U.S. patent application number 12/868557 was filed with the patent office on 2011-09-08 for lung disease targets and uses thereof.
This patent application is currently assigned to CELERA CORPORATION. Invention is credited to Bruno DOMON, Tao HE, Elizabeth JOSELOFF, Aiqun LI.
Application Number | 20110219464 12/868557 |
Document ID | / |
Family ID | 42797720 |
Filed Date | 2011-09-08 |
United States Patent
Application |
20110219464 |
Kind Code |
A1 |
DOMON; Bruno ; et
al. |
September 8, 2011 |
LUNG DISEASE TARGETS AND USES THEREOF
Abstract
The present invention provides a method for diagnosing and
detecting diseases associated with lung. The present invention
provides one or more proteins or fragments thereof, peptides or
nucleic acid molecules differentially expressed in lung cancer
(LCAT) and antibodies binds to LCATs. The present invention
provides that LCATs are used as targets for screening agents that
modulates the LCAT activities. Further the present invention
provides methods for treating diseases associated with lung.
Inventors: |
DOMON; Bruno; (Zurich,
CH) ; JOSELOFF; Elizabeth; (Sliverspring, MD)
; LI; Aiqun; (Gaithersburg, MD) ; HE; Tao;
(North Potomac, MD) |
Assignee: |
CELERA CORPORATION
Alameda
CA
|
Family ID: |
42797720 |
Appl. No.: |
12/868557 |
Filed: |
August 25, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11905216 |
Sep 28, 2007 |
7807392 |
|
|
12868557 |
|
|
|
|
10941087 |
Sep 15, 2004 |
|
|
|
11905216 |
|
|
|
|
60502656 |
Sep 15, 2003 |
|
|
|
60530410 |
Dec 18, 2003 |
|
|
|
60570505 |
May 13, 2004 |
|
|
|
60576801 |
Jun 4, 2004 |
|
|
|
Current U.S.
Class: |
800/13 ;
424/139.1; 435/235.1; 435/243; 435/331; 435/375; 435/419; 435/6.14;
435/7.1; 436/501; 514/21.2; 514/21.92; 530/350; 530/387.2;
530/387.3; 530/387.9; 530/391.3; 530/391.7; 536/23.5; 536/24.5 |
Current CPC
Class: |
G01N 33/57423 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
800/13 ;
435/235.1; 435/331; 435/419; 435/243; 435/375; 436/501; 435/7.1;
530/350; 514/21.2; 514/21.92; 536/23.5; 536/24.5; 530/387.9;
530/387.3; 530/387.2; 530/391.3; 530/391.7; 424/139.1;
435/6.14 |
International
Class: |
A01K 67/00 20060101
A01K067/00; C12N 7/01 20060101 C12N007/01; C12N 5/10 20060101
C12N005/10; C12N 1/00 20060101 C12N001/00; C12N 5/071 20100101
C12N005/071; G01N 33/566 20060101 G01N033/566; C12N 5/12 20060101
C12N005/12; C07K 14/47 20060101 C07K014/47; C07K 2/00 20060101
C07K002/00; A61K 38/17 20060101 A61K038/17; A61K 38/02 20060101
A61K038/02; C07H 21/00 20060101 C07H021/00; C07H 21/02 20060101
C07H021/02; C07K 16/18 20060101 C07K016/18; C07K 16/42 20060101
C07K016/42; A61K 39/395 20060101 A61K039/395; C12Q 1/68 20060101
C12Q001/68; A61P 35/00 20060101 A61P035/00 |
Claims
1. An isolated protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS:1-778 and 1830-2100.
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:779-1829; b) nucleotide sequences that encode a protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:1-778 and 1830-2100; 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 lung cancer cells or stimulating
apoptosis of lung 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 lung
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 lung 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:1-778 and 1830-2100; 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:779-1829
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 lung 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 lung cancer, the method comprising detecting the presence,
abundance, or activity of the protein of claim 1 in a sample and
determining or predicting the effectiveness of the treatment or
selecting the treatment for administration based on the presence,
abundance, or activity of the protein.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the fields of molecular biology
and oncology. Specifically, the invention provides a molecular
marker and a therapeutic agent for use in the diagnosis and
treatment of lung diseases.
BACKGROUND OF THE INVENTION
[0002] Lung cancer is the second most prevalent type of cancer for
both men and women in the United States and is the most common
cause of cancer death in both sexes. Lung cancer can result from a
primary tumor originating in the lung or a secondary tumor which
has spread from another organ such as the bowel or breast. The
five-year survival rate for lung cancer continues to be poor at
8-15% survival indicating a large unmet need with regard to more
effective treatments and better diagnosis. The estimated total lung
cancer deaths in the U.S. in 2003 are 157,200 and the total
estimated new cases in 2003 are 171,900. Primary lung cancer is
divided into three main types; small cell lung cancer; non-small
cell lung cancer; and mesothelioma. Small cell lung cancer is also
called "Oat Cell" lung cancer because the cancer cells are a
distinctive oat shape. There are three types of non-small cell lung
cancer. These are grouped together because they behave in a similar
way and respond to treatment differently to small cell lung cancer.
The three types are squamous cell carcinoma, adenocarcinoma, and
large cell carcinoma. Squamous cell cancer develops from the cells
that line the airways. Adenocarcinoma also develops from the cells
that line the airways. However, adenocarcinoma develops from a
particular type of cell that produces mucus (phlegm). Large cell
lung cancer has been thus named because the cells look large and
rounded when they are viewed under a microscope. Mesothelioma is a
rare type of cancer which affects the covering of the lung called
the pleura. Mesothelioma is often caused by exposure to
asbestos.
[0003] Secondary lung cancer is cancer that has started somewhere
else in the body (for example, the breast or bowel) and spread to
the lungs. Choice of treatment for secondary lung cancer depends on
where the cancer started. In other words, cancer that has spread
from the breast should respond to breast cancer treatments and
cancer that has spread from the bowel should respond to bowel
cancer treatments.
[0004] The stage of a cancer indicates how far a cancer has spread.
Staging is important because treatment is often decided according
to the stage of a cancer. The staging is different for non-small
cell and for small cell cancers of the lung.
[0005] Non-small cell cancer can be divided into four stages. Stage
I is very localized cancer with no cancer in the lymph nodes. Stage
II cancer has spread to the lymph nodes at the top of the affected
lung. Stage III cancer has spread near to where the cancer started.
This can be to the chest wall, the covering of the lung (pleura),
the middle of the chest (mediastinum) or other lymph nodes. Stage
IV cancer has spread to another part of the body.
[0006] Since small cell lung cancer can spread quite early in
development of the disease, small cell lung cancers are divided
into only two groups. These are: limited disease, that is cancer
that can only be seen in one lung and in nearby lymph nodes; and
extensive disease, that is cancer that has spread outside the lung
to the chest or to other parts of the body. Further, even if
spreading is not apparent on the scans, it is likely that some
cancer cells will have broken away and traveled through the
bloodstream or lymph system. To be safe, it is therefore preferred
to treat small cell lung cancers as if they have spread, whether or
not secondary cancer is visible. Because surgery is not typically
used to treat small cell cancer, except in very early cases, the
staging is not as critical as it is with some other types of
cancer. Chemotherapy with or without radiotherapy is often
employed. The scans and tests done at first will be used later to
see how well a patient is responding to treatment.
[0007] Procedures used for detecting, diagnosing, monitoring,
staging, and prognosticating lung cancer are of critical importance
to the outcome of the patient. For example, patients diagnosed with
early lung cancer generally have a much greater five-year survival
rate as compared to the survival rate for patients diagnosed with
distant metastasized lung cancer. New diagnostic methods which are
more sensitive and specific for detecting early lung cancer are
clearly needed.
[0008] Lung cancer patients are closely monitored following initial
therapy and during adjuvant therapy to determine response to
therapy and to detect persistent or recurrent disease of
metastasis. There is clearly a need for a lung cancer marker which
is more sensitive and specific in detecting lung cancer, its
recurrence, and progression.
[0009] Another important step in managing lung cancer is to
determine the stage of the patient's disease. Stage determination
has potential prognostic value and provides criteria for designing
optimal therapy. Generally, pathological staging of lung cancer is
preferable over clinical staging because the former gives a more
accurate prognosis. However, clinical staging would be preferred
were it at least as accurate as pathological staging because it
does not depend on an invasive procedure to obtain tissue for
pathological evaluation. Staging of lung cancer would be improved
by detecting new markers in cells, tissues, or bodily fluids which
could differentiate between different stages of invasion.
[0010] 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
[0011] The present invention is based on the identification of
certain cell surface proteins (including shed proteins) or
cytosolic proteins that are differentially expressed in lung
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 lung disease. The
differentially expressed proteins of the present invention and the
nucleic acids encoding said proteins and the fragments of said
proteins are referred to herein as lung cancer associated target,
LCAT proteins or LCAT nucleic acids or LCAT peptides,
respectively.
[0012] The present invention provides peptides and protein
differentially expressed in lung cancer (hereinafter LCAT). Based
on the site of protein localization, e.g., surface or cytosolic,
and protein characterization, e.g. receptor or enzyme, specific
uses of these LCATs are provided. Some of the LCATs of the present
invention serve as targets for one or more classes of therapeutic
agents, while others may be suitable for antibody therapeutics.
