%,
renal cell carcinoma 40%, hepatocellular 25%, pharyngeal 30%,
gastric 40%.
[0451] Transglutaminase-2 (TGM2): TGM2 protein was overexpressed in
seventeen out of twenty four pancreatic tumors and seven out of
seven metastatic tissues. Pathology study noted overexpression in
capilliary endothelium including neovascular capillaries. TGM2
protein was also overexpressed in four out of ten ovarian tumors
and overexpressed in non small cell lung tumor, prostate tumor.
TGM2 was overexpressed by 2 pathology grades in esophageal 10%,
renal 30%, liver 10%, pharyngeal 20%, gastric 10%.
[0452] Neutral Amino Acid Transporter (ASCT2): ASCT2 protein was
overexpressed in sixteen out of twenty seven pancreatic tumors
(60%) and four out of seven pancreatic metastatic tissues. The
protein was also overexpressed in 40% prostate cancer and 30%
ovarian cancer tissues. Neutral amino acid transporter is also
overexpressed by 2 pathology grades in: esophageal 10%
hepatocellular 75%
[0453] CD166: CD166 protein was overexpressed in 10% pancreatic,
colon and ovarian tumor, in 50% overexpressed in breast tumors.
[0454] CD46: CD46 protein was overexpressed in 10% pancreatic tumor
and in 30% non-small cell lung tumors.
[0455] Kunitz Type inhibitor-2 (HAI-2): HAI-2 was overexpressed in
40% pancreatic tumors and in 10% breast and ovarian tumors.
[0456] Na/K ATPase Beta-3: Na/K ATPase Beta-3 was overexpressed in
pancreatic tumor, ovarian tumor.
[0457] GS3786: GS3786 was overexpressed in pancreatic tumor and
prostate tumor.
[0458] Diminuto-like: Diminuto-like protein is overexpressed (by 2
pathology grades relative to normal comparative cell type) in 20%
pancreatic, 40% prostate, 20% breast and 10% colon tumors
9. IHC Staining of Frozen Tissue Sections
[0459] 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 longterm
storage.
[0460] Sections are fixed by immersing in acetone jar for 1-2
minutes at room temperature, followed by drying at room
temperature. 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). 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 phosphatase 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
[0461] Cultured tumor cells are labeled with 100 .mu.Ci .sup.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 washing
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
[0462] 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
[0463] Lipofectamine was purchased from Invitrogen (Carlsbad,
Calif.) and GeneSilencer from Gene Therapy Systems (San Diego,
Calif.). Synthetic siRNA oligonucleotides were from Dharmacon
(Lafayette, Colo.), Qiagen (Valencia, Calif.) or Ambion (Austin,
Tex.) RNeasy 96 Kit was purchased from Qiagen (Valencia, Calif.).
Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous One
Solution Cell Proliferation Assay were both purchased from Promega
(Madison, Wis.). Function blocking antibodies were 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 was purchased from Molecular probes (Eugene,
Oreg.).
RNAi
[0464] RNAi was performed by using Smartpools (Dharmacon), 4--for
Silencing siRNA duplexes (Qiagen) or scrambled negative control
siRNA (Ambion). Transient transfections were 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 was isolated by using the RNeasy 96
Kit (Qiagen) according to manufacturer's instructions and
expression of mRNA was quantitated by using TaqMan technology.
Protein expression levels were examined by flow cytometry and
apoptosis and proliferation assays were performed daily using
Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous One
Solution Cell Proliferation Assay (see protocols below).
[0465] i) RNAi Transfections--Lipofectamine 2000
[0466] Transient transfections were carried out on sub-confluent
pancreatic 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 was transfected using
lipofectamine according to manufacturer's instructions. Cells were
plated in 96 well plates in antibiotic free medium. The next day,
the transfection reagent and siRNA were prepared for transfection
as follows: Each 0.1-1 ul of lipofectamine 2000 and 10-150 mM siRNA
were resuspended 25 ul serum-free media and incubated at room
temperature for 5 minutes. After incubation, the diluted siRNA and
the lipofectamine 2000 were combined and incubated for 20 minutes
at room temperature. The cells were then washed and the combined
siRNA-Lipofectamine 2000 reagent added. After further 4 hours
incubation, 50 ul serum containing medium was added to each well. 1
and 4 days after transfection, expression of mRNA was quantitated
by RT-PCR using TaqMan technology and protein expression levels
were examined by flow cytometry. Apoptosis and proliferation assays
were performed daily using Apop-one homogeneous caspase-3/7 kit and
CellTiter 96 AQueous One Solution Cell Proliferation Assay (see
protocols below).