[0013] Accordingly, the present invention provides a method for
diagnosing or detecting lung disease in a subject comprising:
determining the level of one or more LCAT proteins, or any
fragment(s) thereof, in a test sample from said subject, wherein
said LCAT protein comprises a sequence selected from a group
consisting of SEQ ID NOS: 1-778; wherein a differential level of
said LCAT 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 lung disease.
[0014] The present invention also provides a method for detecting
lung cancer in a subject comprising: determining the level of one
or more LCAT peptide(s) comprising a peptide sequence selected from
a group consisting of SEQ ID NOS: 1830-2100 in a test sample from
said subject, wherein a differential level of said LCAT peptide(s)
in said sample to the level of said LCAT peptide(s) in a test
sample from a healthy subject, or the level of said LCAT peptide(s)
established for a healthy subject, is indicative of lung
disease.
[0015] The present invention further provides a method for
detecting lung disease in a subject comprising: determining the
level of one or more LCAT nucleic acid(s), or any fragment(s)
thereof, in a test sample from said subject, wherein said LCAT
nucleic acid(s) encode a LCAT protein sequence selected from a
group consisting of SEQ ID NOS: 1-778; wherein a differential level
of said LCAT 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 lung disease.
[0016] The invention also provides methods for detecting the LCAT
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.
[0017] 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: 1-778.
[0018] The present invention further provides a composition
comprising an antibody that binds to a protein selected from a
group consisting of SEQ ID NOS: 1-778, and an acceptable
carrier.
[0019] The present invention further provides a method for treating
lung disease, comprising administering to a patient in need of said
treatment a therapeutically effective amount of one or more
antibody(ies) of this invention.
[0020] The present invention further provides a method for treating
lung disease comprising (i) identifying a subject having lung
disease and (ii) administering to a said patient a therapeutically
effective amount of one or more antibody(ies) of this
invention.
[0021] The present invention further provides a method to screen
for agents that modulate LCAT protein activity, comprising the
steps of (i) contacting a test agent with a LCAT protein and (ii)
assaying for LCAT protein activity, wherein a change in said
activity in the presence of said agent relative to LCAT protein
activity in the absence of said agent indicates said agent
modulates said LCAT protein activity.
[0022] The present invention further provides a method to screen
for agents that bind to LCAT proteins, comprising the steps of (i)
contacting a test agent with a LCAT protein and (ii) measuring the
level of binding of agent to said LCAT protein.
[0023] The invention also provides diagnostic methods for human
disease, in particular for lung diseases, its metastatic stage, and
therapeutic potential.
[0024] 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.
[0025] The invention also provides a method for monitoring the
disease progression and the treatment progress.
[0026] The invention further provide a method of diagnosis by an
array, wherein the array is immobilized with two or more LCAT
proteins, peptides or nucleic acid molecules. The proteins,
peptides or nucleic acid molecules include but are not limited to
the SEQ ID NOS: 1-2100.
[0027] The invention also provides monoclonal or polyclonal
antibodies and composition thereof reactive with antigenic portion
of LCAT protein, peptides or fragments thereof in a form for use in
lung disease diagnosis.
[0028] The invention further provides an immunogenic antibody for
treating lung disease or diseases associated with lung
diseases.
[0029] The present invention provides a method for screening agents
that modulate LCAT activity, comprising the steps of (a) contacting
a sample comprising LCAT with an agent; and (b) assaying for LCAT
activity, wherein a change in said LCAT activity in the presence of
said agent relative to LCAT activity in the absence of said
compound indicates said agent modulates LCAT. The agents include
but are not limited to protein, peptide, antibody, nucleic acid
such as antisense RNA, RNAi fragments, small molecules.
[0030] The present invention further provides a method for treating
lung diseases, comprising: administering to a patient with one or
more agents in a therapeutically effective amount to treat lung
diseases.
[0031] The present invention provides a method for treating lung
diseases, comprising: identifying a subject having lung diseases;
and administering to a patient to one or more antibodies in a
therapeutically effective amount to treat lung diseases.
[0032] The present invention further provide method for diagnosis
and treatment for lung cancer.
[0033] 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
CL001546CDR
[0034] The CD-R named CL001546CDR contains the following two text
(ASCII) files:
1) File SEQLIST.sub.--1546.TXT provides the Sequence Listing. The
Sequence Listing provides the protein sequences (SEQ ID NOS:
1-778); transcript sequences (SEQ ID NOS: 779-1829) and peptide
sequences (SEQ ID NOS: 1830-2100) as shown in Table 1. File
SEQLIST.sub.--1546.TXT is 7752 KB in size. 2) File
TABLE1.sub.--1546DIV.TXT, which is 288 KB in size, provides Table
1.
[0035] The material contained on the CD-R labeled CL001546 CDR is
hereby incorporated by reference pursuant to 37 CFR 1.77(b)(4).
TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110219464A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Description of Table 1
[0036] Table 1 (provided on the CD-R) discloses the LCAT proteins,
transcripts, and peptides sequences.
[0037] The transcript/protein information includes:
[0038] a protein number (1 through 778)
[0039] a Celera protein internal identification number for the
protein encoded by the Celera transcript (hCP and/or UID)
[0040] a public protein accession number (Genbank e.g., RefSeq NP
number, Swiss-prot, or Derwent) for the protein
[0041] an art-known gene/protein name
[0042] a Celera transcript internal identification number (hCT
and/or UID)
[0043] a public transcript accession number (Genbank e.g., RefSeq
NM number, or Derwent)
[0044] a Celera hCG and UID internal identification numbers for the
gene
[0045] an art-known gene symbol
[0046] Celera genomic axis position (indicating start nucleotide
position-stop nucleotide position)
[0047] the chromosome number of the chromosome on which the gene is
located
[0048] 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
[0049] alternative gene/protein name(s) and/or symbol(s) in the
OMIM entry
[0050] Table 1 (provided on the CD-R) also discloses the peptides
which correspond to the protein, the lung cancer cell lines and
lung tumor tissues ("source"), 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.
Over expressed singleton indicates that the peptide peak in
diseased sample was detected and there was no peak detected in
control samples. Under expressed singleton indicates that the
peptide peak was detected in the control sample and there was no
peak in the diseased sample.
DESCRIPTION OF FIGURES
[0051] FIG. 1. Tissue Factor expression in various tumor types by
immunohistochemistry.
[0052] FIG. 2. Kunitz-type 1 protease inhibitor expression in
various tumor types by immunohistochemistry.
[0053] FIG. 3. Expression validation of CD 49f by mRNA and FACS
[0054] FIG. 4. Flow cytometry validation in lung tissues: 15
matched pairs of lung tumors and normal tissue have been used for
analyzing expression by FACS for 10 targets and over expression in
EpCAM positive cells was observed in at least 50% of the lung
tumors for 6 lung targets.
[0055] FIG. 5. CD 73 mRNA/protein correlation in lung cell
lines
[0056] FIG. 6. Overexpression of mRNA for CD90 in Lung Cancer
[0057] FIG. 7. Expression validation of CD 98 by mRNA and FACS.
[0058] FIG. 8. CD98: Overexpression of mRNA in lung tumors.
[0059] FIG. 9. Quantitative FACS validation for CD49f. Beas-2B is a
control cell whereas A549, H2291, Calu-1 and H358 are lung tumor
cells. "ABC" indicates the copy number of cells detected by FACS
(.times.10.sup.6).
[0060] FIG. 10. CD59 FACS expression in lung cell lines and lung
tumor tissues. MFI is mean fluorescent intensity raw data.
[0061] FIG. 11. Protein Expression by QFACS for CD98 in Lung Tumor
and Normal Tissue
[0062] FIG. 12. Evaluation of Level and Homogeneity of CD98
Expression in Tumor Tissue by IHC
[0063] FIG. 13. Protein Expression by QFACS for CD147 in Lung Tumor
and Normal Tissue
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
General Description
[0064] 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 lung disease by determining the level of one or more LCAT
protein(s) or any fragment(s) thereof is wherein the level of LCAT
protein(s) are determined by contacting one or more antibody(ies)
that specifically bind to the antigenic regions of the LCAT
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.
[0065] A preferred method for detecting lung disease by determining
the level of one or more LCAT peptide(s) is wherein the level of
LCAT peptides(s) are determined by contacting one or more
antibody(ies) that specifically bind to the antigenic regions of
the LCAT 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.
[0066] A preferred method for detecting lung disease by determining
the level of one or more LCAT nucleic acid(s) is wherein the level
of said LCAT 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.
[0067] The methods for detecting lung disease provided by the
present invention may be used for diagnosing the presence of
disease in a patient, monitoring the presence of lung disease in
patients undergoing treatment and testing for the reoccurrence of
lung disease in patients that were successfully treated for lung
disease; preferably wherein the lung disease is lung cancer. The
test sample may be, but is not limited to, a biological sample such
as tissue, blood, serum or biological fluid.
[0068] The present invention is based on the discovery of
protein(s) and peptide(s) that are differentially expressed in lung
cancer samples versus normal lung samples. These proteins and
peptide, and the encoding nucleic acid molecules are associated
with lung diseases, hereinafter the LCAT protein, peptide or
nucleic acids.
[0069] 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 lung tissues or cell lines in an effort to
discover and identify novel molecules associated with the
disease.
[0070] Based on these discoveries, the present invention provides
proteins, peptides, nucleic acids that are differential in lung
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 lung
diseases. The present invention provides a detection reagent, for
markers for lung diseases at various stages, comprises LCAT
sequences isolated from human lung diseased tissue, sera, cell
lines, blood or biological fluids.
[0071] The present invention provides a method for treating lung
diseases targeting at LCAT. The treatment includes administration
of a therapeutically effective amount of composition comprising,
but not limit to, an antibody, an immunogenic peptide which induces
T cell response, a small molecule, a protein or a nucleic acid
molecule. The composition further comprises an agonist or
antagonist to LCAT.
[0072] 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.
[0073] The present invention further provides the target for
screening an agent for LCAT, wherein the agent is compounds of
small molecules, proteins, peptides, nucleic acids, antibodies or
other agonists or antagonists.
LCAT Peptide/Proteins and Peptides
[0074] The present invention provides isolated LCAT peptide and
protein molecules that consisting of, consisting essentially of, or
comprising the amino acid sequences of the LCAT peptides and
proteins disclosed in the Table 1 (encoded by the nucleic acid
molecules shown in Table 1), as well as all obvious variants of
these peptides that are within the art to make and use. Some of
these variants are described in detail below.
[0075] In one embodiment LCAT peptides include, but are not limited
to, the amino acid sequence of SEQ ID NOS: 1830-2100 and variants
thereof. A LCAT protein includes, but is not limited to, the amino
acid sequences of SEQ ID NOS: 1-778 and variants thereof. LCAT
proteins may be differentially expressed in lung cell line, blood,
tissue, serum or body fluids.
[0076] 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.
[0077] The LCAT 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).
[0078] As used herein, a "peptide" is defined as amino acid
sequences between 5-20 amino acids derived from LCAT proteins such
as SEQ ID NOS: 1-778 or variants thereof. The peptide
differentially expressed in either lung diseased 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:
1830-2100, or variants thereof.
[0079] As used herein, a "protein" is full-length protein
differentially expressed in lung diseased cell line, tissue, blood,
serum or body fluids. A protein includes, but is not limited to,
the amino acid sequences of SEQ ID NOS: 1-778.
[0080] 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)
[0081] 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.
[0082] 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 LCAT 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.
[0083] The isolated LCAT proteins and peptide can be purified from
cells that naturally express it, purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods. Sambrook et al., Molecular Cloning: A
Laboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., (2001). Experimental data as provided in
Table 1 indicates expression in human lung cell lines and lung
tumor tissues. For example, a nucleic acid molecule encoding the
LCAT 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.
[0084] A LCAT 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.
[0085] 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 LCAT 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.
[0086] A chimeric or fusion LCAT 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 LCAT-encoding nucleic acid
can be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the LCAT protein or peptide.
[0087] As mentioned above, the LCAT peptide or the LCAT protein has
obvious variants of the amino acid sequence, such as naturally
occurring mature forms of the LCAT, allelic/sequence variants of
the LCAT, non-naturally occurring recombinantly derived variants of
the LCATs, and orthologs and paralogs of the LCAT proteins or
peptides. Such variants can readily be generated using art-known
techniques in the fields of recombinant nucleic acid technology and
protein biochemistry.
[0088] It is understood, however, that LCAT and variants exclude
any amino acid sequences disclosed prior to the invention.
[0089] 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 LCAT 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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 LCAT peptides of the present invention as
well as being encoded by the same genetic locus as the LCAT peptide
provided herein (see Table 1).
[0094] Allelic variants of a LCAT 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 LCAT
peptide as well as being encoded by the same genetic locus as the
LCAT peptide provided herein. Genetic locus can readily be
determined based on the genomic information provided in Table 1,
such as the genomic sequence mapped to the reference human. As used
herein, two proteins (or a region of the proteins) have significant
homology when the amino acid sequences are typically at least about
70-80%, 80-90%, and more typically at least about 90-95% or more
homologous. A significantly homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence that will hybridize to a LCAT peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0095] Paralogs of a LCAT peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the LCAT 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 LCAT peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0096] Orthologs of a LCAT peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the LCAT 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 LCAT 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.
[0097] Non-naturally occurring variants of the LCAT 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 LCAT peptide. For example, one class of substitutions is
conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a LCAT 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).
[0098] Variant LCAT 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.
[0099] 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.
[0100] 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 LCAT 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)).
[0101] The present invention further provides fragments of the
LCATs, in addition to proteins and peptides that comprise and
consist of such fragments, particularly those comprising the
residues identified in Table 1. As used herein, a fragment
comprises at least 8, 10, 12, 14, 16, 18, 20 or more contiguous
amino acid residues from a LCAT. Such fragments can be chosen based
on the ability to retain one or more of the biological activities
of the LCAT 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 LCAT, 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).
[0102] 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 LCATs are described in basic
texts, detailed monographs, and the research literature, and they
are well known to those of skill in the art.
[0103] 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.
[0104] 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)).
[0105] Accordingly, the LCATs 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 LCAT is fused
with another compound, such as a compound to increase the half-life
of the LCAT (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature LCAT, such as a
leader or secretory sequence or a sequence for purification of the
mature LCAT or a pro-protein sequence.
Protein/Peptide Uses
[0106] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in Table 1; to raise antibodies or to elicit
another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein (or its binding partner or ligand) in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a LCAT-effector
protein interaction or LCAT-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.
[0107] 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. 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, LCATs 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 LCAT. Experimental data as provided in Table 1
indicate that the LCATs of the present invention are expressed at
differential level in various lung cell lines and lung tissues, for
example, SEQ ID NOS: 54-154 are overexpressed in all tested cell
lines and tumor tissues, whereas other proteins or peptides are
underexpressed in selected cell lines or tissues (for example, SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 70). In one example, protein
(SEQ ID NO:27) or peptide (SEQ ID NO: 1847) is overexpressed in one
cell line (HBE4-E6/E7), yet underexpressed in another cell line
(Calu-3).
[0108] A large percentage of pharmaceutical agents are being
developed that modulate the activity of LCAT proteins, particularly
members of the LCAT subfamily (see Background of the Invention).
The structural and functional information provided in the
Background and Figures and Table 1 provide specific and substantial
uses for the molecules of the present invention, particularly in
combination with the expression information provided in Table 1.
Experimental data as provided in Table 1 indicates expression in
human lung cell lines and lung tumor tissues. Such uses can readily
be determined using the information provided herein, that which is
known in the art, and routine experimentation.
[0109] 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 LCATs that
are related to members of the LCAT subfamily. Such assays involve
any of the known LCAT functions or activities or properties useful
for diagnosis and treatment of LCAT-related conditions that are
specific for the subfamily of LCATs that the one of the present
invention belongs to, particularly in cells and tissues that
express the LCAT. Experimental data as provided in Table 1 indicate
that the LCATs of the present invention are expressed at
differential level in various lung cell lines and lung tissues, for
example, SEQ ID NOS: 54-154 are overexpressed in all tested cell
lines and tumor tissues, whereas other proteins or peptides are
underexpressed in selected cell lines or tissues (for example, SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 70). In one example, protein
(SEQ ID NO:27) or peptide (SEQ ID NO: 1847) is overexpressed in one
cell line (HBE4-E6/E7), yet underexpressed in another cell line
(Calu-3).
[0110] 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 LCAT, as a biopsy or expanded in cell culture. Experimental
data as provided in Table 1 indicates expression in human lung cell
lines and lung tumor tissues. In an alternate embodiment,
cell-based assays involve recombinant host cells expressing the
LCAT protein.
[0111] The polypeptides can be used to identify compounds or agents
that modulate LCAT activity of the protein in its natural state or
an altered form that causes a specific disease or pathology
associated with the LCAT. Both the LCATs 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 LCAT. These compounds can be further
screened against a functional LCAT to determine the effect of the
compound on the LCAT 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 LCAT to a desired
degree.
[0112] 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 LCAT protein and a molecule that
normally interacts with the LCAT protein, e.g. a substrate or or an
extracellular binding ligand or a component of the signal pathway
that the LCAT protein normally interacts (for example, a cytosolic
signal protein or another LCAT). Such assays typically include the
steps of combining the LCAT protein with a candidate compound under
conditions that allow the LCAT 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 LCAT 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.
[0113] 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).
[0114] One candidate compound or agent is a soluble fragment of the
LCAT that competes for substrate binding. Other candidate compounds
include mutant LCATs or appropriate fragments containing mutations
that affect LCAT 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.
[0115] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) LCAT
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate LCAT 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 LCAT protein dependent signal cascade can be
assayed. Any of the biological or biochemical functions mediated by
the LCAT can be used as an endpoint assay. These include all of the
biochemical or biochemical/biological events described herein, in
the references cited herein, incorporated by reference for these
endpoint assay targets, and other functions known to those of
ordinary skill in the art or that can be readily identified using
the information provided in Table 1. Specifically, a biological
function of a cell or tissues that expresses the LCAT can be
assayed. Experimental data as provided in Table 1 indicate that the
LCATs of the present invention are expressed at differential level
in various lung cell lines and lung tissues, for example, SEQ ID
NOS: 54-154 are overexpressed in all tested cell lines and tumor
tissues, whereas other proteins or peptides are underexpressed in
selected cell lines or tissues (for example, SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 70). In one example, protein (SEQ ID NO: 27) or
peptide (SEQ ID NO: 1847) is overexpressed in one cell line
(HBE4-E6/E7), yet underexpressed in another cell line (Calu-3).