[0467] ii) RNAi Transfections-GeneSilencer
[0468] Transient transfections were carried out on sub-confluent
pancreatic 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 was transfected using GeneSilencer
according to manufacturer's instructions. Cells were plated in 96
well plates in antibiotic free medium. The next day, the
transfection reagent and the synthetic siRNA were prepared for
transfection as follows: predetermined amount of Gene Silencer was
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 were combined and incubated at room temperature for 5-20
minutes. After incubation, the siRNA-Gene Silencer reagent was
added to each well and incubated in a 37.degree. C. incubator for 4
hours before an equal volume of serum containing media was added
back to the cultured cells. The cells were then incubated for 1 to
4 days before mRNA, protein expression and effects on apoptosis and
proliferation were examined.
Testing of Functional Blocking Antibodies
[0469] Sub-confluent pancreatic 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
[0470] 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
[0471] 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. One example
of the proliferation assay is MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethanoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium) proliferation assay.
Cell Invasion
[0472] 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 nm excitation
and 520 nm emission.
Receptor Internalization
[0473] 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 hr 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-.mu.l samples are taken for colorimetric readings.
mRNA Expression
[0474] 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 manufacturers instructions. Probe and primer
concentrations are 900 nM and 250 nM, respectively, in a 25 .mu.l
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. 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.
[0475] 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.
[0476] 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.
Results
[0477] The assay was performed on analysis of Mucin 4 knowdown.
Mucin 4 siRNA induced apoptosis of ASPC-1 cells 3 days after
transfection, and level of apoptosis was dose-dependent on muscin
siRNA (n=3). The level of apoptosis on day 3 was similar to that
given by TRAIL+actinomycin D positive control (FIG. 5). Moreover,
FIG. 6 also shows that Mucin 4 siRNA inhibited the proliferation of
ASPC-1 cells (n=3). FIG. 7 shows that the level of inhibition was
dose-dependent on the concentration of Mucin 4 siRNA (n=3). The
result shows that Mucin 4 siRNA can inhibit about 50% of viable
ASPC-1 cells 3 days after transfection (n=2). Both SMARTPool Mucin
4 siRNA and Mucin 4 duplex1 siRNA can induce an inhibition of
proliferation and an increase in apoptosis 3 days after
transfection (FIG. 8). Additional assays were also performed on
other targets: Apoptosis occurred when Na+/K+ transporting ATPase
beta-3 chain (PA-088), GS3786(PA-078), CD49c(PA-009), siRNA were
administrated in Cacaspase 3/7 analysis in ASPC-1 cell line.
Apoptosis occurred when Na+/K+ transporting ATPase beta-3 chain,
GS3786, and Celera protein RNAi were administrated in Cacaspase 3/7
analysis in BXPC-3 cell line. Specifically, Na+/K+ transporting
ATPase beta-3 chain increased apoptosis of ASPC-1 pancreatic cancer
cells in a dose-dependent manner and can decrease proliferation of
ASPC-1 pancreatic cancer cells. GS3786 siRNA increased apoptosis of
ASPC-1 pancreatic cancer cells in a dose-dependent manner. It
increased apoptosis of BXPC-3 pancreatic cancer cells and decreased
proliferation of ASPC-1 and BXPC-3 pancreatic cancer cells. CD49c
siRNA increased apoptosis of BXPC-3 pancreatic cancer cells in a
dose-dependent manner and decreased proliferation of BXPC-3
pancreatic cancer cells in a dose-dependent manner. (FIG. 11 and
FIG. 12]
13. In Vivo Studies by Using Antibodies
[0478] Treatment of Pancreatic Cancer Cells with Monoclonal
Antibodies.
[0479] Pancreatic 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-PCAT 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 PCAT Protein with
Monoclonal Antibodies.
[0480] NIH 3T3 expressing PCAT protein are treated with different
concentrations of anti-PCAT MAbs. Cell growth inhibition is
monitored.
In Vivo Treatment of NIH 3T3 Cells Overexpressing PCAT with
Anti-PCAT Monoclonal Antibodies.
[0481] NIH 3T3 cells transfected with either a PCAT expression
plasmidor 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-PCAT monoclonal antibody of the IG2A subclass is injected
intraperitoneally. Tumor occurrence and size are monitored for 1
month period of treatment.
[0482] 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=US20090138977A1).
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=US20090138977A1).
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