[0116] Binding and/or activating compounds can also be screened by
using chimeric LCAT 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
LCAT. 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 LCAT 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 LCAT (e.g. binding partners and/or
ligands). Thus, a compound is exposed to a LCAT polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble LCAT polypeptide is also added to the
mixture. If the test compound interacts with the soluble LCAT
polypeptide, it decreases the amount of complex formed or activity
from the LCAT. This type of assay is particularly useful in cases
in which compounds are sought that interact with specific regions
of the LCAT. Thus, the soluble polypeptide that competes with the
target LCAT 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 LCAT 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 LCAT-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 LCAT-binding
protein and a candidate compound are incubated in the LCAT
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 LCAT protein target molecule, or which are
reactive with LCAT protein and compete with the target molecule, as
well as LCAT-linked assays which rely on detecting an enzymatic
activity associated with the target molecule.
[0120] Agents that modulate one of the LCATs 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 LCAT protein activity identified according to
these drug screening assays can be used to treat a subject with a
disorder mediated by the LCAT pathway, by treating cells or tissues
that express the LCAT. Experimental data as provided in Table 1
indicates expression in human lung cell lines and lung tumor
tissues. These methods of treatment include the steps of
administering a modulator of LCAT 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 LCAT 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 LCAT
and are involved in LCAT activity. Such LCAT-binding proteins are
also likely to be involved in the propagation of signals by the
LCAT proteins or LCAT targets as, for example, downstream elements
of a LCAT-mediated signaling pathway. Alternatively, such
LCAT-binding proteins are likely to be LCAT 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 LCAT
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 LCAT-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 LCAT 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 LCAT-modulating
agent, an antisense LCAT nucleic acid molecule, an LCAT-RNAi
fragment, a LCAT-specific antibody, or a LCAT-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 LCAT 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 lung cell lines and lung tumor tissues. The method involves
contacting a biological sample with a compound capable of
interacting with the LCAT 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 LCAT 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
LCAT protein in which one or more of the LCAT 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 LCAT 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.
[0130] 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 lung cell lines and lung tumor
tissues. Accordingly, methods for treatment include the use of the
LCAT protein or fragments.
Antibodies
[0131] The present invention provides antibodies specifically bind
to LCAT proteins or fragments thereof, peptides, or antigenic
portion thereof.
[0132] 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.
[0133] The antibody of present invention selectively binds a target
LCAT 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.
[0134] 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.
[0135] 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).
[0136] 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.
[0137] 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.
[0138] "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).
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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 LCAT protein can be used.
Particularly important fragments are those covering functional
domains, some but not all the examples of the domains are
identified in Table 1. Many methods are known for generating and/or
identifying antibodies to a given target peptide. Several such
methods are described by Harlow, Antibodies, Cold Spring Harbor
Press, (1989).
[0145] 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.
[0146] "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).
[0147] 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 LCAT
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.
[0148] 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.
[0149] The protein used here in as the immunogen is not limited to
any particular type of immunogen. In one aspect, antibodies are
preferably prepared from regions or discrete fragments of the LCAT
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: 1830-2100 and fragments of SEQ ID NOS:
1-778. 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .delta.1, .delta.2 or .delta.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .delta.3 (Guss et
al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0155] 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
[0156] 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 LCATs of
the present invention are expressed at differential level in
various lung cell lines and lung tissues, for example, SEQ ID NOS:
54-154 are overexpressed in all tested cell lines and tumor
tissues, whereas other proteins or peptides are underexpressed in
selected cell lines or tissues (for example, SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 70). In one example, protein (SEQ ID NO:27) or
peptide (SEQ ID NO: 1847) is overexpressed in one cell line
(HBE4-E6/E7), yet underexpressed in another cell line (Calu-3).
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.
[0157] 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 lung cell lines and lung
tumor tissues. If a disorder is characterized by a specific
mutation in the protein, antibodies specific for this mutant
protein can be used to assay for the presence of the specific
mutant protein.
[0158] 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 lung cell lines and lung tumor tissues. The
diagnostic uses can be applied, not only in genetic testing, but
also in monitoring a treatment modality. Accordingly, where
treatment is ultimately aimed at correcting expression level or the
presence of aberrant sequence and aberrant tissue distribution or
developmental expression, antibodies directed against the protein
or relevant fragments can be used to monitor therapeutic efficacy.
More detection and diagnostic methods are described in detail
below.
[0159] 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.
[0160] The antibodies are also useful for tissue typing.
Experimental data as provided in Table 1 indicates expression in
human lung cell lines and lung tumor tissues. Thus, where a
specific protein has been correlated with expression in a specific
tissue, antibodies that are specific for this protein can be used
to identify a tissue type.
[0161] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the LCAT peptide to
a binding partner such as a substrate or another antibody binding
to LCATs. 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.
[0162] 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
[0163] The present invention further provides isolated nucleic acid
molecules that encode a LCAT 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 LCAT 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in Table 1,
(SEQ ID NOS: 779-1829), or any nucleic acid molecule that encodes
the protein provided in Table 1, (SEQ ID NOS: 1-778). 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.
[0168] In Table 1, human transcript sequences are provided. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0169] 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.
[0170] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the LCAT
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.
[0171] 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).
[0172] 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 LCAT
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.
[0173] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in Table 1. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence.
[0174] 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.
[0175] 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.
[0176] 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 1 or a fragment of this
sequence. Such nucleic acid molecules can readily be identified as
being able to hybridize under moderate to stringent conditions, to
the nucleotide sequence shown in Table 1 or a fragment of the
sequence. Allelic variants can readily be determined by genetic
locus of the encoding gene.
[0177] 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
[0178] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in Table 1 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in Table 1.
[0179] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in Table 1.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0180] 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.
[0181] 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.
[0182] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0183] 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.
[0184] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0185] 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.
[0186] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0187] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0188] 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 LCATs of the present invention are expressed at
differential level in various lung cell lines and lung tissues, for
example, SEQ ID NOS: 54-154 are overexpressed in all tested cell
lines and tumor tissues, whereas other proteins or peptides are
underexpressed in selected cell lines or tissues (for example, SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 70). In one example, protein
(SEQ ID NO:27) or peptide (SEQ ID NO: 1847) is overexpressed in one
cell line (HBE4-E6/E7), yet underexpressed in another cell line
(Calu-3).
[0189] 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 LCAT protein expression relative to normal
results.
[0190] 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.
[0191] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a LCAT protein, such as
by measuring a level of a LCAT-encoding nucleic acid in a sample of
cells from a subject e.g., mRNA or genomic DNA, or determining if a
LCAT gene has been mutated. Experimental data as provided in Table
1 indicate that the LCATs of the present invention are expressed at
differential level in various lung cell lines and lung tissues, for
example, SEQ ID NOS: 54-154 are overexpressed in all tested cell
lines and tumor tissues, whereas other proteins or peptides are
underexpressed in selected cell lines or tissues (for example, SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 70). In one example, protein
(SEQ ID NO:27) or peptide (SEQ ID NO: 1847) is overexpressed in one
cell line (HBE4-E6/E7), yet underexpressed in another cell line
(Calu-3). More detection and diagnosis methods are described in
detail below.
[0192] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate LCAT nucleic acid
expression.
[0193] 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 LCAT gene, particularly biological
and pathological processes that are mediated by the LCAT in cells
and tissues that express it. Experimental data as provided in Table
1 indicates expression in human lung cell lines and lung tumor
tissues. The method typically includes assaying the ability of the
compound to modulate the expression of the LCAT nucleic acid and
thus identifying a compound that can be used to treat a disorder
characterized by undesired LCAT nucleic acid expression. The assays
can be performed in cell-based and cell-free systems. Cell-based
assays include cells naturally expressing the LCAT nucleic acid or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[0194] The assay for LCAT 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 LCAT 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.
[0195] Thus, modulators of LCAT 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 LCAT mRNA in the presence of the candidate compound
or agent is compared to the level of expression of LCAT 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.
[0196] 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
LCAT nucleic acid expression in cells and tissues that express the
LCAT. Experimental data as provided in Table 1 indicate that the
LCATs of the present invention are expressed at differential level
in various lung cell lines and lung tissues, for example, SEQ ID
NOS: 54-154 are overexpressed in all tested cell lines and tumor
tissues, whereas other proteins or peptides are underexpressed in
selected cell lines or tissues (for example, SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 70). In one example, protein (SEQ ID NO:27) or
peptide (SEQ ID NO: 1847) is overexpressed in one cell line
(HBE4-E6/E7), yet underexpressed in another cell line (Calu-3).
Modulation includes both up-regulation (i.e. activation or
agonization) or down-regulation (suppression or antagonization) or
nucleic acid expression.
[0197] 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
LCAT nucleic acid expression in the cells and tissues that express
the protein. Experimental data as provided in Table 1 indicates
expression in human lung cell lines and lung tumor tissues.
[0198] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds or agents on the
expression or activity of the LCAT 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.
[0199] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in LCAT nucleic acid expression, and
particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in LCAT
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 LCAT 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 LCAT 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 LCAT protein.
[0200] Individuals carrying mutations in the LCAT 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.
[0201] Alternatively, mutations in a LCAT gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0202] 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.
[0203] 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 LCAT 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)).
[0204] 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.
[0205] 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 LCAT gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment.
[0206] 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.
[0207] The nucleic acid molecules are thus useful as antisense
constructs to control LCAT 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 LCAT protein. An
antisense RNA or DNA nucleic acid molecule would hybridize to the
mRNA and thus block translation of mRNA into LCAT protein.
[0208] 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.
[0209] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of LCAT nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired LCAT 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 LCAT protein, such as substrate
binding.
[0210] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in LCAT 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 LCAT protein to treat the individual.
[0211] The invention also encompasses kits for detecting the
presence of a LCAT nucleic acid in a biological sample.
Experimental data as provided in Table 1 indicate that the LCATs of
the present invention are expressed at differential level in
various lung cell lines and lung tissues, for example, SEQ ID NOS:
54-154 are overexpressed in all tested cell lines and tumor
tissues, whereas other proteins or peptides are underexpressed in
selected cell lines or tissues (for example, SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 70). In one example, protein (SEQ ID NO:27) or
peptide (SEQ ID NO: 1847) is overexpressed in one cell line
(HBE4-E6/E7), yet underexpressed in another cell line (Calu-3). For
example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting LCAT nucleic
acid in a biological sample; means for determining the amount of
LCAT nucleic acid in the sample; and means for comparing the amount
of LCAT 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 LCAT protein mRNA
or DNA.
Vectors/Host Cells
[0212] 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.
[0213] 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.
[0214] 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).
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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).
[0219] 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).
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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)).
[0224] 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.).
[0225] 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)).
[0226] 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)).
[0227] 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.
[0228] 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).
[0229] 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.
[0230] 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).
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 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.
[0235] Where secretion of the peptide is desired, which may be
difficult to achieve with multi-transmembrane domain containing
proteins such as LCATs, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0236] Where the peptide is not secreted into the medium, which is
typically the case with LCATs, 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.
[0237] 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
[0238] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a LCAT protein or peptide that can be further purified to
produce desired amounts of LCAT protein or fragments. Thus, host
cells containing expression vectors are useful for peptide
production.
[0239] Host cells are also useful for conducting cell-based assays
involving the LCAT protein or LCAT protein fragments, such as those
described above as well as other formats known in the art. Thus, a
recombinant host cell expressing a native LCAT protein is useful
for assaying compounds that stimulate or inhibit LCAT protein
function.
[0240] Host cells are also useful for identifying LCAT 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 LCAT protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native LCAT protein.
[0241] 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 LCAT protein and identifying
and evaluating modulators of LCAT protein activity. Other examples
of transgenic animals include non-human primates, sheep, dogs,
cows, goats, chickens, and amphibians.
[0242] 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 LCAT
protein nucleotide sequences can be introduced as a transgene into
the genome of a non-human animal, such as a mouse.
[0243] 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 LCAT
protein to particular cells.
[0244] 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.
[0245] 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.
[0246] 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.
[0247] 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, LCAT 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 LCAT protein function,
including substrate interaction, the effect of specific mutant LCAT
proteins on LCAT protein function and substrate interaction, and
the effect of chimeric LCAT proteins. It is also possible to assess
the effect of null mutations, that is, mutations that substantially
or completely eliminate one or more LCAT protein functions.
Detection and Diagnosis
[0248] The present invention provides a method for detecting LCAT
nucleic acids, proteins, peptides and fragments thereof that are
differentially expressed in lung diseases in a test sample,
preferably in a biological sample.
[0249] The present invention further provides a method for
diagnosing the lung 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. While the protein is overexpressed, the
expression of LCAT is preferably greater than about 20%, or
preferably greater than about 30%, and most preferably greater than
about 50% or more of normal lung sample; or 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 LCAT is preferably less than
about 20%, or preferably less than 30%, and most preferably less
than about 50% or less of the normal lung sample; or 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 "biological sample" can be collected from
tissues, blood, sera, cell lines or biological fluids. In one
embodiment, a biological sample comprises cells or tissues
suspected of having diseases (e.g., cells obtained from a
biopsy).
[0251] As used herein, "biological fluids" are plasma, interstitial
fluid, urine, cerebrospinal fluid, and the like, containing cells
or proteins including shed proteins.
[0252] As used herein, a "differential level" is defined as the
level of LCAT proteins or nucleic acids in a test sample either
above or below the level of the ones in control samples, wherein
the level of control samples is obtained either from a control cell
line, a normal tissue or body fluids, or combination thereof, from
a healthy subject.
[0253] 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 lung diseases or
lung associated diseases by using conventional diagnostic
methods.
[0254] As used herein the "diseases" include lung diseases and lung
associated diseases. Preferably, the lung disease is lung
cancer.
[0255] As used herein, "cancer" includes epithelial-cell related
cancers, for example pancreatic, lung, colon, prostate, ovarian,
breast as well as bladder and renal cancer.
Nucleic Acid Detections
[0256] The present invention is not limited to the detection
methods described above. Any suitable detection method that allows
for the specific detection of lung diseased cells, tissues or
organs may be utilized. For example, in some embodiments, the
expression of RNA corresponding to a LCAT 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.).
[0257] In another embodiment, the present invention provides a
method for diagnosing or detecting lung diseases in a subject
comprising: determining the level of one or more LCAT nucleic acid
molecules or any fragment(s) thereof in a test sample from said
subject, wherein said LCAT nucleic acid molecule(s) comprises a
sequence selected from a group consisting of SEQ ID NOS: 779-1829
and a combination thereof; wherein a differential level of said
LCAT 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 lung
diseases.
[0258] 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: 779-1829 and combination
thereof.
[0259] In further embodiments of the present invention, the
presence of particular sequences in the genome of a subject is
detected. Such sequences include LCAT sequences associated with
abnormal expression of LCAT (e.g., overexpression or expression at
a physiological inappropriate time). These sequences include
polymorphisms, including polymorphisms in the transcribed sequence
(e.g., that effect LCAT 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
[0260] The present invention provides methods for diagnosing or
detecting the differential presence of LCAT protein. In some
embodiments (e.g., where LCATs are overexpressed in diseased
cells), LCAT proteins are detected directly. In other embodiments
(e.g., where the presence of a LCATs are underexpressed), LCAT to
the disease antigens are detected non-existence.
[0261] 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 LCAT may be indicative of
a specific stage or sub-type of the same cancer or disease.
[0262] The information obtained is also used to determine prognosis
and appropriate course of treatment. For example, it is
contemplated that individuals with a specific LCAT expression or
stage of lung diseases may respond differently to a given treatment
that individuals lacking the LCAT expression. The information
obtained from the diagnostic methods of the present invention thus
provides for the personalization of diagnosis and treatment.
[0263] In one embodiment, the present invention provides a method
for monitoring lung disease treatment in a subject comprising:
determining the level of one or more LCAT proteins or any
fragment(s) or peptide(s) thereof in a test sample from said
subject, wherein said LCAT protein(s) comprises a sequence selected
from a group consisting of SEQ ID NOS: 1-778, SEQ ID NOS: 1830-2100
and a combination thereof; wherein an level of said LCAT 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.
[0264] In another embodiment, the present invention provides a
method for diagnosing recurrence of lung diseases following
successful treatment in a subject comprising: determining the level
of one or more LCAT proteins or any fragment(s) or peptide(s)
thereof in a test sample from said subject, wherein said LCAT
protein(s) comprises a sequence selected from a group consisting of
SEQ ID NOS: 1-778, SEQ ID NOS: 1830-2100 or a combination thereof;
wherein a changed level of said LCAT 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 lung diseases.
[0265] In yet another embodiment, the present invention provides a
method for diagnosing or detecting lung diseases in a subject
comprising: determining the level of one or more LCAT proteins or
any fragment(s) or peptides thereof in a test sample from said
subject, wherein said LCAT protein(s) comprises a sequence selected
from a group consisting of SEQ ID NOS: 1-778, SEQ ID NOS: 1830-2100
and a combination thereof; wherein a differential level of said
LCAT 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 lung diseases.
[0266] 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 NOS: 1-778 and combination thereof.
[0267] Further, the detecting or diagnosing method comprises
determining level of differential expression of 5, 10, 15, 20, 40,
60, 80, 100 or more LCAT peptides, preferably the peptides are
selected from the group consisting of SEQ ID NOS: 1830-2100 and
combination thereof.
[0268] 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.
[0269] In yet another embodiment, the present invention provides a
detection or diagnostic method of LCATs by using LC/MS. The
proteins from cells are prepared by methods known in the art. 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.
[0270] 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 LCAT protein or peptide are similar to the expression
pattern in Table 1 in a test subject indicates the likelihood of
having lung diseases or diseases associated with lung.
[0271] 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
[0272] 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.
[0273] 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.
[0274] 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 LCAT
proteins, peptides or fragments thereof, in specific cells,
tissues, blood, serum or body fluids.
[0275] 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:
[0276] (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.
[0277] (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.
[0278] (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).
[0279] 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.
[0280] 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.
[0281] 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).
[0282] The biological samples can then be tested directly for the
presence of LCAT by assays (e.g., ELISA or radioimmunoassay) and
format (e.g., microwells, dipstick (e.g., as described in
International Patent Publication WO 93/03367), etc). Alternatively,
proteins in the sample can be size separated (e.g., by
polyacrylamide gel electrophoresis (PAGE)), in the presence or
absence of sodium dodecyl sulfate (SDS), and the presence of LCAT
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.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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 LCAT
targets and the affinity value(Kd) is less than 1.times.10.sup.8
M.
[0288] 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.
[0289] 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 LCAT protein express in situ. The detailed procedure is
shown in the Example.
Array:
[0290] "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.
[0291] An "expression profile" is a representation of gene
expression in a sample. A nucleic acid expression profile is
produced using sequencing, hybridization, or amplification
technologies using transcripts from a sample. A protein expression
profile, although time delayed, minors the nucleic acid expression
profile and is produced using gel electrophoresis, mass
spectrometry, or an array and labeling moieties or antibodies which
specifically bind the protein. The nucleic acids, proteins, or
antibodies specifically binding the protein may be used in solution
or attached to a substrate, and their detection is based on methods
well known in the art.
[0292] A substrate includes but not limited to, paper, nylon or
other type of membrane, filter, chip, glass slide, or any other
suitable solid support.
[0293] The invention also provides an array with a cDNA or
transcript encoding LCAT proteins or peptides or fragments thereof,
antibodies that specifically bind LCAT proteins, peptides or
fragments thereof. Preferably, two or more of the nucleic acid
molecules (e.g., SEQ ID NOS: 779-1829), proteins (e.g., SEQ ID NOS:
1-778) or peptides (e.g., SEQ ID NOS: 1830-2100) are immobilized on
a substrate. Specifically, the following targets are selected for
targeting purpose: Tissue Factor Kunitz Type inhibitor-1 (HAI-1),
CD98, CD147, CD73, CD59, CD90, CD49f, CD46, CD151, and CD55.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] In one embodiment, the detected targets comprise, consist
essentially of or consist of combinations of LCAT 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: 1-778), nucleic acids
(SEQ ID NOS: 779-1829) encoding such proteins, or peptides (SEQ ID
NOS: 1830-2100).
[0306] In one embodiment, the combinations of the protein or
nucleic acid targets for detection of a lung cancer comprise
targets selected from a group consisting of Tissue Factor Kunitz
Type inhibitor-1 (HAI-1), CD98, CD147, CD73, CD59, CD90, CD49f,
CD46, CD151, and CD55. wherein their corresponding sequences are
listed in Table 1.
[0307] In another embodiment, the detection targets are combination
of all the targets of: Tissue Factor Kunitz Type inhibitor-1
(HAI-1), CD98, CD147, CD73, CD59, CD90, CD49f, CD46, CD151, and
CD55.
[0308] In yet another embodiment, the invention provides a
composition comprising a plurality of LCAT 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: 779-1829, or all the
sequences of SEQ ID NOS: 779-1829, 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.
[0309] In yet another embodiment, the invention provides a
composition comprising a plurality of LCAT 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: 1-778 or all sequence of SEQ
ID NOS: 1-778; and wherein said plurality of peptide sequences
comprise two or more sequences of SEQ ID NOS: 1830-2100 or all
sequence of SEQ ID NOS: 1830-2100.
[0310] In yet another embodiment, the compositions can be used in
diagnosing or monitoring the treatment of lung cancer
Treatment
[0311] The following terms, as used in the present specification
and claims, are intended to have the meaning as defined below,
unless indicated otherwise.
[0312] "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.
[0313] 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.
[0314] A "lung disease" includes lung cancer, lung tumor (exocrine
or endocrine), lung cysts as well as lung trauma; preferably lung
cancer.
[0315] A "cancer" is epithelial-cell related cancers including, but
not limited to, pancreatic, lung, colon, prostate, ovarian, breast
as well as bladder and renal cancer.
[0316] The present invention provides an application of treatment
by using antibody, immunogenic peptides as well as other LCAT
agonists or antagonists.
[0317] LCATs are proteins differentially expressed in the lung
diseased cell lines or tissues. The proteins are either cell
surface proteins or cytosolic proteins (see the list in Table 1).
These proteins are associated with the diseases especially lung
diseases, particularly lung cancer; thus, they serve as candidate
targets for the treatment of the diseases.
[0318] 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.
[0319] 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.
[0320] 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
[0321] The antibody of the present invention can be used for
therapeutic reason. It is contemplated that the antibody of the
present invention may be used to treat a mammal, preferably human
with lung diseases.
[0322] In general, the antibodies are also useful for inhibiting
protein function, for example, blocking the binding of the LCAT
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.
[0323] 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 LCAT can
be passively administered alone or in conjunction with other
anti-cancer therapies to a mammal afflicted-with lung 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).
[0324] 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.
[0325] 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.
[0326] 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.
[0327] 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.
[0328] 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.
[0329] 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.
[0330] Depending on the type and severity of the disease, about 1
mug/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 mug/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.
[0331] 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.
[0332] 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.
[0333] 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, diphtheria
toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella
toxin, and pokeweed antiviral protein.
[0334] 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.
[0335] 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.
[0336] 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.
[0337] 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.).
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 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 LCAT 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.
[0346] 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.
[0347] 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
[0348] The LCAT proteins or peptides or fragments thereof of this
invention are also intended for use in producing antiserum designed
for pre- or post-disease prophylaxis. Here the protein, peptides or
fragment thereof, is formulated with a suitable adjuvant and
administered by injection to human volunteers, according to known
methods for producing human antisera. Antibody response to the
injected proteins is monitored, during a several-week period
following immunization, by periodic serum sampling to detect the
presence of antiserum antibodies, using an immunoassay as described
herein.
[0349] The antiserum from immunized individuals may be administered
as a prophylactic measure for individuals who are at risk of
developing lung diseases or cancer. The antiserum is also useful in
treating an individual afflicted with lung diseases or cancer for
post-disease prophylaxis.
[0350] Alternatively, peptides derived form the LCAT 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.).
[0351] 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.
[0352] Alternatively, amino acid sequence variants of the peptide
can be prepared by mutations in the DNA, which encodes the peptide,
or by peptide synthesis.
[0353] 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.
[0354] 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).
[0355] 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.
[0356] The peptides of the present invention are used for treatment
of the lung diseases. Treatment involves administration of the
protective composition after the appearance of the disease.
[0357] 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."
[0358] 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 lung diseases and diseases resulting from unregulated
clonal T cell replication.
[0359] Therefore, the present invention relates to peptides or
modified peptides derived from the protein sequences of the LCAT
proteins that differentially expressed in the lung 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.
[0360] 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.
[0361] The recombinant or natural protein, peptides, or fragment
thereof of LCAT, 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 lung diseases, particularly, cancer. The prophylactic
administration of the lung cancer vaccine should serve to prevent
or attenuate lung diseases, preferably cancer, in a mammal.
[0362] Preparation of vaccine is using recombinant protein or
peptide expression vectors comprising all or part of nucleic acid
sequence of LCAT 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:
779-1829 can be introduced into a mammal either prior to any
evidence of lung diseases or to mediate regression of the disease
in a mammal afflicted with lung 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
LCAT 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 LCAT 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.
[0363] Alternatively all or parts thereof of a substantially or
partially purified the LCAT 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.
[0364] 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.).
[0365] The vaccine formulation of the present invention comprises
an immunogen that induces an immune response directed against the
cancer associated antigens such as the LCATs, 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.
[0366] 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. The vaccine formulations may be
evaluated first in animal models, initially rodents
[0367] In one embodiment mammals, preferably human, at high risk
for lung 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 lung diseases, humans with a history of lung diseases,
particular cancer, or humans afflicted with lung 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 lung
diseases or advanced stage of lung 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.
[0368] 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.
[0369] 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-LCAT immune cells or anti-LCAT antibody
is produced. The presence of anti-LCAT immune cells may be assessed
by measuring the frequency of precursor CTL (cytoxic T-lymphocytes)
against LCAT 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.
[0370] 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, lung diseases patients can be tested.
Conventional methods would be used to evaluate the immune response
of the patient to determine the efficiency of the vaccine.
[0371] In yet another embodiment of this invention all, part, or
parts of the LCAT 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 LCAT antigen activated dendritic cells or
processed dendritic cell antigens may be used as immunogens for
vaccines or for the treatment of lung diseases, particularly lung
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).
[0372] In yet another aspect of this invention T-cells isolated
from individuals can be exposed to the LCAT 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).
[0373] 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.
[0374] 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, 3.sup.rd Ed.; Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (2001).
WORKING EXAMPLES
1. Lung Cancer Cell Line Culture
[0375] Lung cancer cell lines were obtained from ATCC (Manassas,
Va.). Cancer cell lines NCI-H23, NCI-H2291, NCI-H1299, NCI-H441,
Calu-1, and Calu-3 were cultured in RPMI 1640 supplemented with
1.times. sodium pyruvate, 10% FBS, 10 mM Hepes, 1.times.
penicillin-streptomycin-glutamine, and 1.times. non-essential amino
acids. Cancer cell lines NCI-H522 and NCI-H358 were cultured in
RPMI 1640 supplemented with 10% FBS, 10 mM Hepes, 1.times.
penicillin-streptomycin-glutamine, and 1.times. non-essential amino
acids. A549 was cultured in Ham's F12 media supplemented with 10%
FBS, 1.times. penicillin-streptomycin-glutamine, and sodium
bicarbonate. SK-LU-1 was cultured in D-MEM media supplemented with
10% FBS and 1.times. penicillin-streptomycin-glutamine.
[0376] Non-cancer lung cell lines Beas-2B, Bet-1A, and HBE4-E6/E7
were purchased from ATCC. These cell lines were cultured in LHC-9
serum-free media supplemented with 1.times.
penicillin-streptomycin-glutamine.
[0377] Primary normal human bronchial epithelial cells (NHBE) were
obtained from Cambrex. NHBE cells were cultured in LHC-9 media. All
cells were passaged twice a week. Cancer cells were disassociated
with trypsin and the non-cancer cell lines and primary cells were
disassociated with versene. Prior to harvesting for experiments,
cells were cultured for 48 hours until 75-80% confluency and
disassociated with versene. All tissue culture reagents were
purchased from BioFluids.
Tissue Processing
[0378] Lung tumor and distal normal tissue was collected. Tissue
specimens arrived 12-16 hours post surgery in Lung Transport
Buffer. Lung Transport Buffer was composed of LHC-9 media
supplemented with 1.times. penicillin-streptomycin-glutamine,
1.times. fungizone (BioFluids) and Protease Inhibitor Cocktail
(Sigma). Upon arrival, tissues were imaged, weighed, and dimensions
measured (L.times.W.times.H). Tumor tissue was dissected when
necessary for removal of necrotic regions. Tissue was re-weighed
post-dissection.
[0379] Tissue was crudely minced and incubated for 20-30 minutes
with periodic agitation at 37.degree. C. in Enzyme Combination #1
(200 units Collagenase, cat# C5894 Sigma; 126 ug DNAse I, cat#D4513
Sigma (in 10 mM Tris/HCl pH7.5); 50 mM NaCl; 10 mM MgCl2; 0.05%
Elastase, cat# E7885 Sigma). D-PBS was added at 3 times the volume
of the enzyme combination, the tissue finely minced, and
disassociated cells passed through a 200 um filter. The cells were
washed twice with D-PBS. Red blood cells are lysed with PharMLyse
(BD Biosciences) when necessary. Cell number and viability are
determined by PI exclusion (GUAVA). Cells at a total cell number
greater than 20.times.10.sup.6 are sorted using a high-speed sorter
(MoFlo Cytomation) for epithelial cells (EpCAM positive).
[0380] The remaining undigested tissue was incubated for 20-30
minutes with periodic agitation at 37.degree. C. in Enzyme
Combination #2 (1.times. Liberase Blendzyme 1 cat# 988-417 Roche,
1.times. Liberase Blendzyme 3 cat#814-184 Roche, 0.05% Elastase
cat# E7885 Sigma). D-PBS was added at 3 times the volume of the
enzyme combination, and the tissue finely minced until tissue was
completely disassociated. The cells were passed through a 200 um
filter, washed twice with D-PBS, and pooled with cells from Enzyme
Combination #1 digestion.
[0381] Cells were passed through a 70 um filter for single cell
suspension and cell number and viability was determined by PI
exclusion (GUAVA). When needed, red blood cells were lysed with
PharMLyse (BD Biosciences). Cells were incubated in 20 ml of
1.times. PharMLyse in D-PBS for 30 seconds with gentle agitation
and cells pelleted at 300.times.g for 5 minutes at 4.degree. C.
Cells were washed once in D-PBS and cell number and viability were
recalculated by PI exclusion using the GUAVA. Cells at a total cell
number greater than 20.times.10.sup.6 were sorted using a
high-speed sorter (MoFlo Cytomation) for epithelial cells (EpCAM
positive).
2. Cloning and Expression of Target Proteins
[0382] cDNA Retrieval
[0383] Peptide sequences were searched by BlastP against the Celera
Discovery System (CDS) 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
[0384] 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 Mg504.
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
[0385] 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
[0386] 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
[0387] For cloning using this method, PCR primers contained the
following overhangs:
TABLE-US-00001 Forward 5' overhang:
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3' Reverse 5' overhang:
5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'
[0388] 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
[0389] 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 DH5a 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
[0390] Constructs were transformed into one or more of the
following host strains: BL21 SI, BL21 AI, (Invitrogen); Origami B
(DE3), Origami B (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS,
Rosetta-Gami (DE3), Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B
(DE3) pLysS (Novagen). The transformants 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
[0391] 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
[0392] 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.
[0393] 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.
[0394] 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
[0395] Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in a
batchwise or continuous flow process which sequentially adds
.alpha.-amino- and side chain-protected amino acid residues to an
insoluble polymeric support via a linker group. A linker group such
as methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-a-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin. Trifluoroacetic acid
or piperidine are used to remove the protecting group in the case
of Boc or Fmoc, respectively. Each additional amino acid is added
to the anchored residue using a coupling agent or pre-activated
amino acid derivative, and the resin is washed. The full length
peptide is synthesized by sequential deprotection, coupling of
derivitized amino acids, and washing with dichloromethane and/or
N,N-dimethylformamide. The peptide is cleaved between the peptide
carboxy terminus and the linker group to yield a peptide acid or
amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook,
San Diego Calif. pp. S1-S20). Automated synthesis may also be
carried out on machines such as the 431A peptide synthesizer (ABI).
A protein or portion thereof may be purified by preparative high
performance liquid chromatography and its composition confirmed by
amino acid analysis or by sequencing (Creighton (1984) Proteins,
Structures and Molecular Properties, W H Freeman, New York
N.Y.).
5. Antibody Development
Polyclonal Antibody Preparations:
[0396] 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 LCATs from a
Library of scFvs
[0397] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against LCAT 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).
[0398] 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 10.sup.9 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.10.sup.8 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
mug/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage
are prepared as described in PCT publication WO 92/01047.
[0399] 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 rpm. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 mug
ampicillin/ml and 25 mug 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 mum filter (MINISART NML; Sartorius) to
give a final concentration of approximately 10.sup.13 transducing
units/ml (ampicillin-resistant clones).
[0400] Panning of the Library: Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 mug/ml or 10 mug/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.
[0401] Characterization of Binders: Eluted phages 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
[0402] i) Materials: 1) Complete Media No Sera (CMNS) for washing
of the myeloma and spleen cells; Hybridoma medium CM-HAT Cell Mab
(BD), 10% FBS (or HS); 5% Origen HCF (hybridoma cloning factor)
containing 4 mM L-glutamine and antibiotics} to be used for plating
hybridomas after the fusion.
[0403] 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.
[0404] 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-glutamine is dispensed into media to
supplement growth. L-glutamine 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%.
[0405] 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.
[0406] 5) 1 bottle of PEG 1500 in Hepes (Roche, N.J.)
[0407] 6) 8-Azaguanine is stored as the dried powder supplied by
SIGMA at -700.degree. C. until needed. Reconstitute 1 vial/500 ml
of media and add entire contents to 500 ml media (eg. 2
vials/litre).
[0408] 7) Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza
(1.times.) stored in the refrigerator at 4.degree. C.
[0409] 8) Clonal cell medium D (Stemcell, Vancouver) contains HAT
and methyl cellulose for semi-solid direct cloning from the
fusion.
[0410] 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.
[0411] 10) Origen HCF can be obtained directly from Igen and is a
cell supernatant produced from a macrophage-like cell-line. It can
be thawed and aliquoted to 15 ml tubes at 5 ml per tube and stored
frozen at -20.degree. C. Positive Hybridomas are fed HCF through
the first subcloning and are gradually weaned. It is not necessary
to continue to supplement unless you have a particularly difficult
hybridoma clone. This and other additives have been shown to be
more effective in promoting new hybridoma growth than conventional
feeder layers.
[0412] ii) Procedure
[0413] 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.
[0414] 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.
[0415] 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.
[0416] 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, and spun at 800 rpm for 12
minutes.
[0417] 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 a 1 cc needle over 1 minute.
[0418] RPMI-CMNS is added to the PEG cells 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.
[0419] Myeloma/HAT control. P is prepared as follows. Dilute about
1000 P3X63 Ag8.653 myeloma cells into 1 ml of mediu 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% CO.sub.2 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.
[0420] 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;
[0421] 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
[0422] mRNA Expression Validation in Tissues by TAQMAN
[0423] Expression of mRNA was quantitated by RT-PCR using TAQMAN
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.
[0424] 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.
[0425] 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 (CDS)
database. The assays were designed to span exon-exon borders and do
not amplify genomic DNA.
[0426] The TaqMan primers and probe sequences were as designed by
Applied Biosystems (AB) as part of the Assays on Demand product
line or by custom design through the AB Assays by Design
service.
[0427] 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
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.
[0428] 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
[0429] Data were analyzed for fold differences in expression using
an endogenous control for normalization, and measuring expression
was 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.
Tissue Flow Cytometry Analysis
[0430] Post tissue processing, cells were sorted by flow cytometry
known in the art to enrich for epithelial cells. Alternatively,
cells isolated from lung tissue were stained directly with EpCAM
(for epithelial cells) and the specific antibody to LCAT. Cell
numbers and viability were determined by PI exclusion (GUAVA) for
cells isolated from both normal and tumor lung tissue. A minimum of
0.5.times.10.sup.6 cells 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 LCAT 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 LCAT targets were all purchased by BD
Biosciences and PE-conjugated. The isotype control antibody used
for these experiments was PE-conjugated mouse IgG1k.
Western Analysis
[0431] Western blot analysis of target proteins are carried out
using whole cell or tissue extracts prepared. To make cell
extracts, the cells are 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 are centrifuged at 5,000.times.g
for 5 minutes at 4.degree. C. The supernatants are collected and a
protease inhibitor cocktail (Sigma) is added. The Pierce BCA assay
is used to quantitate total protein. Samples are separated by
SDS-PAGE and transferred to either a nitrocellulose or PVDF
membrane. The Western Breeze kit from Invitrogen is used for
Western blot analysis. Primary antibodies are either purchased from
commercially available sources or prepared using one of the methods
described in Section 5. For this application, antibodies are
typically diluted 1:500 to 1:10,000 in diluent buffer. Blots were
developed using Pierce NBT.
Results
[0432] A range of LCAT targets were validated by Taqman mRNA
expression. Targets such as CD73 (FIG. 5), CD98 (FIGS. 7, 8, 11),
CD49f (FIG. 3), CD90 (FIG. 6) are shown to be overexpressed at mRNA
level in lung tumor tissues.
[0433] FACS analysis was performed using LCATs in lung cell line or
lung tumor tissue. Targets such as CD 98 (FIG. 7), CD49f (FIGS. 3 4
and 9), CD59 (FIG. 10), CD73 (FIGS. 4 and 5), CD147 (FIG. 13), as
well as CD151, CD142, CD55 and CD46 (FIG. 4).
7. Detection and Diagnosis of LCAT by Liquid Chromatography and
Mass Spectrometry (LC/MS)
[0434] The proteins from cells are prepared by methods known in the
art.
[0435] 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:
[0436] 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.
[0437] Similar experiments are 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 used 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).
[0438] 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
[0439] 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.
[0440] 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.
[0441] An identical set of tissues 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
[0442] 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
[0443] For each antibody, a positive and negative control sample
was generated using data from the ICAT analysis of the lung cancer
cell lines. Cell lines were selected that are known to express low
levels of a particular target as determined from the ICAT data.
Similarly, a lung tumor line was selected that was determined to
overexpress the target was selected.
Antigen Retrieval
[0444] 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
[0445] 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 incubations to ensure a
moist environment. Sections were rinsed twice for 5 minutes in PBS,
shaking gently.
Develop and Counterstain
[0446] 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; and 1.25 microliters
hydrogen peroxide
[0447] 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.
[0448] 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
[0449] Tissue Factor: In normal tissues, antibody to tissue factor
or CD142 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).
[0450] Kunitz Inhibitor-1: 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 is overexpressed in breast, non-small cell lung, and
prostate, ovarian as well as pancreatic cancers.
[0451] CD98: FIG. 12 shows that CD98 is overexpressed in lung,
kidney, liver, ovary, pancreatic and gastric tumor tissues.
9. IHC Staining of Frozen Tissue Sections
[0452] Fresh tissues are embedded carefully in OCT in plastic mold,
without trapping air bubbles surrounding the tissue. Tissues are
frozen by setting the mold on top of liquid nitrogen until 70-80%
of the block turns white at which point the mold is placed on dry
ice. The frozen blocks were stored at -80.degree. C. Blocks are
sectioned with a cryostat with care taken to avoid warming to
greater than -10.degree. C. Initially, the block is equilibrated in
the cryostat for about 5 minutes and 6-10 mm sections are cut
sequentially. Sections are allowed to dry for at least 30 minutes
at room temperature. Following drying, tissues are stored at
4.degree. C. for short term and -80.degree. C. for long term
storage.)
[0453] 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).
[0454] Following incubation, the sections are washed gently in
Tris-saline for 3-5 minutes and then in Tris-saline/2.5% serum for
another 3-5 minutes. If a biotinylated primary antibody is used, in
place of the secondary antibody incubation, slides are covered with
100 ul of diluted alkaline phosphatase conjugated streptavidin,
incubated for 30 minutes at room temperature and washed as above.
Sections are incubated with alkaline phosphates substrate (1 mg/ml
Fast Violet; 0.2 mg/ml Napthol AS-MX phosphate in Tris-Saline pH
8.5) for 10-20 minutes until the desired positive staining is
achieved at which point the reaction is stopped by washing twice
with Tris-saline. Slides are counterstained 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
[0455] 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
[0456] 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, 500 cells plus serum
(pre- and posttherapy), or 50 .mu.l cells plus mouse serum as a
control. The plates are incubated in a cold room on a shaker for 45
min. Human complement of a 1:5 dilution (resuspended in 1 ml of
ice-cold water and diluted with 3% human serum albumin) is added to
each well at a volume of 100 .mu.l Control wells include those for
maximum release of isotope in 10% Triton X-100 (Sigma) and for
spontaneous release in the absence of complement with medium alone.
The plates are incubated for 2 h at 37.degree. C., centrifuged for
3 min, and then 100 .mu.l of supernatant is removed for
radioactivity counting. The percentage of specific lysis is
calculated as follows: % cytotoxicity=[(experimental
release-spontaneous release)/(maximum release-spontaneous
release)].times.100. A doubling of the CDC to >20% is considered
significant.
12. In Vitro Assays in Cell Lines
RNAi
[0457] RNAi is performed by using Smartpools (Dharmacon), 4-for
Silencing siRNA duplexes (Qiagen) or scrambled negative control
siRNA (Ambion). Transient transfections are carried out in
triplicate by using either Lipofectamine 2000 from Invitrogen
(Carlsbad, Calif.) or by using GeneSilencer from Gene Therapy
Systems (San Diego, Calif.) in methods described below. 1 to 4 days
after transfections, total RNA is isolated by using the RNeasy 96
Kit (Qiagen) according to manufacturer's instructions and
expression of mRNA is quantitated by using TaqMan technology.
Protein expression levels are examined by flow cytometry and
apoptosis and proliferation assays are performed daily using
Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous One
Solution Cell Proliferation Assay (see protocols below).
[0458] i) RNAi Transfections-Lipofectamine 2000
[0459] Transient transfections are carried out on sub-confluent
lung cancer cell lines as previously described. Elbashir, S. M. et
al. (2001) Nature 411: 494-498; Caplen, N. J. et al. (2001) Proc
Natl Acad Sci USA 98: 9742-9747; Sharp, P. A. (2001) Genes and
Development 15: 485-490. Synthetic RNA to gene of interest or
scrambled negative control siRNA is transfected using lipofectamine
according to manufacturer's instructions. Cells are plated in 96
well plates in antibiotic free medium. The next day, the
transfection reagent and siRNA are prepared for transfections as
follows: Each 0.1-1 ul of lipofectamine 2000 and 10-150 mM siRNA
are resuspended 25 ul serum-free media and incubated at room
temperature for 5 minutes. After incubation, the diluted siRNA and
the lipofectamine 2000 are combined and incubated for 20 minutes at
room temperature. The cells are then washed and the combined
siRNA-Lipofectamine 2000 reagent added. After further 4 hours
incubation, 50 ul serum containing medium is added to each well. 1
and 4 days after transfection, expression of mRNA is quantitated by
RT-PCR using TaqMan technology and protein expression levels are
examined by flow cytometry. Apoptosis and proliferation assays are
performed daily using Apop-one homogeneous caspase-3/7 kit and
CellTiter 96 AQueous One Solution Cell Proliferation Assay (see
protocols below).
[0460] ii) RNAi Transfections-GeneSilencer
[0461] Transient transfections are carried out on sub-confluent
lung cancer cell lines as previously described. Elbashir, S. M. et
al. (2001) Nature 411: 494-498; Caplen, N. J. et al. (2001) Proc
Natl Acad Sci USA 98: 9742-9747; Sharp, P. A. (2001) Genes and
Development 15: 485-490. Synthetic RNA to gene of interest or
scrambled negative control siRNA is transfected using GeneSilencer
according to manufacturer's instructions. Cells are plated in 96
well plates in antibiotic free medium. The next day, the
transfection reagent and the synthetic siRNA are prepared for
transfection as follows: predetermined amount of Gene Silencer is
diluted in serum-free media to a final volume of 20 ul per well.
After resuspending 10-150 mM siRNA in 20 ul serum-free media, the
reagents are combined and incubated at room temperature for 5-20
minutes. After incubation, the siRNA-Gene Silencer reagent is added
to each well and incubated in a 37.degree. C. incubator for 4 hours
before an equal volume of serum containing media was added back to
the cultured cells. The cells are then incubated for 1 to 4 days
before mRNA, protein expression and effects on apoptosis and
proliferation are examined.
Testing of Functional Blocking Antibodies
[0462] Sub-confluent lung cancer cell lines are serum-starved
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
[0463] 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 using an excitation wavelength of 485+/-20 nm and
an emission wavelength of 530+/-25 nm.
Proliferation
[0464] 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.
Cell Invasion
[0465] 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
[0466] 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, 100-0 samples are taken for colorimetric readings.
mRNA Expression
[0467] RNA are obtained as the method set forth above. 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. Percentage knockdown is then
calculated relative to the no addition control.
13. In Vivo Studies by Using Antibodies
[0468] Treatment of Lung Cancer Cells with Monoclonal
Antibodies.
[0469] Lung 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-LCAT 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 LCAT Protein with
Monoclonal Antibodies.
[0470] NIH 3T3 expressing LCAT proteins are treated with different
concentrations of anti-LCAT MAbs. Cell growth inhibition is
monitored.
In Vivo Treatment of NIH 3T3 Cells Overexpressing LCAT with
Anti-LCAT Monoclonal Antibodies.
[0471] NIH 3T3 cells transfected with either a LCAT 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-LCAT monoclonal antibody of the IG2A subclass is injected
intraperitoneally. Tumor occurrence and size are monitored for 1
month period of treatment.
[0472] 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=US20110219464A1).
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=US20110219464A1).
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